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Characterization of Abelson murine leukemia virus-transformed midgestation embryonic cells and their normal homologues

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Characterization of Abelson murine leukemia virus-transformed midgestation embryonic cells and their normal homologues
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Siegel, Michael L., 1949-
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ix, 220 leaves : ill. ; 29 cm.

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Bone marrow ( jstor )
Cell lines ( jstor )
Cells ( jstor )
Cultured cells ( jstor )
Histamines ( jstor )
In vitro fertilization ( jstor )
Interleukins ( jstor )
Mast cells ( jstor )
Rats ( jstor )
Receptors ( jstor )
Dissertations, Academic -- Immunology and Medical Microbiology -- UF ( mesh )
Immunology and Medical Microbiology thesis Ph.D ( mesh )
Leukemia Virus, Murine ( mesh )
Muridae -- embryology ( mesh )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida, 1986.
Bibliography:
Includes bibliographical references (leaves 189-218).
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Also available online.
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Typescript.
General Note:
Vita.
Statement of Responsibility:
by Michael L. Siegel.

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CHARACTERIZATION OF ABELSON MURINE LEUKEMIA VIRUS-
TRANSFORMED MIDGESTATION EMBRYONIC CELLS
AND THEIR NORMAL HOMOLOGUES




By

MICHAEL L. SIEGEL


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY


UNIVERSITY OF FLORIDA


1986








ACKNOWLEDGEMENTS


This dissertation and my accomplishments over the past

several years are the result of a team effort. I wish to

acknowledge the contributions of all of my "teammates," and

hope that I slight none by inadvertent oversight.

First, I would like to thank my friend, mentor, and

supervisory chairperson, Edward J. Siden. His profound

powers of perception and his ability to assimilate new

observations into an existing body of knowledge have set

ideals which I shall take with me. His friendship and

camaraderie will always be of value to me.

Second, I wish to acknowledge and thank my supervisory

committee, J. Bert Flanegan, Carlo Moscovici, Steve Russell,

and Roy Weiner, who collectively guided me through my

dissertation research, sometimes despite my reluctance and

protestations.

My family has been a constant source of support,

cheer, and inspiration. My wife Jeremie has coped with

every crisis I brought home and has survived more mood

fluctuations in the last few years than most people endure

in a lifetime. David and Rebecca have also endured the

journey with few complaints and good humor, and it is to

them I owe the preservation of my humor. I must also thank

my father, Seymour Siegel, who inspired me to strive to be

the best at whatever I tried (even if I was a garbage man),








and my mother, Frances Heifer Siegel, who showed me the

value of patience and determination. My brother, Victor,

is also acknowledged for encouraging me to read and learn.

I owe much of my recent success to the support of my

fellow graduate students. In particular, I express my

thanks to Randy Horwitz, who has helped me stay young,

sharpened my cynical wit, shared my most profane moments,

and provided me with friendship which has endured almost

six years in Gainesville.

Finally, I wish to recognize the faculty and staff

(past and present) of the Department of Immunology and

Medical Microbiology who have made my experience more

fulfilling. In particular, I wish to thank Ken Berns, who

encouraged me to return to graduate studies after a seven

year hiatus. I am also indebted to Catherine and Richard

Crandall, George Gifford, and Michael Boyle, for a

seemingly endless supply of reagents and advice. Last, but

not least, I thank Muriel Reddish, Patrice Boyd, Ellen

Boukari and their superb staffs, without whom the work

would probably have taken six additional years.


iii









TABLE OF CONTENTS


Page

ACKNOWLEDGEMENTS ii

LIST OF TABLES vi

LIST OF FIGURES vii

ABSTRACT viii

CHAPTERS

I INTRODUCTION AND REVIEW OF THE LITERATURE 1

Introduction: Hematopoietic Cell Differentiation
and Tumor Models 1
Mast Cell Function, Origin, Ontogeny, and
Heterogeneity 6
Connective Tissue Mast Cells 18
Mucosal Mast Cells 19
Basophils 25
Mastocytomas 26
Culture-Derived Mast Cells 28
Other In Vitro-Derived Metachromatic Cells 53
Relationship of In Vivo- and In Vitro-Derived
Mast Cells 56
Epilogue 59

II ABELSON MURINE LEUKEMIA VIRUS-INFECTED CELLS
FROM MIDGESTATION PLACENTA EXHIBIT MAST CELL
AND LYMPHOID CHARACTERISTICS 61

Introduction 61
Materials and Methods 63
Results 77
Discussion 95

III CHARACTERIZATION OF MAST CELLS DERIVED FROM
MIDGESTATION EMBRYONIC TISSUES IN LIQUID
CULTURE 1.05

Introduction 105
Materials and Methods 106
Results 117
Discussion 135








IV ISOLATION, ENUMERATION, AND CHARACTERIZATION OF
IN VITRO MAST CELL PRECURSORS DERIVED FROM
MIDGESTATION EMBRYONIC PLACENTA 144

Introduction 144
Materials and Methods 146
Results 154
Discussion 172

V SUMMARY AND CONCLUSIONS 183

REFERENCES 189

BIOGRAPHICAL SKETCH 219









LIST OF TABLES


Page


Table II-I Lineage-Specific Antibodies Used in Surface
Marker Analysis

Table 11-2 Histamine Content of Embryonic Tumor Cell
Lines and Control Tumor Cell Lines

Table 11-3 Analysis of Lineage-Specific Surface
Determinants on A-MuLV-Transformed
Embryonic and Control Tumor Cell Lines

Table 11-4 Analysis of Surface Membrane Receptors for
IgE and IgG on A-MuLV-Transformed Embryonic
Cell Lines and on Control Tumor Cell Lines

Table 11-5 Metachromatic Granules in A-MuLV-Transformed
Embryonic Cell Lines and Control Tumor Cell
Lines

Table 11-6 Interleukin 3 Content of Conditioned Media
and Cell Lysates of A-MuLV-Transformed
Embryonic Cells and Control Tumor Cells


Table III-1



Table IV-1


Effect of Cocultivation of Culture-Derived
Mast Cells with Adherent Cells and Their
Conditioned Media

Frequency of Mast Cell Precursors in Adult
Bone Marrow from Homozygous and
Heterozygous Mice


Table IV-2 Expression of Surface and Cytochemical
Markers on Colony-Derived Mast Cells


Table IV-3

Table IV-4


Sorting of Control Cells by Rosetting

Sorting of Bone Marrow Cells by Surface
Determinants


133



162


165

167


169






LIST OF FIGURES


Page


Figure II-1



Figure 11-2


Detection of Cell Surface Determinants
on Abelson Murine Leukemia Virus-
Transformed Embryonic Cells

Detection of Surface Receptors for IgE
on A-MuLV-Transformed Embryonic Cells


Figure 11-3 Virus-Transformed Cells Contain Abelson
Murine Leukemia Virus-Specific DNA
Sequences


Figure 11-4



Figure III-i



Figure III-2


Figure III-3



Figure III-4



Figure III-5


Figure III-6




Figure III-7



Figure IV-1


Tumors Isolated from Mice Injected with
Cell Lines 10P12 and 11PO Contain
A-MuLV-Specific DNA Sequences

Progression of Hematopoietic Lineage
Markers in Long-Term Mast Cell Cultures
Derived from Embryonic Tissues

Metachromatic Granules of Long-Term,
Culture-Derived Embryonic Mast Cells

Progression of Hematopoietic Lineage
Markers In Long-Term Mast Cell Cultures
Derived from Adult Bone Marrow

Population Dynamics of Bone Marrow-
Derived Mast Cells Infected with
A-MuLV

Expression of Ly5 Antigen on A-MuLV-
Infected Mast Cells

Mixed Population of RA3-3Al-Positive
Lymphoid Cells and RA3-3Al-Negative
Cultured Mast Cells in Long-Term Bone
Marrow Cultures

Abelson Murine Leukemia Virus-Infected
Mast Cells Express v-abl Gene
Product

Colonies in Long-Term Agar Cultures of
Embryonic Cells in Conditioned Media


Figure IV-2 Frequency of Mast Cell Precursors in
Midgestation Embryonic Tissues


94



120


121



124



126


128




130



131


156


158


Figure IV-3


Frequency of Placental Mast Cell
Precursors in the Third Trimester of
Gestation


vii


160












Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

CHARACTERIZATION OF ABELSON MURINE LEUKEMIA VIRUS-
TRANSFORMED MIDGESTATION EMBRYONIC CELLS
AND THEIR NORMAL HOMOLOGUES

By

Michael L. Siegel

May 1986

Chairman: Edward J. Siden
Major Department: Immunology and Medical Microbiology

The embryonic origin and ontogeny of mast cells is

poorly understood, despite a growing body of literature

relevant to that area of study. We have systematically

investigated the development of mast cells in the embryonic

mouse, beginning our studies with the observation of mast

cell characteristics of midgestation embryonic placental

cells transformed with the defective retrovirus, Abelson

murine leukemia virus. Unlike previously reported Abelson

virus-transformed cells, the placental cell lines exhibited

many of the characteristics of culture-derived mast cells,

including differentiation antigens, high affinity receptors

for IgE, and metachromatic granules containing histamine

and sulfated proteoglycans. Some of the cell lines also

expressed the B220 marker previously reported to be

specific for cells of the B lymphoid lineage. We also


viii










developed a simple, sensitive, nonfluorometric, nonisotopic

assay to detect membrane receptors for immunoglobulins.

The observation of the mast-like, Abelson virus-

transformed cell lines led us to investigate the presence

of mast cell precursors in normal midgestation embryonic

tissues. We found embryonic precursors to mast cells in

homologous, noninfected tissues and conducted a detailed,

systematic analysis of the differentiation of mast cells in

liquid cultures over the course of several weeks of

selection and enrichment. We also studied the effects of

Abelson virus infection and adherent cell cytokines on

lymphoid differentiation antigens in mast cell cultures.

Mast cell precursors in embryonic tissues of mid- and

late gestation were quantitated by a clonal assay. We

described the embryologically earliest reported mast cell

precursors in the mouse and report that the mouse embryo is

a rich reservoir of such precursors, containing

proportionately at least as many such cells as adult bone

marrow. We have observed that mast cells which

differentiate in agar culture, like some of the Abelson

virus-transformed cell lines, express the B220 determinant.

We have also described preliminary experiments in which we

selected mast cell precursors in bone marrow on the basis

of surface membrane determinants.









CHAPTER I
INTRODUCTION AND
REVIEW OF THE LITERATURE

Introduction: Hematopoietic Cell Differentiation
and Tumor Models


The ontogeny of the hematopoietic system of the mouse

can be viewed as a progression of finite, genetically

programmed stages in the maturation of pluripotent stem

cells into the terminally differentiated state of each of

the various blood lineages. Pluripotent hematopoietic stem

cells, defined by their ability to reconstitute lethally

irradiated recipients (Till and McCulloch, 1961), are first

detected in the murine yolk sac between eight and twelve

days of gestation (Tyan, 1968). Beginning with day ten and

throughout the remainder of gestation, cells with the same

differentiative capacity are found in the fetal blood and

liver (Moore and Metcalf, 1970). In the adult, pluripotent

hematopoietic stem cells are found in the bone marrow (Till

and McCulloch, 1961) and spleen (Nakahata and Ogawa, 1982).

The mechanisms involved in the differentiation of

pluripotent hematopoietic stem cells into mature,

functional blood elements are, for the large part, unknown.

These pathways may involve the interaction of pluripotent

or committed progeny stem cells with other cells or

macromolecular products in their inductive environment

(Kincade et al., 1981a), resulting in the cell's commitment

to one of several genetically programmed, phenotypically

distinguishable chains of events; alternately, random
I











stochastic processes may play a role in the differentiation

of hematopoietic cells (Nakahata et al., 1982a; Suda et

al., 1984).

The differentiation pathways of hematopoietic cells

and the molecular events involved in normal hematopoiesis

are best understood and defined by delineating discrete

cellular intermediates. Observation and identification is

frequently hampered by hematopoietic tissue heterogeneity,

short life span, and low frequency of cells of interest.

These difficulties are overcome in part by virus-induced

transformation of such cells, resulting in relatively

homogeneous populations of adequate size and frequently

unlimited growth potential. Although transformed

homologues of normal cells are frozen at a particular stage

of development by the action of the transforming gene

product, virus infection may also induce phenotypic changes

which are unparalleled in the course of normal

differentiation. To avoid this pitfall, it is therefore

prudent to confirm that the characteristics of tumor cell

models of early blood progenitors mimic their naturally

occurring counterparts.

Abelson murine leukemia virus (A-MuLV) is a

replication-defective retrovirus which arose by

recombination of portions of the genome of the replication-

competent, thymotropic Moloney murine leukemia virus and a

cellular gene, c-abl (Shields et al., 1979; Goff et al.,











1980) in a prednisolone-treated BALB/cCR mouse (Abelson and

Rabstein, 1970). A-MuLV is capable of rapid transformation

of bone marrow-derived thymus-independent lymphoid cells in

vivo (Sklar et al., 1975; Premkumar et al., 1975) and in

vitro (Rosenberg et al., 1975; Baltimore et al., 1979;

Rosenberg and Baltimore, 1976a, 1980).

Although most of the in vivo transformants reportedly

have been of the B cell phenotype (Premkumar et al., 1975),

plasmacytomas have also been reported (Potter et al.,

1973). T cell lymphomas (Cook, 1982), myelomonocytic

leukemias (Raschke et al., 1978), and mast cell tumors

(Mendoza and Metzger, 1976; Risser et al., 1978; Pierce et

al., 1985) have also resulted from in vivo infections,

indicating that Abelson virus may affect the growth and

differentiation of multiple hematopoietic lineages.

Abelson murine Leukemia virus is capable of

transforming both hematopoietic and nonhematopoietic cells

in vitro. Rosenberg and Baltimore tRosenberg et al.,

1975; Rosenberg and Baltimore, 1976a, 1976b) have developed

an in vitro culture system for the transformation and

clonal proliferation of lymphoid cells from murine

hematopoietic tissues. Under well-defined conditions,

permanent cell lines with pre-B lymphoid characteristics

(Siden et al., 1979) have been generated. These cell lines

are believed to exhibit the earliest known differentiation

markers and immunoglobulin gene organizational structures











of pre-B cells (Boss et al., 1979; Siden et al., 1979; Alt

et al., 1981, 1984). Using identical conditions for

transformation of mouse placenta and fetal liver, Waneck

and Rosenberg (1981) described colonies of cells expressing

various differentiated erythroid characteristics,including

cessation of growth. Unlike its Moloney leukemia virus

ancestor, A-MuLV transforms the NIH/3T3 fibroblast cell

line (Scher and Siegler, 1975).

Modifications of the original Rosenberg and Baltimore

in vitro transformation protocol have resulted in the

transformation of phenotypically disparate lineages.

Whitlock and colleagues (1983) and Serunian and Rosenberg

(1986) have reported the transformation of more

differentiated B lineage cells from in vitro-derived

"normal" populations. More recently, permanent cell lines

expressing mast cell characteristics have been reported

following transformation of midgestation embryonic placenta

(Siegel et al., 1985) and third trimester fetal liver

targets (Pierce et al., 1985).

Recent reports from several laboratories indicate that

retroviruses may alter the growth factor requirement of

cells of several lineages. Rapp and colleagues (1985)

reported the development of factor-independent cell lines

following transformation of interleukin 2-dependent T

lineage and interleukin 3-dependent myeloid cell lines by

recombinant viruses bearing the v-myc oncogene. Abelson











virus was similarly capable of abrogating the interleukin

3-dependence of both myeloid (Greenberger et al., 1979;

Cook et al., 1985) and mast (Pierce et al., 1985; Chapter

III, this dissertation) lineages. Taken with the

observation of erythropoietin-independent evthroid cells

(Waneck and Rosenberg, 1981) and interleukin 3-independent

mast cells (Pierce et al., 1985; Siegel et al., 1985) from

A-MuLV-infected primary cell populations, the data suggest

that A-MuLV may alter the requirements of these cells for

growth factors while permitting the cells to differentiate.

Although the precise role of the v-abl oncogene in

maintaining factor-independent proliferation is not yet

known, it is interesting to note that the cellular

homologue, c-abl, is transcribed at its highest level in

the developing embryo at the same time that the number of

A-MuLV targets reaches its peak. At least two retroviral

oncogene products have recently been shown to have

analogous structures in normal cells. The epidermal growth

factor receptor exhibits striking similarity to the erb B

oncogene product of avian erythroblastosis virus (Downward

et al., 1984), while the v-sis oncogene of simian sarcoma

virus encodes a protein structurally and immunologically

related to platelet-derived growth factor (Doolittle et

al., 1983; Robbins et al., 1983; Waterfield et al., 1983).

Although the function of c-abl has not been established, it

is known that all of the hematopoietic lineages sensitive











to A-MuLV transformation are also sensitive to the

proliferative effects of interleukin 3 (reviewed in Iscove

and Roitsch, 1985; Rennick et al., 1985). Seminal studies

on the culture of mast cells revealed that cells of this

lineage would proliferate in the presence of media

conditioned by lectin- or antigen-stimulated T lymphocytes

or by the myelomonocytic leukemia cell line WEHI-3B

(Hasthorpe, 1980; Nabel et al., 1981; Nagao et al., 1981;

Schrader, 1981; Tertian et al., 1981). More recently,

interleukin 3, the proliferative factor in the conditioned

media, was purified to homogeneity (Ihle et al., 1983;

Razin et al., 1984a). Subsequent studies performed with

the glycoprotein product of cloned interleukin 3 gene have

substantiated the proliferative activity of the factor

(Yokota et al., 1984; Rennick et al., 1985).

Mast Cell Function, Origin, Ontogeny, and Heterogeneity

Contemporary knowledge and interest in the mast cell

has its roots in the midnineteenth century. The earliest

description of these cells is found in the work of von

Recklinghausen (1863), who observed and illustrated the

mast cell in the unstained mesentery of the frog. Credit

for the discovery of the mast cell, however, is generally

assigned to Paul Ehrlich, a young physician-scientist who

was then interested in the differential staining affinities

of certain tissue cells and their organelles. Ehrlich

(1877) first described mast cell-like cells in several











species as elements which stained atypically red-violet

with the blue basic analine dye dahlia. The term mast cell

is derived from Ehrlich's nomenclature (Mastzellen), which

he assigned to granular cells which were abundant in well-

nourished tissues of frogs (Ehrlich, 1879). In the same

work, Ehrlich first used the term "Metachromasie," or

metachromasia, to describe the anomalous staining of these

connective tissue cells. Aside from their role as

histochemical curiosities, however, few references to mast

cell derivation, functions, and heterogeneity were

published in the ensuing fifty years (Selye, 1965).

Mature mast cells have been attributed with a number

of physiologic functions, many of which have been reviewed

in the recent literature (Austen, 1984; Shanahan et al.,

1984; Katz et al., 1985a; Lagunoff, 1985). The most widely

known mast cell function is the anaphylactic response,

which was first described by Selye in 1937 (cited in Selye,

1965). The response was originally experimentally induced

in rats by intraperitoneal injection of egg white and

produced hyperemia and edema of the lips, ears, paws, and

genitalia which were aggravated by adrenalectomy and

ablated by stressors like formaldehyde, which induce

adrenocortical hyperplasia. The release of histamine

during anaphylactoid inflammation was hypothesized at this

time, although release of the mediator was not

experimentally associated with mast cells until 1954











(Benditt et al., 1954). Other substances shown to be

released during the anaphylactic response include heparin

and related proteoglycans, leukotrienes, prostaglandins, 5-

hydroxytryptamine and other amines, and neutral proteases

(reviewed in detail in Austen, 1984).

Mast cells have been directly or indirectly implicated

in a number of other physiologic roles. Histamine has been

associated with modulation and regulation of the immune

system (Askenase et al., 1981) including reduction of T

cell effector function (Plaut et al., 1973; Schwartz et

al., 1980) and decreased lymphokine production (Rocklin,

1976). Mast cells have also been implicated in delayed-

type hypersensitivity reactions (Askenase, 1977), immune

complex formation (Benveniste et al., 1972), natural

cytotoxicity (Farram and Nelson, 1980; Henderson et al.,

1981), and parasite resistance (Capron et al., 1978), as

well as elaboration of a factor akin to interleukin 1 which

potentiates inflammation and collagenase activity in

synovial cells (Van Den Hoof and Tichelar-Guttar, 1983;

Yoffee et al., 1984, 1985).

Nonimmune functions have also been ascribed to mast

cells. "Microenvironmental hormones" (Lewis and Austen,

1981) produced by mast cells have been implicated in tissue

growth and repair (Kahlson and Rosengren, 1968) and

thyroxine secretion by the thyroid (Melander, 1977).

Histamine was shown to be required for timely blastocyst











implantation (Nalbandov, 1971; Dey and Johnson, 1980a,

1980b; Dey, 1981), as were prostaglandins (Kennedy, 1977),

but the number of detectable mast cells in the gravid

uterus was shown to decrease after implantation

(Shelesnyak, 1960; Brandon and Bibby, 1979). Mast cell

association with nerve tissue was first noted in 1878

(cited in Selye, 1965) and intimate contact between nerve

endings and mast cell plasma membranes was documented by

Weisner-Menzel and colleagues (1981) and Newson and

colleagues (1983). Histamine release has been attributed

to stimulation of cutaneous nerves (Kiernan, 1972), thus

establishing a neuroendocrine-mast cell axis. Mast cells

in alimentary tract mucosa have been implicated in

promotion of gut mobility (Erjavek et al., 1981; Fjellner

and Hagermark, 1981); histamine-induced gastric secretion

was shown to be blocked by histamine H2 receptor agonists

(Soll et ai., 1981) and enhanced by glucocorticoids

(Sathiamoorthy et al., 1976). The latter observation is

contradictory to published reports of glucocorticoid-

induced suppression of intestinal anaphylaxis (King et al.,

1985), and may indicate different modes of action of

steroids on sensitized and nonsensitized mast cells. Thus,

Daeron and colleagues (1982) noted that glucocorticoids

inhibited antigen-induced, but not calcium ionophore

A23187-induced, histamine release from mast cells.











The derivation of mast ceils in mammalian tissues has

been the subject of considerable investigation, and

sometimes heated debate, since the early nineteen sixties.

Because the developmentally earliest described mast cells

were observed in connective tissue, and a gradation of

"immature" to "mature" forms of mast cells could be

isolated from this source, several investigators proposed

that mast cells were derived directly from connective

tissue precursors (Burton, 1963; Michels, 1963). Combs and

colleagues (.1965) observed the development of mast cells in

embryonic rats between fifteen and twenty-three days post

coitum. The cells appeared to arise in undifferentiated

mesenchymal tissue and progressed through a gradation of

intermediates to characteristic mast cells. Asboe-Hanson

(1971) further noted that mast ceils in the skin appeared

to differentiate locally from mesenchymal elements.

Based on observations of profound mastocytosis

associated with immune and neoplastic lymphocyte

proliferation, a second faction proposed the hematopoietic

origin of mast ceils. Accordingly, Ginsburg (1963) removed

thymuses from mice and cultured them with embryonic skin

monolayers. The mast cells so derived led the author

(Ginsburg, 1963; Ginsburg and Lagunoff, 1967) to propose

that mast cells were derived from thymocytes, an

observation subsequently confirmed in the rat (Ishizaka et

al., 1976). Burnet (1965, 1975, 1977), observing that mast











cells exhibited evidence of thymic origin or dependence,

participated in immunologic reactions, were similarly

activated by lectins and immune stimuli, and were

relatively amitotic, speculated that the mast cells were
"post-mitotic" T lineage cells.

Other hematopoietic cell types have been postulated to

be mast cell by precursors in vivo and in vitro cultivation

techniques. Desaga and colleagues (1971) performed

repeated peritoneal lavages on rats to deplete them of

mature serosal mast ceils. Mast cell-deficient

peritoneal exudate cells, cytochemically identified as

monocytes at the beginning of in vitro growth, developed

into metachromatic, granulated mast cells within two days

of harvest. Similarly, Czarnetzki and Behrendt (1981)

reported that peritoneal exudate cells from mast cell

depleted rats (injected with sterile water

intraperitoneally) resembled mononuclear phagocytes both

morphologically and cytochemically before and shortly after

culture in L-cell conditioned media. The in vitro-

propagated population, however, differentiated into mast

cells which were identified morphologically and which

contained granules with histamine and alpha-naphthol

acetate esterase.

The hematopoietic origin of some, and perhaps all,

mast cells was defined in great detail by Kitamura and

colleagues in a series of reports beginning less than ten











years ago. Initially, they demonstrated that irradiated,

mast cell-deficient mice could be reconstituted by

injection of bone marrow from untreated donors (Kitamura et

al., 1977). Since irradiation did not eradicate all

recipient mast cells prior to reconstitution, a donor

strain with phenotypically distinct mast cell granules

(beige) was used to definitively demonstrate the origin of

the cells which were detected. Similar experiments, using

unirradiated, genetically mast cell-deficient W/Wv mice,

demonstrated that adult bone marrow (Kitamura et al., 1978;

Hatanaka et al., 1979), blood (Kitamura et al., 1979a) and

spleen (Kitamura et al., 1979d) were rich reservoirs of

mast cell precursors which were also found in smaller

numbers in thymus, lymph node, and Peyer's patches

(Kitamura et al., 1979d). Mast cells were also detected in

fetal liver populations despite the apparent lack of mature

mast cells in that tissue (Kitamura et al., 1979c).

The hematopoietic nature of the in vivo mast cell

precursor was further defined by the same group. fn

preliminary studies, Kitamura and colleagues (1981) showed

that genetically mast cell-deficient W/Wv mice could be

reconstituted with cells from individual spleen colonies of

normal (C57BL/6) mice which had been irradiated and

subsequently reconstituted with bone marrow cells. Having

assured themselves of the clonality of each donor spleen

colony (by injecting mixed phenotypically distinct beige











and wild type bone marrow cells into the primary,

irradiated recipients and screening the colonies for cells

with only one type of granule), the authors were able to

conclude that the colony forming unit of the spleen (Till

and McCulloch, 1961) was the ultimate progenitor cell for

mast cells in the spleen, stomach, caecum, and skin as well

as for peripheral blood granulocytes and erythrocytes.

These results were corroborated by Sonada and colleagues

(1983), who demonstrated that late (twelve days post

injection) spleen colonies, which included both erythroid

and myeloid elements, contained mast cell precursors which

differentiated in secondary recipient skin.

The ontogeny of embryonic mast ceils in mice is

meagerly defined in the literature. As previously

mentioned, in vivo mast cell precursors are present,

despite the absence of more mature forms, in the mouse

fetal liver thirteen and more days post coitum (Kitamura

et al., 1979c). Embryonic mast cell ontogeny was better

defined in the rat. Csaba and Kapa (1960) demonstrated the

presence of mast cells, which incorporated exogenous

heparin, in the thymus, spleen, lymph nodes, myocardium,

and kidney of day sixteen to seventeen rat embryos. By

studying sections through rat embryos between fifteen and

twenty-three days of gestation, Combs and colleagues (1965)

placed mast cells into four stages of differentiation based

upon their staining characteristics with alcian blue and











safranin, dyes which preferentially bind to the

glycosaminoglycan components of mast cell granules. By

this technique, alcian blue binds to poorly sulfated

glycosaminoglycans like chondroitin sulfate, while safranin

binds to highly sulfated molecules like heparin. A

gradation of cells, beginning with large, lymphocyte-like

elements with few, aician blue-stained granules (Stage I),

and progressing through characteristic mature mast cells

with small nuclei and large numbers of safranin-staining

granules (Stage IV). was documented. Mast cell stages were

also differentiated by nuclear characteristics (mitotic

figures), granule heparin (periodic acid-Schiff staining),

granule glycosaminoglycan synthesis (sodium sulfate uptake),

granule histamine (diazotized parabromoaniline reaction),

and granule protease (phenylproprionyl naphthol AS

reaction) content. The first recognizable mast cells

(Stage I) were found in the head mesenchyme at fifteen days

of gestation. Mast cell numbers rapidly increased during

the sixteenth day of gestation, with Stage II mast cells

found in the connective tissue of the dorsal vertebrae.

Subcutaneous (Stage III) mast cells were identified in

tissues of embryos eighteen and nineteen days post coitum.

Mature Stage IV mast cells were first present shortly

before birth and were indistinguishable from adult

connective tissue mast ceils.











The study of mast cells, which has accelerated

dramatically in the past quarter century, has revealed that

Ehrlich's "Mastzellen" are heterogeneous both

evolutionarily interspeciess) and functionally

(intraspecies). Mast cell heterogeneity has been the topic

of numerous general reviews in recent years (Enerback,1981;

Bienenstock et al., 1982, 1983; Pearce, 1982, 1983;

Shanahan et al., 1984; Jarrett and Haig, 1984; Austen,

1984: Katz et al., 1985a; Lagunoff, 1985; Pearce et al.,

1985). From this sea of literature, the differences

between mast ceils of various species are apparent. The

multipotent biogenic compound of mammalian mast cells,

histamine, has not been detectable in fishes and

amphibians, while serotonin is apparently unique to rodent

and dopamine to bovine mast cells. Heparin proteoglycan

from porcine, bovine, rat, and human sources is

heterogeneous in molecular weight and charge (Stevens and

Austen, L981). The proteolytic enzymes of mast cells are

also phylogeneticallv disparate; rodent mast cells contain

an alpha-chymotrypsin-like activity, while dog, human, and

turtle mast cells have trypsin-like activity and bird and

fish mast cells have no esteroprotease activity (Woodbury

and Neurath, 1980; Lagunoff, 1985). Ultrastructurally, the

granules of human mast cells appear to be organized in

crystalline scrolls, while rat mast cell granules are











dense, homogeneous spheres which dissociate into fibrillar

structures in hypertonic salt solution (Lagunoff, 1972).

The initial observation of intraspecies mast cell

heterogeneity is generally attributed to Maximow (1906),

who reported that the rat intestine was replete with mast

cells which differed from other rat mast cells in

morphology and stain affinity. These differences were

reinvestigated by Enerback, who, sixty years after

Maximow's observations, published a series of reports which

described in detail the differences in morphology, stain

affinities (Enerback, 1966a, 1966b), and sensitivity to

degranulating agents (Enerback, 1966c, 1966d) between

dermal mast cells (representative of the connective tissue

or serosal subset) and intestinal mast cells

(representative of mucosal or atypical mast cells). Thus,

mucosal mast ceils were shown to be smaller, possess uni-

or bilobed nuclei, and be less granulated than serosal mast

cells, and the granules of the former population were far

more heterogeneous in size than those of the latter.

Enerback also observed that the mucosal mast cells stained

red with acidic toluidine blue, while serosal mast cells

stained purple. He noted that standard formaldehyde

fixatives used to preserve serosal mast cell granules were

ineffective on mucosal mast cells, and selected and

adapted several fixatives (such as Carnoy's and Mota's

preparations) to more adequately preserve the more labile











mucosal mast cell granules. Mucosal mast cells also

required higher concentrations of thiazine dyes, like

toluidine blue, and azure A dyes, as well as prolonged

staining times, when compared to serosal mast cells, while

the granules of the former cells had higher affinity to

copper phthalocyanine dyes such as Astra blue at ph 0.3.

Enerback therefore concluded that the mucosal mast and

connective tissue mast cells differed not only

morphologically, but biochemically as well, and offered

that the mucosal mast cells contained less highly sulfated

mucopolysaccharides than the dermal cells. Finally,

Enerback noted that in rats systemically exposed to the

histamine releasing agent 48/80, serosal mast cells in the

mesentery, tongue, and skin were degranulated and therefore

undetectable, while mast cells in the duodenal mucosa were

unaffected and perhaps greater in number. While being

unable to explain the latter observation, Enerback was able

to conclude that mucosal mast cells differed from their

serosal counterparts functionally as well.

The differences between mucosal and serosal mast

cells, first noted by Maximow and then Enerback, have

since been appended by the observations of numerous

investigators and extend beyond those mentioned to surface

markers, histamine content, IgE receptors and

internalization of bound IgE, proteoglycan composition,

proteases, sensitivity to histamine secretagogues, effects











of neuropeptides and endorphins, thymus dependence, and

life span. These characteristics have been surveyed in

detail in reviews previously cited. We will therefore only

briefly survey the literature which is cogent to the

ultimate topic of this discussion, the in vitro, culture-

derived mast cell.

Connective Tissue Mast Cells

Although typical mast cells have been isolated from a

variety of connective tissue sources throughout the rodent

body, the most frequently studied member of this subset is

that which is isolated, free of extraneous tissues, from

the serosal surfaces of the peritoneal cavity. As

previously discussed, by injecting bone marrow into mast

cell-deficient hosts, Kitamura and colleagues (1977) were

able to demonstrate the relationship of hematopoietic

precursors to the serosal mast cells. The ultimate

precursor cell in the bone marrow was shown to be the

colony-forming unit of the spleen (Kitamura et al., 1981;

Sonoda et al., 1983). From the bone marrow, mast cell

precursors migrate through the blood (Kitamura et al.,

1979a; Zucker-Franklin et al., 1981; Sonoda et al., 1983)

and subsequently proliferate and differentiate in

connective tissue (Hatanaka et al., 1979; Kitamura et al.,

1979b,1979d). Connective tissue mast cells in rodents have

been found to proliferate and differentiate independently of

thymic influences. Thus, the athymic nude mouse has mast











cells in its connective tissues (Wlodarski, 1976; Reed et

al., 1982). Aldenborg and Enerback (1985) recently reported

that congenitally athymic rnu/rnu rats have at least as many

(or more) peritoneal mast cells as normal controls for the

first fourteen weeks of life; adult rnu/rnu rats, however,

have fewer peritoneal mast cells than their wild type

counterparts. These results may indicate that peritoneal

mast cell populations may be subject to thymic influences

later in life or may simply reflect a separate, thymus-

independent defect inherent in the athymic rat. Further

studies will be necessary to elucidate the apparent

contradiction.

The proteoglycan composition of serosal mast cell

granules has been the subject of study since the initial

discovery of heparin in the canine liver by Jorpes in 1937

(cited in Selye, 1965). Subsequently, heparin has been

identified in rat peritoneal mast cells (Tas and

Berndsen, 1977; Yurt et al., 1977; Stevens and Austen,

1982), human lung mast cells (Metcalfe et al., 1979), and

mouse peritoneal mast cells (Razin et al., 1982c).

Mucosal Mast Cells

Following the development of improved methods for

their fixation and staining by Enerback (1966a, 1966b), the

study of the atypical, or mucosal, mast cell accelerated

significantly. Early reports of similarities between

cultured, thymus-derived mast cells of the mouse (Ginsburg,











1963; Ginsburg and Lagunoff, 1967) and the rat (Ishizaka et

al., 1976) and Enerback's atypical mast cells of the lamina

propria spurred erroneous theories that the mucosal mast

cell was derived from the thymus (Burnet, 1965, 1975,

1977). Based upon observations of mucosal mastocytosis

following experimental infection of rats and mice with the

nematodes Nippostrongylus brasiliensis and Trichinella

spiralis, a lymphoid origin of these cells was also

proposed by other investigators (Rose et al., 1976; Befus

and Bienenstock, 1979; Mayrhofer, 1979a,1979b; Nawa and

Miller, 1979).

The derivation of mucosal mast cells was ultimately

resolved by Crowle (1982), who reconstituted mucosal mast

cell-deficient mice with cells derived from a variety of

hematopoietic tissues. Crowle observed that W/Wv mice

could be reconstituted by bone marrow and spleen cells but

not by thymocytes or thymus grafts, while athymic mice

could be reconstituted by thymus grafts, thymocytes or

splenocytes. Crowle proposed that the W/Wv mice were

defective in mucosal mast cell precursors which were

present in bone marrow (and spleen) of normal mice, while

athymic mice possessed the precursor population and needed

a thymus-related component to effect differentiation.

Crowle concluded that mucosal mast cells were derived from

bone marrow and required a thymic influence for

accumulation in mucosal surfaces. These relationships











were further reinforced by the same investigator (Crowle

and Reed, 1984). Reconstitution of athymic mice was

ablated by pretreatment of wild type mouse bone marrow

cells or splenocytes with anti-Thy 1 and complement, while

similar treatment of beige bone marrow or spleen cells

still resulted in the detection of some mast cells, albeit

fewer, in the mucosa of thymus-intact W/Wv mice.

The thymic dependence of mucosal mast cells, in

contrast to the thymus-independent growth and development

of serosal mast cells, has been documented by a number of

other investigators. Prior to the reports of Crowle

(1982,1984), Ruitenberg and Elgersma (1976) observed that

nude mice infected with Trichinella spiralis experienced no

intestinal mucosal mast cell response unless reconstituted

by thymus or parasite-immune thoracic duct cell grafts,

concluding that thymus-derived T-lineage cells were required

for mucosal mastocytosis. These results were substantiated

by a number of other researchers (Olson and Levy, 1976;

Mayrhofer and Bazin, 1981; Reed et al., 1982). Similar

studies were performed in the rat. Mayrhofer (1979a) noted

that the number of mucosal mast cells in Nippostrongylus

brasiliensis-infected rats increased in a pattern similar to

primary and secondary immune responses. Adult thymectomy or

chronic thoracic duct drainage several months prior to

nematode challenge (to deplete mature T cells) resulted in

significantly depressed intestinal mastocytosis, while











thymectomy shortly before challenge was ineffective in

ablating the mast cell response (Mayrhofer, 1979b). These

last observations indicated that the thymus, per se, is not

the immediate source of mast cells or mast cell growth

factors. Similar depression of mucosal mast cell response

and poor clearance of intestinal parasites were observed in

B rats, thymectomized, irradiated animals which were

reconstituted with bone marrow of T cell-depleted (chronic

thoracic duct drainage) donors (Mayrhofer and Fisher, 1979).

Interestingly, contrary to the reports of Crowle (Crowle,

1982; Crowle and Reed, 1984), unchallenged B rats, as well

as athymic nu/nu mice, were reported to have normal numbers

of mucosal mast cells when compared to appropriate controls

(Mayrhofer and Bazin, 1981).

Three other independent lines of evidence fortified

the hypothesis that mucosal mast cells were dependent on a

T cell-derived proliferation-differentiation factor.

First, the primary mucosal mast cell response to

Nippostrongylus brasiliensis in the rat was enhanced by

adoptive transfer of immune T cells (Nawa and Miller,

1979). Second, mucosal mastocytosis was demonstrated in a

variety of other immune scenarios, including the

inflammatory reactions of ulcerative colitis, Crohn's

disease, and pulmonary fibrosis (Askenase, 1980). Third,

Guy-Grand and colleagues (1984) recently reported the

direct stimulation of intestinal mucosal mast cell











precursors in BALB/c mice bearing the myelomonocytic

leukemia WEHI-3, a constitutive producer of the mast ceil

growth factor interleukin 3, which is identical to the mast

cell growth factor produced by activated T lymphocytes

(Yung et al., 1981).

A variety of other distinguishing characteristics have

been ascribed to the cells alternately called mucosal mast

cells, atypical mast cells, and histaminocytes (Code,

L977). Early studies of mucosal mast cells were performed

on tissue sections or on heterogeneous populations of cells

isolated from the gut mucosa. The previously cited

methodologies of Enerback (1966a, 1966b) were later

optimized by the inclusion of techniques which further

stabilized the granules (neutral formalin fixation) and

enzymatically (with trypsin) stripped stain-retarding

proteins from glutaraldehyde-treated preparations (Wingren

and Enerback, 1983). The recent development of methods for

isolating such cells from the small intestine, which

exploited the mucosal mastocytosis induced by parasitic

infection, was reported by Befus and colleagues (1982a),

and made it possible to analyze mucosal mast cells in the

absence of extraneous elements and to confirm some previous

observations. In contrast to peritoneal mast cells,

isolated mucosal mast cells are smaller and have a shorter

lifespan. Mucosal mast cells have fewer granules which

contain nonheparin, lower sulfated proteoglycans (as vet











biochemically undefined), less histamine, and serotonin

(Bienenstock et al., 1983).

The response of mucosal mast cells to a variety of

secretagogues has been the subject of a number of studies.

Similar to serosal mast cells, mucosal mast cells are

responsive to the degranulation effects of IgE and antigen,

IgE and anti-IgE, concanavalin A, ionomycin, and compounds

23187 and Br-X537A, albeit with the release of less of

their total histamine content (Befus et al., 1982a, 1982b;

Pearce et al., 1982). Enerback's early observations on the

insensitivity of rat mucosal mast cells to degranulation by

compound 48/80 in vivo were confirmed by other

investigators in canine (Lorenz et al., 1969; Rees et al.,

1981) and murine (Enerback, 1981) models. This

unresponsiveness was confirmed in isolated mucosal mast

cells and extended to the secretagogue Bee Venom Peptide

401 (Befus et al., 1982a, 1982b), for which membrane

receptors were found to be absent on mucosal, but not

serosal, mast cells (Pearce et al., 1982). The same group

also reported that, unlike serosal mast cells, mucosal mast

cells were unresponsive to enhanced, antigen-induced

secretion of histamine mediated by phosphatidyl serine.

Mucosal mast cells were also shown to be distinct from

serosal mast cells in their responsiveness to secretary

antagonists disodium chromoglycate, theophylline, and

AH9679, while both subsets were equally sensitive to the











degranulation inhibitory effects of Doxantrazole (Befus et

al., 1982b; Pearce et al., 1982, 1985).

Basophils

Cells with membrane receptors for IgE are not limited

to the mast cell lineage. Both lymphocytes (Gonzales-

Molina and Spiegelberg, 1978) and macrophages (Melewicz et

al., 1982) have been reported to bind IgE; the affinity of

the membrane receptors of the non-mast cells, however, was

ten to one-hundred times less than that of mast cells

(Ogawa et al., 1983). The best known of the mast-like

cells, however, are the basophils (basophilic

granulocytes), polymorphonuclear leukocytes which are

present in the blood of several mammalian species (Lagunoff

and Chi, 1980). Like mast cells, basophils contain

metachromatic granules and express surface membrane

receptors for IgE. Histamine is released by antigenic

challenge of IgE-bearing basophils (Lagunoff and Chi,

1980). The granules of basophils of several species have

been reported to contain chondroitin sulfate proteoglycan

(Olsson et al., 1970; Orenstein et al., 1978; Metcalfe et

al., 1980b) similar in its degree of sulfation to the

proteoglycan of mucosal mast cells (Tas and Berndsen,

1977). Basophilic granulocytes, however, are apparently

absent from mouse peripheral blood (Lagunoff and Chi,

1980). The relationship of these cells to culture-derived











mast cells in several species, including the mouse, will be

further discussed in a later section of this review.

Mastocytomas

The study of mastocytomas has contributed to the

understanding of mast cell growth, differentiation, and

function, and has provided the bridge between complex in

vivo investigations and better-defined, clonal population

analyses of in vitro cultures. Spontaneous mastocytoma,

while common in such species as dogs (Cobb et al., 1975;

Yoffee et al., 1984, 1985), is a more infrequent condition

in other species, notably rats and mice (Lagunoff, 1985).

Efrati and colleagues (1957) described human mastocytoma

cells as large, lymphocyte-like elements similar to the

early mast cell progenitors described in lower mammals

(Maximow, 1906).

The link between in vivo and in vitro mast cell

studies was established in 1959 when Schindler and colleagues

(1959) reported the successful adaptation of the

methylcholanthrene-induced, murine mastocytoma P815 (Dunn

and Potter, 1957) to growth in culture. The latter

investigators had isolated the neoplasm from a disseminated

disease with foci in the spleen and subcutaneous tissue and

had subsequently adapted it to a highly transplantable

ascites form from which a number of observations were made.

Variations in cell morphology, granule size and density,

and nuclear morphology were noted in the descriptive study.











In vitro analysis (Schindler et al., 1959) demonstrated

that the cells synthesized histamine and serotonin.

Through extensive culture, some sublines of P815 lost both

granules and intracellular histamine content; in some

sublines, however, the condition was reversed by addition

of sodium butyrate to the culture medium (Mori et al.,

1979).

Focal mastocytomas have frequently been associated

with the presence of lymphocytes (Galli and Dvorak, 1979;

Askenase, 1980), supporting the once popular hypothesis

that the latter cells were precursors to the former

(Burnett, 1965, 1975, 1977). It was also noted that some

mastocytomas, like basophils and culture-derived mast

cells, synthesize chondroitin sulfate proteoglycan rather

than the heparin (Lewis et al., 1973) found in serosal mast

cells.

Some mastocytomas have been reported during the course

of experiments analyzing Abelson murine leukemia virus-

induced lymphomagenesis (Mendoza and Metzger, 1976; Risser

et al., 1978). The mastocytomas generally arose in

peritoneal oil granulomas evoked by tetramethylpentadecane

(Pristane) in mice which were inoculated with Abelson virus

thirty to forty days after administration of the oil

(Pierce et al., 1985). The tumors were generally

transplantable into syngeneic hosts and frequently could be

adapted to growth in vitro. In a yet unexplained, but













perhaps related note, Hasthorpe (1980) reported the

isolation and adaptation to in vitro growth of a factor-

dependent mast-like cell line from splenocytes of a DBA-2

mouse infected with Friend ervthroleukemia virus. Although

electron microscopy established the presence of budding C-

type virus particles on the highly granular cells,

Hasthorpe's FMP1.1 cell line was nontumorigenic upon

intraperitoneal or subcutaneous administration to syngeneic

hosts and could not proliferate in the absence of exogenous

growth factor in vitro.

Culture-Derived Mast Cells

The growing body of knowledge concerning mast cells is

due, in part, to the development of methodologies for the

selection, enrichment, and maintenance of culture-derived

mast cells. These cells possess many of the

characteristics of their in vivo correlates, the mucosal

mast cells, as demonstrated in the following text. A note

of caution, however, must be interjected into the seemingly

logical flow between naturally occurring in vivo mast cells

and culture-derived mast cells. Although similar by a

variety of criteria including morphology, biochemistry,

function, and growth factor dependence, culture-derived

mast cells are not "normal" in the sense that they have

been produced in anatomically foreign, although perhaps

physiologically sufficient, conditions. They may therefore











be considered models of their in vivo homologues until

definitive evidence permits us to conclude that the two

populations are completely identical. We will therefore

continue to use the term "culture-derived" mast cells, and

similarly distinctive terms, to maintain the tenor of this

caveat throughout the following discussion.

Adherent Feeder Layer Studies

The earliest reports of culture-derived mast cells by

Ginsburg (Ginsburg,1963; Ginsburg and Sachs, 1963) involved

a complex system of mouse thymocytes cultured on feeder

layers of mouse embryonic fibroblasts. It was apparent to

the authors that the monolayer was essential for

proliferation of mast cells. Culture of thymocytes in the

absence of the feeder layer failed to produce mast cells.

On the other hand, culture of embryonic skin fibroblasts

from eighteen day fetuses, without additional thymocytes,

infrequently resulted in mast cell outgrowth. The tissue

source of the mast cells, therefore, was disputable, and

quite possibly both thymocytes and embryonic feeder layers

contributed progenitors to the mast cell culture. The

issue was better defined several years later when the same

group reported that irradiated embryonic fibroblast

monolayers, which could no longer produce mast ceils when











cultured alone, could support the growth of culture-derived

mast cells from cocultured thymocytes (Ginsburg and

Lagunoff, 1967).

Thus, the culture-derived mast cell era was ushered in

with the observation that at least some mast cell

progenitors were present in lymphoid tissue and that

adherent cells were required for in vitro mast cell

differentiation and growth. In seminal attempts to clone

mast cell precursors in soft agar, Pluznik and Sachs (1965)

reported that embryonic feeder layers were again required

for outgrowth of mast cells from disaggregated splenocytes.

Ishizaka and colleagues (1977). investigating rat thymus-

derived mast cells in a system modeled after that of

Ginsburg, observed that clonal expansion was more prolific

in the presence of a feeder layer, although mast cells were,

indeed, isolated from cultures containing only thymocytes.

In the latter case, however, the mast cells were observed

to be associated with islands of fibroblast-like adherent

cells which arose in the thymocyte cultures.

The origin of mast cells in feeder layer cultures was

better resolved, almost twenty years after its initial

identification, by the definition of two morphologically

distinct populations of mast cells in mixed cultures of

adult lymphoid and embryonic feeder layer cells (Ginsburg

et al., 1982). Mast cells derived from the feeder layer

were morphologically similar to those found in connective











and serosal tissues, while those of lymphoid origin (lymph

node and thoracic duct) resembled mucosal mast cells, being

smaller in size with sparser, but larger granules than the

former cells. Pure mucosall mast cells" (in fact, culture-

derived mast cells of lymphoid origin) could be grown on

selected feeder layers which were free of serosal type

precursors. The mucosall mast cells" persisted, however,

only in the presence of T cell-derived factors. In

contrast, the mast cells derived from embryonic feeder

layers continued to persist, albeit without further

expansion, for six months or longer in the absence of

exogenous factors.

The same group (Davidson et al., 1983) later reported

that lymph node cells derived from unimmunized, horse

serum-immunized, and helminth-infected mice, grown in the

presence of conditioned media (from antigen-stimulated

mesenteric lymph node cells) but in the absence of

irradiated embryonic mouse fibroblast feeder layers,

proliferated (as large, vacuolated cells) but failed to

develop granules. When the undifferentiated, culture-

derived cells were transferred to fibroblast monolayers,

however, the cells developed metachromatic granules

containing histamine within seven days. Intimate contact

between the two populations of viable cells was apparently

essential to granule maturation, as neither fibroblast

conditioned media, fibroblast homogenates, glutaraldehyde-











fixed fibroblasts, nor separation of fibroblasts from

"large lymphocyte" mast ceil precursors by a membrane could

effect the change.

Despite the initial success in culturing mast cells

from lymphoid tissue cocultivated with adherent cell feeder

layers, reports in the literature of the technique's use

were limited to those of the previously cited groups. Two

factors probably contributed to the limited use of adherent

cell monolayers in mast cell culture. First, the system

was quite complex, requiring considerable time and

extensive subculture (or irradiation) of feeder layers to

eliminate the contribution of connective tissue type mast

cell precursors. Secondly, and perhaps more significantly,

the development of culture-derived mast cells in media

conditioned by activated lymphocytes by at least three

independent groups provided the opportunity to maintain

mast cells in the absence of a continuous monolayer of

feeder cells. As previously noted, however, even in the

absence of fibroblast feeder layers, islands of adherent

cells are observed in early cultures of lymphoid and

hematopoietic tissue-derived mast cells (confirmed in our

studies; see Chapters III and IV). Since the adherent

ceils are present in such cultures before the selection and

enrichment of mast cells, it is, at this juncture,

plausible to speculate that the adherent cells may assume a

transient, maturational role in mast cell differentiation.











Conditioned Media-Dependent Mast Cells

The development of techniques for culturing mast cells

from hematopoietic and lymphoid tissues has led to an

exponential increase in mast cell research and literature

citations. With the burst of scientific activity, however,

has come a concurrent increase in the number of terms used

to describe culture-derived mast cells, including P

(persisting) cells (Schrader and Nossal, 1980), histamine-

containing granular cells (Sredni et al., 1983), mucosal

mast cells, basophil/mast cells, and atypical mast cells.

Despite the discrepancy of terms, however, the long term

suspension cultures of mast cells appear to be strikingly

similar. Mast cells have thus been derived from mouse bone

marrow (Tertian et al., 1980, 1981; Nagao et al., 1981;

Razin et al., 1981a, 1982a,b,c; Schrader, 1981; Schrader et

al., 1981; Galli et al., 1982b; Crapper and Schrader, 1983;

Sredni et al., 1983; Wedling et al., 1983, 1985; Yung et

al., 1983; Suda et al., 1985), spleen (Hasthorpe, 1980;

Schrader and Nossal, 1980; Schrader et al., 1980, 1981;

Schrader, 1981; Tertian et al., 1981; Crapper and Schrader,

1983; Sredni et al., 1983; Pharr et al., 1984), fetal liver

(Nabel et al., 1981; Razin et al., 1984b), peripheral blood

(Crapper and Schrader, 1983; Suda et al., 1985), thymus

(Tertian et al., 1981; Schrader, 1981; Davidson et al.,

1983), lymph nodes (Ginsburg et al., 1978; Crapper and

Schrader, 1983), and intestinal mucosa (Schrader et al.,











1983b). Similar cells have been cultured from rat bone

marrow (Haig et al., 1982, 1983), peripheral blood (Zucker-

Franklin et al., 1981; Czarnetzki et al., 1983), and thymus

(Ishizaka et al., 1976, 1977) as well as human fetal liver

(Razin et al., 1981b), umbilical cord blood (Ogawa et al.,

1983), and adult peripheral blood (Denburg et al., 1983;

Czarnetzki et al., 1984).

The development of in vitro methods for mast cell

culture provided additional means of detecting embryonic

mast cell precursors. As previously noted, Ginsburg (1963)

observed occasional mast cell outgrowth in cultures of day

eighteen embryonic mouse skin. Similar experiments with

the rat model demonstrated that embryonic rat thymus,

isolated between eighteen and twenty days post coitum and

cocultured with adult rat thymocvtes or thymocyte

conditioned media, contained cells capable of

differentiating into mast cells (Ishizaka et al., 1976). A

third group (Nabel et al., 1981; Galli et al., 1982a) was

able to culture murine mast cells derived from day thirteen

fetal liver suspensions cultured in lymphocyte conditioned

media. Using the adherent cell system, Ginsburg and

colleagues (1982) were also able to demonstrate that mast

cells could be derived in culture from disaggregated mouse

embryos between ten and thirteen days of gestation. It was

thus apparent that the precursors of culture-derived mast

cells were present in the mouse embryo at ten days post











coitem, several days before mast ceils per se were

observable in the embryo (Kitamura et al., 1979c).

Interleukin 3 From Lymphocyte-Conditioned Media And Other

Sources

The analysis of culture-derived mast cells and their

precursors evolved from studies of hematopoietic cell

growth factors produced by lymphocytes. Reports of mast

cell-supporting factors (which, for the sake of convention,

we will commonly call interleukin 3) in the supernatants of

mitogen-stimulated splenocytes began to surface at the

beginning of the present decade (Burgess et al., 1980;

Hasthorpe, 1980). Since that time, a number of other

investigators have utilized media conditioned by a variety

of means to support the differentiation and growth of

culture-derived mast cells. Such media have thus been

derived from splenocytes activated by concanavalin A

(Clark-Lewis and Schrader, 1981; Tertian et al., 1981; Yung

et al., 1981; Schrader et al., 1981; Nakahata et al.,

1982b; Yung and Moore, 1982; Sredni et al., 1983), by

pokeweed mitogen (Hasthorpe, 1980; Nakahata et al., 1982b,

Wedling et al., 1983, 1984; Pharr et al., 1984), by

phytohemagglutinin A (Ogawa et al., 1983), by bacterial

lipopolysaccharide (Nakahata et al., 1982b), and by mixed

lymphocyte reactions augmented by lectin (Razin et al.,

1981a, 1982a, 1982c). Conditioned media with analogous

activity have been elicited from concanavalin A-stimulated











mesenteric lymph node cells of parasitized animals

(McMenamin et al., 1985). Phytohemagglutinin A- or

concanavalin A-stimulated human blood lymphocytes also

produced activities which supported the growth of human

basophilic cells and a growth factor-dependent mouse cell

line (Tadokoro et al., 1983; Stadler et al., 1985).

The phenotype of the murine lymphoid cell which

produces interleukin 3 was deduced from the activities of

conditioned media of a number of related T cell clones. In

contrast to Lyt 1+2+, Lyt 1-2+, and Lyt 1-2- cells, which

did not support culture-derived mast cell growth, the

supernatants of Lyt 1+2- T cell clones, corresponding to

the inducer T lymphocyte subset, supported the

proliferation of such cells (Nabel et al., 1981). The

observations of Nabel and colleagues were subsequently

confirmed by other investigators (Fung et al., 1984; Yokota

et al., 1984), and by the observation that rat mesenteric

lymph node cells expressing differentiation markers of

helper T lymphocytes (OX19+, W3/25+, 0X8-) were responsible

for the production of a factor with analogous activity to

mouse interleukin 3 (McMenamin et al., 1985).

A number of permanent cell lines also produce factors

which support the growth of culture-derived mast cells.

The best known of these cell lines is the myelomonocytic

WEHI-3 line, which constitutively produces high levels of

interleukin 3 (Nagao et al., 1981; Schrader et al., 1981;











Yung et al., 1981; Yung and Moore, 1982). Mast cell-

promoting activities have also been demonstrated in

conditioned media from concanavalin A-stimulated T cell

hybridoma cells (Clark-Lewis and Schrader, 1981), cloned T

cell lines (Nabel et al., 1981), iectin-stimulated T

leukemias (Yung et al., 1981; Yung and Moore, 1982; Metcalf

and Kelso, 1985), and B lymphoma cells (Clark-Lewis et al.,

1982).

The biologically active factor in lymphocyte and WEHI-

3 conditioned media has been given a variety of names. It

was first termed "multi-CSF" by Burgess and colleagues

(1980), due to its ability to support the differentiation

of multiple hematopoietic lineages in vitro. Schrader and

colleagues (Schrader and Nossal, 1980; Schrader, 1981;

Schrader et al., 1981; Clark-Lewis and Schrader, 1981)

reported that "P cell stimulating factor" (PSF), which

supported the growth of persisting, mast-like cells in

vitro, was present in concanavalin A spleen conditioned

media. Ihle and colleagues (1981, 1982) proposed the name

"interleukin 3", or EL 3, for the factor which induced the

enzyme 20-alpha-hydroxysteroid dehydrogenase in nude mouse

spleen cells as well as effecting a number of

differentiative and supportive activities in multiple cell

lineages. Although Ihle's terminology has achieved the

greatest usage in recent literature, alternative

nomenclature for the same activity has been proposed by











Bazill and colleagues ("multi-hematopoietic cell growth

factor", MCGF; Bazill et al., 1983) and Iscove

("multilineage hematopoietic growth factor", multi-HGF;

Iscove, 1985).

interleukin 3, derived from a variety of sources, has

been purified to homogeneity (Yung and Moore, 1982; Ihle et

al., 1982b; Bazill et al., 1983; Clark-Lewis et al., 1984)

and, more recently, the genes for IL 3 have been molecularly

cloned and expressed (Fung et al., 1984; Yokota et al.,

1984; Rennick et al., 1985). Extensive reviews of the

literature describing interleukin 3 (per se, and the

related activities called by various other names) have been

published recently and should be consulted for additional

information (Clark-Lewis et al., 1985; Ihle, 1985; Iscove,

1985; Schrader et al., 1985; Whetton et al., 1985; Yung and

Moore, 1985).

Interleukin 3-Independent Mast Cells

Several reports of interleukin 3-independent mast cells

have appeared in the literature in recent years.

Schrader's group (Schrader and Crapper, 1983; Schrader et

al., 1983a) observed the emergence of factor-independent

variants from factor-dependent cells. In one experiment,

factor-dependent ceils were plated in agar in the absence

of exogenous interleukin 3. From these cultures, several

colonies of autonomous culture-derived mast cells (P cells)

arose which, after several weeks, were subsequently adapted











to growth in interleukin 3-free liquid media. The

autonomous colonies secreted interleukin 3 into the culture

media, but also retained their receptors for the factor.

Furthermore, the autonomous cells generated more colonies

when plated at low density in the presence of exogenous

interleukin 3 in agar than in the absence of the growth

factor. It seems likely that the autogenouss" cells were

not truly factor-independent, but rather were variants

which were able to proliferate, albeit at lower efficiency,

at the low levels of interleukin 3 provided by an autocrine

mechanism.

Similar factor-independent culture-derived mast cells

have been reported by a second group (Ball et al., 1983;

Conscience and Fisher, 1985). Several long-term bone

marrow-derived cultured mast cell lines were found, after

eleven months in culture, to contain variants which

proliferated at higher rates than similar cultures in the

presence of interleukin 3. The more proliferative cells

were able to continue cell growth in the absence of

exogenous interleukin 3, but the doubling time was

increased 160 percent when compared to cells of the same

line maintained in conditioned media. In contrast to the

autogenous cells of Schrader, the media conditioned by the

factor-independent mast cells described in the more recent

studies failed to support the growth of other factor-

dependent culture-derived mast cells. It is possible,











however, that the cells were able to maintain their growth

in the presence of interleukin 3 at levels below those

detected in the assay. Interestingly, both sets of factor-

independent mast cells were tumorigenic in syngeneic mice.

Although no retroviral particles were observed in one of

these cell lines (Ball et al., 1983), the observations are,

by the criteria of tumorigenicity and factor independence,

similar to those of a recent report of Abelson murine

leukemia virus-transformation of culture-derived mast cells

(Pierce et al., 1985). Two possible mechanisms could

reconcile the yet unexplained results. First, the

activation of a latent replication-defective viral genome

in the long-term culture-derived mast cells of Ball and

colleagues could be the missing link. Under such

circumstances, no viral particles would be detected, but

the cells could become both factor independent and

tumorigenic by virtue of the viral transforming gene

product. Similarly, the activation of a cellular homologue

of a viral transforming gene, like c-abl (the normal

function of which is unknown), could be invoked to activate

the mechanisms necessary to generate the phenotype of the

factor-independent, tumorigenic mast cells.

Mast Cell Precursors

The development of in vitro techniques for the

differentiation and maturation of mast cells from

phenotypically immature progenitors permitted the analysis











of mast cells based upon a number of criteria. First, the

number of culture-derived mast cell precursors in

particular tissues was determined quantitatively. Prior to

the development of mast cell culture techniques which

utilized sources of interleukin 3 for mast cell

proliferation, Pluznik and Sachs (1965) enumerated mast

cell clones in soft agar with feeder layers. The authors

reported approximately thirty mast cell colony forming

units per million spleen cells seeded, while the frequency

of culture-derived mast ceil precursors in embryos, thymus,

and lymph nodes were five per million, three per hundred

million, and less than one per fifty million cells,

respectively. Schrader and colleagues (1981) cloned mast

cell precursors from mouse bone marrow in soft agar with

WEHI-3 conditioned media and found thirty to two-hundred

progenitors per million Thy 1-negative cells. These

results were contradicted by a more recent report (Sredni

et al., 1983) which found 600 to 700 bone marrow

precursors, 400 to 500 spleen precursors, 25 to 30 thymus

precursors, and 100 to 200 lymph node precursors per

million seeded cells. The latter results, however, were

generated in a system using concanavalin A-activated

splenocyte conditioned media as a source of interleukin 3

and different strains of mice, thereby making comparison

difficult. Interestingly, the latter authors also observed

that athymic nude mice had as many mast cell precursors as











syngeneic wild type controls. Since athymic mice do not

exhibit the profound mucosal mastocytosis found in

appropriate controls (Olson and Levy, 1976), the last

observations could be interpreted to indicate that athymic

mice lack the inductive conditions required for the

proliferation of seemingly normal numbers of mast cell

precursors.

Mast cell precursors were enumerated by Nakahata and

colleagues (1982), using a modification of the semisolid

methylcellulose media culture system previously used to

identify erythroid and myeloid precursors. In the latter

and subsequent report using this system (Pharr et al.,

1984), which used pokeweed mitogen activated spleen

conditioned media for a source of interleukin 3, the

investigators found between twenty and 140 mast cell

precursors per million BDF1 mouse spleen cells and 200 mast

cell precursors per million bone marrow cells. Suda and

colleagues (1985), using the Nakahata culture system,

demonstrated that W/Wv mice, which are severely deficient in

mast cells of both serosal and mucosal subsets, had the

same number of peripheral blood mast cell precursors as

wild type mice (approximately thirty per million nucleated

cells), thus indicating that the mast cell defect was in a

homing or developmental step, rather than at the stem cell

or migratory level.

A third method for the enumeration of culture-derived











mast cell precursors from various tissues was reported by

Crapper and Schrader (1983). Using limiting dilution

analysis of cells in liquid culture containing WEHI-3

conditioned media, The authors were able to enumerate mast

cell precursors in bone marrow, spleen, mononuclear

peripheral blood cells, and lymph nodes. All of the data

recorded in the latter experiments concurred with the

previously cited results of Schrader and colleagues (1981)

as well as those reported by the Nakahata group (Nakahata

et al, 1982b; Pharr et al., 1984). Furthermore, Crapper

and Schrader were able to substantiate the findings of Suda

and colleagues that mast ceil deficient mice had similar

numbers of culture-derived mast cell precursors (in bone

marrow and spleen) when compared to appropriate wild-type

controls, although the former authors used wf/Wf and the

latter authors used W/Wv.

The development of techniques for the propagation of

culture-derived mast cells has also permitted the

characterization of such cells at various stages of

differentiation. Thus, Ginsburg and colleagues (Ginsburg,

1963; Ginsburg and Sachs, 1963; Ginsburg and Lagunoff,

1967; Ginsburg et al., 1982; Davidson et al., 1983)

reported a progression of characteristics of cultured mast

cells, starting with large, mononuclear "stem" cells.

After six to ten days in culture, large, lymphocyte-like
"mastoblasts" with round and bilobed nuclei and a narrow











rim of metachromatic cytoplasm, similar to those described

by Maximow (1906) in stained tissue sections, arose and

became dominant in cell culture. The cytoplasm of the

monoblasts continued to increase in size as the nucleus

became more distinctly indented, with a chromophobic region

in the concave aspect of the nucleus. At twelve to

thirteen days in culture, a foamy region in the cytoplasm

was seen to spread, sometimes encompassing the entire

cytoplasm. Metachromatic material first appeared in the

foamy region as faint, amorphous substance in vacuoles,

later increasing in size and staining intensity to well-

defined granules. These latter cells were described as

"young mast cells", possessing granular, metachromatic

cytoplasm, with actively mitotic, kidney-shaped, round or

oval nuclei; such ceils dominated the cultures between days

twelve and twenty-two. After three weeks in culture, the

majority of the cells were mature, round mast cells with

round to oval, eccentric or centered amitotic nuclei and

abundant, metachromatic cytoplasmic granules, similar in

morphology and histochemistry to mucosal mast cells.

Similar staging of rat culture-derived mast cell precursors

and intermediates has been reported (Ishizaka et al., 1976;

Zucker-Franklin et al., 1981; Sterry and Czarnetzki, 1982;

Czarnetzki et al, 1983), corroborating in situ observations

(Combs et al.. 1965).











Characteristics of Culture-Derived Mast Cells

The development of refined analytical methods, first

applied to other hematopoietic cells, aided in the

characterization of culture-derived mast cells. Yung and

colleagues (1983) analyzed bone marrow cells by

centrifugation techniques and reported that interleukin 3-

responsive cells could be isolated by nature of their

median buoyant density (1.033) from interleukin 2-

responsive cells (1.075). The authors also noted that the

buoyant densities of long-term bone marrow-derived cultured

mast ceils (1.062 to 1.095 g/ml) were similar to those

determined by Pretlow and Cassidy (1970), who analyzed

heterogeneous populations of freshly isolated peritoneal

mast cells and reported that immature mast cells have a

median buoyant density of 1.087. Interleukin 3-

responsive cells were separated by sedimentation velocity

analysis from the in vitro precursors of macrophages (CFU-

M) and the pluripotent colony-forming cell of the spleen

(CFU-S), but not the bipotent precursor of granulocytes and

macrophages (CFU-GM).

Culture-derived mast cells and their precursors have

been characterized for the expression of a broad variety of

hematopoietic differentiation markers in attempts to assign

them to a particular lineage. Bone marrow and intestinal

precursors to culture-derived mast cells were observed to

lack the T lineage antigens Thy 1, Lyt 1 and Lyt 2











(Schrader et al., 1983b; Guy-Grand et al., 1984). Like

their precursors, more differentiated culture-derived mast

ceils are deficient in Thy 1, Lyt 1, and Lyt 2 ( Ginsburg

et al., 1981; Nabel et al., 1981; Schrader, 1981; Schrader

et al., 1981; Tertian et al., 1981; Davidson et al., 1983;

Sredni et al., 1983; Ghiara et al., 1985), although

Schrader and colleagues (1982) reported that Thy 1 may be

transiently expressed on these cells. Similarly, culture-

derived mast cells lack surface immunoglobulin, a B lineage

marker (Ginsburg et al., 1981; Schrader et al., 1981;

Tertian et al., 1981; Sredni et al., 1983), NK-1, a marker

of natural killer cells (Nabel et al., 1981), complement

receptors and MAC-1, a differentiation antigen of

mononuclear phagocytes which is also expressed by some

natural killer and T lymphoma cells (Tertian et al., 1981).

Like their in vivo correlates, murine culture-derived

mast cells express surface receptors for IgE (Ginsburg et

al., 1978: Nagao et al., 1981; Schrader, 1981; Schrader et

al., 1981; Tertian et al., 1981; Ginsburg et al., 1982;

Nakahata et al., 1982b; Sredni et al., 1983; Wedling et

al., 1983, 1985), which induce the anaphylactic release of

histamine when cross-linked by IgE and homologous antigen

(Ginsburg et al., 1978; Sredni et al., 1983) or anti-IgE

(Ginsburg et al., 1982). The number of IgE receptors per

cell has been estimated to be 2 to 3 x 105, similar to the

number on serosal and mucosal mast cells (Razin et ai.,











1981a; Ginsburg et al., 1982). Some investigators have

also noted the presence of receptors for IgG (Schrader,

1981; Tertian et al., 1981); the cells, however, did not

phagocytose opsonized or unopsonized targets (Schrader,

1981; Sredni et al., 1983). Liquid cultured mast cells in

conconavalin A-stimulated spleen conditioned media also

express the lymphocyte marker Ly 5 (Nabel et al., 1981;

Tertian et al., 1981). Contradictory observations of

histocompatability Class II (Ia) antigens, or the lack

thereof, on culture-derived mast cells were resolved by

Wong and colleagues (1982), who showed that such cells,

grown in the presence of immune interferon (interferon

gammma, found in the supernatants of concanavalin A-

stimulated splenocytes) expressed the marker while cells

grown in the absence of interferon (as in WEHI-3

conditioned media) were devoid of Ia. Culture-derived mast

cells were also shown to express Class I histocompatability

antigens and receptors for peanut agglutinin (Schrader,

1981; Schrader et al., 1981; Tertian et al., 1981).

Russell and colleagues developed a panel of rat

monoclonal antibodies against murine mononuclear phagocytes

which could discriminate between culture-derived mast ceils

and connective tissue mast cells (Leblanc et al., 1982).

The same group observed that culture-derived mast cells

expressed the phenotype Bl.1-/B23.1+/ B54.2+, while in

contrast peritoneal mast cells were B1.1+/ B23.1-/B54.2+











(Katz et al., 1983). Although the Forsmann glycolipid

recognized by monoclonal antibody B1.1 is undetectable on

culture-derived mast cells, the latter cells do express the

antigen precursor, globotetrasylceramide (Katz et al.,

1985b), and may therefore be deficient or defective in the

glycosyltransferase required for the synthesis of the

mature antigen.

Culture-derived mast cells have been extensively

characterized biochemically as well. The histamine content

of cultured mast cells, like that of mucosal mast cells,

has been estimated between 450 and 500 nanograms per

million cells, at least ten-fold less than the histamine

content of comparable numbers of peritoneal mast cells

(Nabel et al., 1981; Nagao et al., 1981; Razin et al.,

1981a; Galli et al., 1982b; Sredni et al., 1983; Wedling et

al., 1985). Mouse culture-derived mast cell (and mucosal

mast cell) granules stain blue when treated with alcian

blue and safranin (Ginsburg and Lagunoff, 1967), indicating

the presence of weakly sulfated mucopolysaccharides,

whereas serosal mast cell granules, containing strongly

sulfated heparin proteoglycan, stain red. Razin and

colleagues (1982c) analyzed the proteoglycan of murine

culture-derived mast cells and found they contained

glucuronic acid-N-acetylgalactosamine-4,6-disulfate, or

chondroitin sulfate proteoglycan E, a unique

glycosaminoglycan which could not be detected in basophilic











leukemia cells, peritoneal mast cells, or chondrocytes.

Chondroitin sulfate proteoglycan E was shown in this study

to be chemically distinct from heparin by a number of

criteria including sensitivity to enzymatic degradation and

molecular weight (chondroitin sulfate proteoglycan E has an

estimated molecular weight of 200 kilodaltons, in contrast

to heparin, which has a molecular weight of 750

kilodaltons). These results have been confirmed in the

literature (Razin et al., 1983; Sredni et al., 1983).

Mouse culture-derived mast cell granules also contain a

number of other in vivo mast cell-associated biological

mediators, including serotonin (5-hydroxytryptamine) and

dopamine (Tertian et al., 1981).

Arachidonic acid metabolites, the prostaglandins and

leukotrienes, are important biological mediators

associated with metachromatic cells. IgE-dependent

activation of mouse culture-derived mast cells results in

the synthesis and release of leukotriene C4 (Razin et al.,

1982b, 1983), a component of the slow releasing substance

of anaphylaxis (Austin, 1984). These studies also showed

that bone marrow-derived cultured mast cells generated

approximately twenty-five times more leukotriene C4 than

prostaglandin D2 upon activation by calcium ionophore

A23187 or IgE receptor-mediated pathways. In contrast, rat











peritoneal mast cells preferentially synthesized and

released prostaglandin D2 in forty-fold excess over

leukotriene C4.

Culture-derived mast cells are ultrastructurally

distinct from serosal mast cells. Cells of the former

category thus possess granules which are more heterogeneous

in size and electron density than the latter (Ginsburg and

Luganoff, 1967; Ginsburg et al., 1978; Nabel et al., 1981;

Galli et al., 1982b; Wedling et al., 1985). Granules in

culture-derived mast cells are ovoid and are frequently

associated with small vessicles (Sredni et al., 1983) and a

well-developed Golgi apparatus (Ginsburg and Lagunoff,

1967). The substance of mouse culture-derived mast cell

granules is crystalline (Razin et al., 1982a), while that

of human origin (resembling basophils, rather than mast

cells) is more particulate (Razin et al., 1981b). Mouse

bone marrow-derived mast cells form membrane channels after

activation with IgE and anti-IgE through which granules may

reach the cell surface (Razin et al., 1982a), similar to

human lung mast cells (Caulfield et al., 1980). The

cytoplasmic membrane of culture-derived mast cells is

characterized by numerous, fine protrusions (Galli et al.,

1982b; Wedling et al, 1985) which are absent from

peritoneal mast cell membranes.

The response of culture-derived mast cells to

secretagogues is similar to that of mucosal mast cells, but











not serosal mast cells. As previously discussed,

leukotriene C4 is synthesized and released as a result of

activation of the IgE receptor-mediated pathway. In

addition, histamine, chondroitin sulfate proteoglycan E,

and beta-hexosaminadase are released by immune activation

(Razin et al., 1983). Like both serosal and mucosal mast

cells, culture-derived mast cells are induced to

degranulate by the calcium ionophore A23187 (Razin et al.,

1982a, 1982b; Sredni et al., 1983; Robin et al., 1985).

Culture derived mast cells, however, mimic mucosal mast

cells in their lack of response to compound 48/80 (Sredni

et al, 1983). In contrast, mouse peritoneal mast cells are

degranulated by compound 48/80.

Many of the characteristics previously described for

mouse culture-derived mast cells have been reported in

analogous rat and human systems. Ishizaka and colleagues

(1976) cultured rat thymocytes in the presence of rat

embryonic fibroblast monolayers and observed the outgrowth

of cells with receptors for IgE and metachromatic granules.

Haig and colleagues (1982, 1983) grew rat bone marrow-

derived cultured mast cells in the presence of media

conditioned by mesenteric lymph node cells. The

investigators observed that, similar to culture-derived

murine mast cells, rat culture-derived mast cells were

smaller than peritoneal mast cells, possessed sparse

granules of heterogeneous size, and expressed surface











receptors for IgE. Rat culture-derived mast cell granules

stained blue by the alcian blue-safranin technique and were

metachromatic when stained with toluidine blue. The

granule proteoglycan was identified as non-heparin,

although no report of its precise chemical composition has

been published to date. Such cells also contained

immunochemically detectable levels of rat mast ceil

protease II, which was previously described as a marker of

mucosal, but not serosai mast cells. Rat mesenteric lymph

nodes (Denburg et al., 1980) and peripheral blood (Zucker-

Franklin et al., 1981) have also been shown to contain

precursors of culture-derived mast cells.

Reports of mast cells derived from human tissues are

clouded by difficulties in distinguishing between the

various types of basophilic cells when compared to in vivo

correlates, namely mucosal mast ceils, connective tissue

mast cells, and basophils. Granulated cells with receptors

for IgE and low levels of histamine (50 to 450 nanograms

per million cells), thus resembling mouse culture-derived

mast cells, have been observed in cultures of human fetal

liver grown in unconditioned media (Razin et al., 1981b).

Adherent human blood mononuclear ceils and pleural exudate

cells, which were propagated with L-ceil conditioned media,

exhibited similar characteristics (Czarnetzki et al., 1983,

1984; Kruger et al., 1983). Ceils with high affinity

receptors for [gE and slightly higher levels of histamine











(480 to 1600 nanograms per million cells) were cultured

from human umbilical cord blood grown in phytohemagglutinin

A-stimulated human T cell conditioned media (Ogawa et al.,

1983). Horton and O'Brien (1983) reported the culture of

granulated cells with centrally placed, round or indented

nuclei from human bone marrow harvested from a patient with

systemic mastocytosis. The culture-derived mast cells

showed no growth advantage with a number of conditioned

media, but required an adherent, bone marrow-derived feeder

layer to persist.

Other In Vitro-Derived Metachromatic Cells

As noted previously, some of the confusion in the

terminology applied to culture-derived mast ceils has been

generated by the appearance of characteristics of one or

more of the in vivo basophilic cell correlates. Thus,

although basophils in mice are lacking (Lagunoff and Chi,

1980) or extremely rare (Dvorak et al., 1982), a report of

cloned, basophil-like cell lines with IgE receptors has

appeared in the literature (Galli et al., 1982a). The cell

line in question, derived from mouse splenocytes cultured

in concanavalin A-stimulated splenocyte conditioned media,

lacked histamine and had both natural killer cell

differentiation markers and natural killer activity.

In a series of studies on the rat, Czarnetzki and

colleagues described the growth of connective tissue-like

mast cells in vitro. In the earliest study (Czarnetzki et











al., 1979), mast cell-free peritoneal exudate cells were

harvested from rats which were previously injected with

sterile water (intraperitoneally). The peritoneal cells

were cultured in L-cell conditioned media with sodium

butyrate. The mast cells which grew out of this

population, although initially possessing blue-staining

granules by the alcian biue-safranin technique, later had

red staining granules and released histamine in response to

compound 48/80. Despite their serosal mast cell

characteristics, these culture-derived mast ceils were

similar to the classical description of mucosal mast cells

in that they contained low levels of histamine (500

nanograms per million cells) and survived for only short

periods. Similarly described cells were subsequently

isolated by the same group from rat peritoneal cells

(Czarnetzki and Behrendt, 1981), and rat mononuclear

phagocytes (Czarnetzki et al., 1981, 1982; Sterry and

Czarnetzki, 1982).

In related studies, in vitro cultivated human

peripheral blood mononuclear cells (Denburg et al., 1983)

and guinea pig bone marrow ceils (Denburg et al., 1980)

developed into metachromatic cells with segmented nuclei

which were more characteristic of basophils than mast

cells. The human cells, in particular, possessed the

polymorphonuclear structure with mature chromatin, Golgi

and microtubules which are more characteristic of basophils











than mast cells. This conclusion, however, directly

disagreed with Zucker-Franklin (1980), who contended that

human mast cells and basophils share common ultrastrucural

organization.

Tadokoro and colleagues (1983) cultured cells with

metachromatic granules and lobulated nuclei from normal

human bone marrow in conditioned media from lectin-

stimulated blood lymphocytes. The culture-derived cells

contained 500 to 2000 nanograms of histamine per million

cells and were responsive to IgE-anti-IgE- and calcium

ionophore-mediated histamine release but were refractory to

the effects of compound 48/80. The authors concluded that

their conditioned media contained a basophil-promoting

activity which furthermore had a molecular weight of 25 to

40 kilodaltons and was distinct from interleukin 2. The

same group recently reported that media conditioned by

phytohemagglutinin A- and concanavalin A-stimulated human

blood lymphocytes could support the interleukin 3-dependent

mouse cell line 32Dci as well as promote the growth of

human culture-derived basophils (Stadler et al., 1985).

Furthermore, the interleukin 3 and basophil-promoting

activities, which were also found in media conditioned by

the growth of E-rosetting T lymphocytes and the MoT cell

line, were biochemically distinct by at least five

different criteria. The isolated human interleukin 3 was

shown to promote the growth of mast cells which were unable











to proliferate in the presence of mouse interleukin 3 (in

WEHI-3 conditioned media). Thus, in the human system, the

culture-derived mast cell-basophil dilemma is no longer

solely a matter of terminology and mistaken identity, but,

in fact, appears to involve multiple growth promoters and,

most likely, multiple progenitor cells.

Relationship of In Vivo- and In Vitro-Derived Mast Cells

A small body of evidence supports the theory that

culture-derived mast cells are more than circumstantially

related to mucosal, and perhaps serosal, mast cells. A

number of characteristics, including morphology,

histochemical fixation and staining, dependence of

proliferation on T cell-derived factors, biogenic amine

content, protease content, presence of receptors for IgE,

and sensitivity to secretogogues, have been noted in this

review and cited by many of the authors as proof of the

relationship between culture-derived mast cells and the

mast cells of the mucosal surfaces. There is preliminary

evidence that culture-derived mast cells have natural

cytotoxic activity against tumors such as WEHI-3 and Meth A

which is enhanced by interleukin 3 (Ghiara et al., 1985).

Investigators have noted that in vivo-derived mast cells

exhibited similar tumoricidal activity (Farram and Nelson,

1980) and that in vivo-derived cells with the











Thy l-/Lyt 1-/ Lyt 2- phenotype, which are demonstrably

cytotoxic, are sensitive to the proliferative activities of

interleukin 3 (Djeu et al., 1983; Lattime et al., 1983).

Evidence of more direct relationships between in

vitro-derived mast cells and their in vivo correlates has

been elusive. Several investigators have associated the

high incidence of culture-derived mast cell precursors and

mucosal mast cells in the intestine of normal mice (Crapper

and Schrader, 1983; Guy-Grand et al., 1984). The role of

antigenic stimulation and T cell function in the

proliferation of mucosal mast cells per se has been

thoroughly described in the literature (for reviews see

Jarrett and Haig, 1984; Shanahan et al., 1984; Bienenstock

et al., 1983). Guy-Grand and colleagues (1984) also showed

that the number of mast cells which could be cultured from

intestinal mucosa increased with antigenic stimulation and

WEHI-3 tumor burden, implicating the role of interleukin 3

in the in vivo proliferation of mast cell precursors. The

results, however, associated the in vivo and in vitro mast

cell precursors by existence in the same tissue, and did

not directly show that the populations involved were

identical.

The most suggestive evidence to date of the

relationship between in vivo- and in vitro-derived mast

cells involves the demonstration that culture-derived mast

cells, when injected into mast cell-deficient mice,











populated both mucosal and connective tissue-serosal

compartments. Nakano and colleagues (1985) injected

culture-derived mast cells and partially purified (30 to 40

percent) peritoneal mast cells into W/Wv mice by various

routes. At high levels of inoculum (105 to 106 cells),

intravenously- or intraperitoneally-injected cultured mast

cells populated the spleen and stomach mucosaa and muscle),

while at lower levels of inoculum (102 to 104cells), only

intraperitoneallv injected cells were able to populate the

same anatomical sites. Both cultured and peritoneal mast

cells were relatively inefficient at populating the skin,

however, possibly due to the presence of mast cells, or

their precursors, which were already present in the skin

(Kitamura et al., 1977). An interesting note to the Nakano

studies was the evolution of mucosal or serosal mast cell

characteristics from injected cells (regardless of origin)

depending on the anatomical site of subsequent lodging.

Thus, the granules of mast cells isolated from the

peritoneal cavity, spleen, skin, and gastric muscularis

propria of reconstituted animals stained preferentially

with safranin (which has an affinity for highly sulfated

mucopolysaccharides like heparin) and the fluorescent dye

berberine sulfate (which also binds to heparin), and were

ultrastructurally homogeneous in size and electron density,

while mast ceils identified in the glandular stomach mucosa

stained preferentially with alcian blue and were unstained











by berberine sulfate. Mast cell phenotype, therefore, may

be functionally regulated at the level of the tissue

microenvironment in which a multipotent mast cell precursor

or intermediate develops.

Epilogue

Despite an apparent wealth of literature available on

the subject, the potential still exists for scholarly.

significant contributions to the body of knowledge which

describes mast cells. The lineage relationship between the

mast cells found in vivo mucosall and serosal) is still

poorly defined, ond only recently have preliminary studies

approached the relationship between the aforementioned

cells and their putative correlate, the culture-derived

mast cell. Little is known of the phenotype of the cells

which give rise to mast cells in culture, and the ontogeny

of the mast cell in early embryonic tissues is documented

in scant and unsystematic reports.

In the course of the remaining chapters of this

dissertation, we describe our recent contributions to the

study of the mast cell. Beginning with the observation of

cell lines with basophilic granules, we have characterized

Abelson murine leukemia virus-transformed mast cell-like

lines of midgestational, embryonic origin using panels of

monoclonal antibodies as well as biochemical and molecular

biological techniques (Chapter II). Although mast cells

were not detected in homologous, uninfected tissues,











culture-derived mast cells could be propagated from

embryonic sources in the presence of exogenously supplied

interleukin 3. These studies (and parallel experiments on

adult bone marrow-derived mast ceils) also provide the

first detailed analysis of hematopoietic marker expression

of cultures progressing from heterogeneous to homogeneous

populations of mast cells (Chapter III). We have

subsequently analyzed the frequency of mast cell precursors

in embryonic placenta, nonplacental embryonic tissues, and

adult tissues, demonstrating the earliest reported mast

cell precursors as well as a heretofore unreported rich

source of such cells, the placenta (Chapter IV). In the

same chapter, we have characterized the cell surface of

mast cells grown in semisolid agar media and have presented

encouraging preliminary results of experiments designed to

sort mast cell precursors on the basis of differentiation

antigen expression.








CHAPTER II
ABELSON MURINE LEUKEMIA VIRUS-INFECTED CELLS FROM
MIDGESTATION PLACENTA EXHIBIT MAST CELL AND LYMPHOID
CHARACTERISTICS

Introduction

Abelson murine leukemia virus (A-MuLV) is a

replication-defective transforming retrovirus which was

isolated from a tumor in a steroid-treated BALB/c mouse

inoculated with the Moloney murine leukemia virus (Abelson

and Rabstein, 1970). Molecular analysis of the A-MuLV

genome has revealed that the virus arose by recombination

between the thymotropic Moloney virus genome and a cellular

gene termed c-abl (Goff et al., 1980; Shields et al.,

1979). The recombinant virus can infect and immortalize

hematopoietic cells in vivo and in vitro, and can transform

certain fibroblast cell lines in vitro (Scher and Siegler,

1975). The virus has demonstrated the ability to transform

in vivo mature cells of the B lineage (Potter et al., 1973;

Premkumar et al., 1975) as well as those of the T (Cook,

1982), myelomonocytic (Raschke et al., 1978; Ralph et al.,

1976), and mast cell (Risser et al., 1978; Mendoza and

Metzger, 1976) lineages. In contrast, in vitro infection

of adult or embryonic primary hematopoietic tissues

followed by clonal selection in semisolid media results in

the immortalization of cells exhibiting characteristics of

the earliest stages of B lymphoid differentiation

(Rosenberg et al., 1975; Rosenberg and Baltimore, 1976b;











Boss et al., 1979; Siden et al., 1979; Alt et al., 1981).

Abelson virus can also induce agar colony-forming ceils

which express erythroid characteristics (Waneck and

Rosenberg, 1981); the latter cells, however, fail to

proliferate as permanent cell lines in liquid culture.

Based upon Abelson virus's propensity to immortalize B

lineage precursors in vitro, experiments were designed to

study early embryonic lymphoid precursors. The

midgestation embryonic placenta has been reported to be the

earliest source of B cell precursors (Melchers and

Abramczuk, 1980; Melchers, 1979) in the mouse. The

successful development of permanent cell lines from

midgestation embryonic placenta transformed in vitro by A-

MuLV has recently been reported (Siegel et al., 1985).

Primary agar colony counts indicated that the frequency of

A-MuLV targets is highest at ten days of gestation. Unlike

previously reported A-MuLV-transformed embryonic cell

lines, the genomes of the placental cells contain a

germline immunoglobulin heavy chain locus characteristic of

nonlymphoid cells and perhaps very immature lymphoid cell

precursors.

We proceeded to analyze this novel group of A-MuLV

embryonic transformants to better ascribe them to cells of

a particular lineage. This chapter summarizes our efforts

to characterize the A-MuLV transformants derived from

midgestation embryonic tissues and presents several











significant observations. First, unlike previously

reported Abelson virus-transformed embryonic cells, the

placental cell lines isolated in our laboratory exhibit

many characteristics of culture-derived and mucosal mast

cells including differentiation antigens, high affinity

receptors for igE, and metachromatic granules containing

histamine and sulfated proteoglycans. Second, we describe

the development of a simple, sensitive, nonisotopic,

nonfluorometric method to detect membrane receptors for

immunoglobulins. Third, all of the cell lines analyzed had

at least one, and sometimes more than one integrated A-MuLV

provirus. Finally, the cell lines proliferated independent

of exogenous mast cell growth factor, and no growth factor

could be detected in either cell lysates or the

supernatants of exponentially growing cultures maintained

at high cell density.





Materials and Methods



Cell Lines

Cloned A-MuLV-transformed embryonic cell lines were

established in our laboratory as previously reported

(Siegel et al., 1985). All had been adapted to growth in

liquid culture and had been successfully maintained in 10P

(RPMI 1640 (GIBCO, Grand Island, NY) with 5x10-5 M 2-









mercaptoethanol (Sigma Chemical Co., St. Louis, MO)) and 10

percent heat-inactivated fetal bovine serum (Sterile

Systems, Logan, UT)) for at least two years with twice

weekly passages prior to analysis. The nomenclature used

to designate each cell line included the number of days of

gestation (detection of vaginal plug on day 0) followed by

a suffix ("P" for placenta of (BALB/cAN x B10.BR/SgSn)Fl

origin, "PC" for placenta of (BALB/cAN x CBA/J) origin, and

a clone number. Cell line 10P12, for example, was the

twelfth clone isolated from 10 day placental cells derived

from matings of BALB/cAN females and B10.BR males.

Mouse tumor cell lines used as experimental controls in

the studies were chosen to represent the major

hematopoietic lineages:

1. B lymphoid

a. FLEI-4, an pre-B cell line derived by E.J.

Siden by infection of day 15 BALB/c fetal

liver with A-MuLV strain P120;

b. 18-81 (Siden et al., 1979), a pre-B cell line

induced by infection of bone marrow ceils with

A-MuLV strain P120;

2. T lymphoid

a. RLd 11, a radiation-induced leukemia

obtained from N. Rosenberg;

b. B2-4-4, a Moloney virus-induced leukemia

obtained from N. Rosenberg;











c. YAC-1, a Moloney leukemia virus-induced

lymphoma obtained from R. Weiner (University

of Florida);

3. Monocyte-macrophage

a. WEHI-3, a myelomonocytic leukemia obtained

from M. Norcross (University of Florida);

b. P388D1, a methylcholanthrene-induced monocytic

tumor obtained from American Type Culture

Collection (Rockville, MD);

4. Basophil-mast cell

a. P815, a methylcholanthrene-induced mastocytoma

obtained from S. Noga (University of Florida);

b. CB6ABMC4, an A-MuLV-induced mastocytoma

obtained from M. Potter (National Cancer

Institute, Bethesda, MD);

c. BALABMC20, an A-MuLV-induced mastocytoma

obtained from M. Potter.

All of the above cell lines were maintained in 10P with

twice-weekly passage.

Cell line DA-1, which was used in the analysis of

interleukin 3 production, was generously supplied by J.N.

Ihle (National Cancer Institute, Frederick, MD) and was

maintained in a one-to-one mixture of WEHI-3 conditioned

media and enriched media (EM; Razin et al., 1984a).











Analysis of Histamine Content

Histamine content was determined by a modification of

the enzymatic-isotopic microassay of Taylor et al. (1980).

Histamine methyltransferase (HMT) reagent was prepared from

BALB/cAN mouse brain which was homogenized in iced 5mM

sodium phosphate buffer, pH7.9 (10 ml per gram of brain)

with an iced, sintered glass homogenizer. The crude

homogenate was transferred to an Oak Ridge type tube and

cleared centrifugally at 50,000xg for ten minutes at 4C.

Histamine standard solutions and tumor cell lysates were

prepared in PIPES-BSA which contained 25mM PIPES, pH 7.4

(Sigma Chemical Co., St. Louis, MO), 0.4 mM magnesium

chloride, 5 mM potassium chloride, 120 mM sodium chloride,

1 mM calcium chloride, 5.6 nm glucose (Fisher Scientific

Co., Fairlawn, NJ), 0.1 percent BSA (Sigma); standard

solutions and cells were boiled for ten minutes and cleared

for five minutes in a microfuge (Brinkmann Instruments,

Westbury, NY). Immediately before starting the reaction,

HMT-SAMe was formulated: 500 microliters of HMT reagent

was mixed with 10 microliters (5 microcuries) S-[methyl-

3H]-adenosylmethionine (76.1 Ci per mMol; New England

Nuclear, Boston, MA) and 5 microliters unlabeled S-

adenosylmethionine (SAMe, Sigma Chemical Co.; 16 micrograms

per ml). Each reaction mixture consisted of 10 microliters

of HMT-SAMe and 20 microliters of histamine standard

solution or tumor cell lysate. Following incubation for











two hours at 37C, each reaction was stopped with 0.1 ml of

1 M sodium hydroxide and saturated with solid sodium

chloride. The contents of each tube were extracted with

0.25 ml chloroform in the presence of 10 microliters of

unlabeled SAMe (16 micrograms per ml)and the phases were

separated by brief centrifugation. The aqueous phase was

carefully removed with a pasteur pipet and the organic

phase was re-extracted with an equal volume of 1 M sodium

hydroxide and unlabeled SAMe. Aliquots (150 microliters)

of the organic phase were added to toluene-based

scintillation fluid (10 ml) and counted on a Beckman liquid

scintillation counter (Beckman Instruments, Palo Alto, CA).

All samples and standards were prepared in duplicate or

triplicate. Histamine content was determined by comparison

of tritiated counts in dilutions of tumor lysates to those

in histamine standard solutions and were expressed as

nanograms (ng) per 104 cells.

Cytological Staining

Staining procedures were performed on air dried

cytocentrifuge (Shandon Scientific Company, Ltd., London,

U.K.) preparations on 25x75 mm precleaned glass slides.

Slides were fixed in methanol for one to two minutes prior

to staining with either Wright's Giemsa (Schalm et al.,

1975) or May-Gruenwald Giemsa (Thompson and Hunt, 1966).

Metachromatic cell granules were identified by staining for

five minutes with 0.1% w/v toluidine blue in 30% v/v











ethanol, pH 0.5 after fixation for two minutes in Mota's

fixative (lead subacetate in acidic ethanol)(Yam et al.,

1971). All stained slides were coverslipped with Permount

(Fisher Scientific Co.) prior to observation by light

microscopy.

Antibodies

Lineage-specific determinants on the surfaces of

placental and control cell lines were probed with a panel

of monoclonal (mAb) and polyvalent antibodies whose

significant features are summarized in Table II-l. All

monoclonal antibody preparations, unless otherwise noted,

were used as filtered hybridoma supernatants from

stationary phase cultures grown (in our facility) in

Dulbecco's Modified Eagle's Minimal Essential Media (GIBCO)

with 10 percent heat-inactivated fetal bovine serum and

5x10-5 M 2-mercaptoethanol. Preliminary work with mAb

B23.1 was performed with partially purified antibodies

generously supplied by P. LeBlanc (University of Florida);

later studies were performed with filtered hybridoma

supernatants which had been grown in RPMI 1640 with 10

percent fetal bovine serum, generously supplied by G. Place

in the laboratory of S. Russell (University of Florida).

Monoclonal antibody FT-1 was generously supplied by M.

Kasai (National Institute of Health, Tokyo, Japan); the

nature of its production and processing have been

previously reported (Kasai et al., 1983).












Table II-1.


Designation
(isotype)


Lineage-Specific Antibodies Used in Surface
Marker Analysis


Specificity (Literature Citation)


14.8
(IgG2b)

RA3-2C2
(IgM)


RA3-3A1
(IgM)


M6
(IgM)


anti-Asialo GM1
polyclonall)






T24/31.7
(IgG)


5H1
(IgM)





B23.1
(IgM)


200 Kd Ly5 antigen on lymphoid cells from spleen,
lymph node, and bone marrow (Kincade et al., 1981b).

Ly5 antigen on lymphoid cells from spleen, bone
marrow, lymph node, and plasma cells but not on
thymocytes or CFU-S (Coffman and Weissman, 1981a).

220 Kd Ly5 variant (B220) on B lineage cells on
spleen, lymph node, and bone marrow, but not on
thymocytes (Coffman and Weissman, 1981b).

Probably recognizes Dolichus biflorus agglutinin
receptor on early fetal thymocytes and on some
thymic leukemia cells (Kasai et al., 1983).

Neutral glycolipid asialo GM1 on early fetal
thymocytes, some fetal liver cells, and few adult
bone marrow, spleen, and lymph node cells as well
as some thymic leukemia cells and natural killer
cells. Not on adult and embryonic, Thy-i positive
thymocytes (Habu et al., 1980; Kasai et al., 1980;
Young et al., 1980).

Thy 1 glycoprotein on thymocytes and T cells from
spleen but not on bone marrow prothymocytes
(Dennert et al., 1980).

Abelson transforming antigen on bone marrow targets
of A-MuLV, on most thymocytes, some bone marrow and
spleen cells, fetal liver erythrocytes and pre-B
cells, and bone marrow pre-B cells, but not on lymph
node cells, CFU-S or stem cells committed to myeloid
lineages (Shinefeld et al., 1980).

Antigen on resident and elicited macrophages, adherent
cultured bone marrow cells and macrophage-like cell
lines as well as on culture-derived mast cells but not
on resident peritoneal mast cells (Katz et al., 1983;
Leblanc et al., 1982).











Rabbit anti-asialo GM1 was received from W.W. Young

(University of Virginia Medical Center) as a delipidated

serum. Rabbit anti-rat immunoglobulins was purchased as a

lyophilized powder (IgG fraction of rabbit anti-rat IgG

(heavy and light chains). Miles Laboratories, Elkhardt, IN)

and reconstituted per manufacturer's specifications.

Rabbit anti-mouse immunoglobulins was purchased from

Gateway Biologicals (St. Louis, MO).

Normal rat serum was prepared from cardiac blood of an

unimmunized animal. Normal mouse serum was prepared from

pooled specimens of multiple unimmunized BALB/cAN mice.

Cell Surface Markers

Cell surface differentiation antigens were detected by

a modification of the method of Uchanska-Ziegler and

colleagues (1982). Formalin-treated, heat killed

Staphylococcus aureus (S.aureus) Cowan I (The Enzyme

Center, Inc.,Boston, MA, and later a generous gift of Dr.

Michael D.P. Boyle) were first coated with rabbit anti-rat

IgG (RAMIgG, Miles Laboratories, Elkhart, IN) and then with

rat monoclonal antibodies specific for mouse

differentiation markers. Either hybridoma cell culture

media or partially purified immunoglobulins were used as a

source of the latter antibodies.

Mouse cells (1x105 or fewer) selected for analysis

were pelleted in round-bottom, PVC microtiter wells

(Dynatech, Alexandria, VA) and resuspended in a small











volume (5 microliters) of S.aureus-RAMIgG-monoclonal

antibody sandwiches. Following a thirty-minute incubation

on ice, the contents of each well were washed six times and

a sample was prepared as a cytocentrifuge mount. Slides

were stained with May-Gruenwald Giemsa or Wright's Giemsa.

Fc Receptor Assays

Receptors for the Fc domain of mouse IgE and IgG were

detected by a novel modification of the S. aureus method

used to detect other surface antigens (Siden and Siegel,

1986). Indicator bacteria were prepared by incubating 25

microliters of packed S. aureus or Escherichia coli (E.

coli) with 0.175 ml of 2,4,6-trinitrobenzene sulfonic acid

(3.7 mg per ml in 0.28M cacodylate buffer, pH6.9) for 10

minutes at room temperature. The 1.5 ml microfuge tube

containing the reactants was wrapped in foil to retard

photodecomposition and taped to a rotator during

incubation. After four washes with 0.01 M phosphate-

buffered 0.15 M sodium chloride, pH 7.3 (PBS; Mishell and

Shiigi, 1980), the TNP-S. aureus or TNP-E. coli were

resuspended to their original volume in balanced salt

solution (BSS; Mishell and Shiigi, 1980) with 10 mM HEPES

(pH 7.35), 0.1% w/v sodium azide, 5x10-5 M 2-

mercaptoethanol (Sigma Chemical Co.) and 1% v/v fetal

bovine serum (H1OBNFl).

Hybridoma supernatants containing mouse IgE anti-

(DNP)2 (IgELa2, American Type Culture Collection) and











partially purified mouse IgG anti-TNP (generous gift of M.

Rittenberg, Oregon Health Center University) were cleared

by centrifugation at 12,000xg for 15 minutes at 4C. The

latter was diluted in Dulbecco's modified Eagle's minimal

essential medium (GIBCO) with 10% v/v heat-inactivated

fetal bovine serum, and both contained 0.1% w/v sodium

azide. Cells to be analyzed were suspended in Hl0BNF1 at

ixl06 per ml, aliquoted at 0.1 ml per well of a 96-well PVC

cluster (Dynatech, Inc., Alexandria, VA) and pelleted by

centrifugation (2 minutes, 10Oxg, 4C). The cluster was

"flicked" and briefly vortexed (five to ten momentary

touches) and the cells were resuspended in 0.1 ml of

cleared IgE or IgG anti-TNP. Clusters were covered with

plastic wrap and placed in a 37C, 5% carbon dioxide

incubator for one hour. Following incubation, the treated

cells were washed twice with Hl0BNF1 by centrifugation.

The cell pellets were dispersed by vortexing and 5

microliters of 10% w/v TNP-S. aureus or TNP-E. coli were

added to each well. The covered microtiter clusters were

placed on ice for thirty minutes and the contents of the

wells were washed six times as previously described. The

pellets were resuspended in 0.1 ml of HO10BNFl. Ten to

thirty microliter samples of each well were

cytocentrifuged, stained with May-Gruenwald Giemsa and

observed by bright field microscopy.











Mice

BALB/cAn mice used in these studies were bred in our

colony. BlO.BR/SgSn and CBA/J mice were purchased from The

Jackson Laboratory (Bar Harbor, ME). For studies involving

timed pregnancies, females were placed in the cages housing

one or two males at ratios of one to three females per

male. Females were observed for vaginal plugs following

overnight cohabitation and the date of detection was noted

as day zero of gestation.

Analysis of A-MuLv Proviral Integration

High molecular weight DNA was extracted from tumor

cells by the method of Steffan et al. (1979). Tumor cells

were harvested from liquid culture by centrifugation and

washed in cold PBS. The cells were resuspended at ten

million per milliliter in TES (10 mM Tris, 5 mM EDTA, 100 mM

sodium chloride, pH 7.5) and added dropwise to an equal

volume of lysis buffer consisting of TES with one percent

w/v SDS (Sigma Chemical Co.) and 0.04 percent Proteinase K

(Fisher Scientific Co.). The lysates were digested

overnight at 37C with gentle mixing. DNA was extracted

from the lysate. twice for thirty minutes with an equal

volume of glass-distilled phenol and twice again with

chloroform containing 4 percent isoamyl alcohol (Fisher

Scientific Co.). The extracted DNA was precipitated in 2.5

volumes of 95 percent ethanol or isopropanol, dissolved in

TIOE1 buffer (10 mM Tris, 1 mM EDTA, pH 7.5), and dialyzed











extensively against the same buffer. Normal embryonic

tissue, prepared in an identical manner, provided control

DNA. All DNA was quantitated spectrophotometrically by the

OD260/OD280 method (Maniatis et al., 1982).

Ten micrograms of DNA were incubated with 10 units of

restriction endonuclease BAM HI (New England Biolabs,

Beverly, MA) in TA buffer (O'Farrell et al., 1980);

completeness of digestion was monitored by the addition of

one microgram of bacteriophage lambda DNA to a duplicate

sample. The digested DNA was mixed with sample buffer (50 mM

Tris, pH 7.5, 5 mM EDTA, 25% w/v Ficoll, 0.05% w/v

bromophenol blue, at 5X concentration) and electrophoresed

for eighteen hours at 40 volts D.C. on an 0.8% agarose gel

in TEA buffer (40 mM Tris-acetate, 2 mM EDTA, pH 7.8).

Both the gel and the electrophoresis buffer contained 0.5

micrograms of ethidium bromide per milliliter. Samples

were organized such that a set of digested cellular DNAs

were pipetted into wells on one side of the gel and

duplicates containing bacteriophage lambda DNA were

pipetted into wells on the other side of the same gel.

Following electrophoresis, the gel was photographed

under ultraviolet light to visualize the restricted DNA and

verify that all of the lambda DNA had been digested to

completion. A ruler placed alongside the gel was

photographed at the same time to provide a scale of DNA

fragment sizes for later reference. The lanes containing











lambda DNA were generally cut away and discarded. The gel

was then exposed to shortwave ultraviolet light for an

additional ten minutes to break the DNA and thereby

facilitate transfer. DNA in the gel was denatured with 0.5

M sodium hydroxide, 0.6 M sodium chloride for one hour,

neutralized in 1 M Tris, pH 7.4, 1.5 M sodium chloride (two

changes of 150 to 200 milliliters for 30 to 45 minutes

each), and transferred to nitrocellulose (Schleicher and

Scheull, Keene, NH) by the method of Southern (1975).

Probes for A-MuLV-related sequences were prepared from

the virus-specific recombinant plasmid pAB3Sub3 (Goff et

al., 1980) by nick translation (Rigby et al., 1977) to a

specific activity of 108 dpm per microgram. The 32p_

labeled sequences were hybridized to the nitrocellulose-

immobilized DNA for twenty hours at 68C by the method of

Wahl (1979). The nitrocellulose blot was then washed

extensively under stringent conditions (0.015 M sodium

chloride, 0.0015 M sodium citrate at 68C). The probed

blots were autoradiographed on XAR-5 film (Eastman-Kodak,

Rochester, NY) with two calcium-tungstate-phosphor

intensifying screens (Cronex Lightning Plus, E.I.DuPont de

Nemours and Co., Wilmington, DE) for two to five days.

Conditioned Media

Embryonic tumor cells were cultured under conditions

similar to those used to generate WEHI-3 conditioned media

(W3CM) (Razin et al., 1984a). Cells from log phase











cultures were seeded at Ix106 per ml in o10P in bacterial-

grade petri dishes (Fisher Scientific Co.). Following four

days (92 to 100 hours) of incubation in humidified five

percent carbon dioxide, the conditioned media were

harvested by centrifugation and filtered through tissue

culture grade 0.2 micron nitrocellulose filters.

Conditioned media were concentrated by stirred-cell

ultrafiltration (Amicon, Danvers, MA), by dialysis against

polyethylene glycol 20,000 (Fisher Scientific), or by

ammonium sulfate (Fisher Scientific Co.) precipitation when

such procedures were desired.

Cell lysates were prepared from ceils grown to 7 to

9x105 per ml as follows: Cell cultures were centrifuged

(ten minutes at 200xg, 40) and the cells were washed twice

in PBS. The cells were resuspended in one milliliter 10P

and subjected to three rounds of alternate freezing (in dry

ice-ethanol) and thawing (at 37C). The lysates, which

contained no viable cells upon microscopic examination,

were cleared centrifugally at 2000xg for fifteen minutes

(4C) and at 12,000xg for fifteen minutes (4C), and were

filtered through 0.45 micron sterile, disposable filters

(Gelman Sciences, Ann Arbor, MI). Lysates were stored at

-20C prior to use.

Assay for Interleukin 3

Interleukin 3-like activity was analyzed by a

modification of the method of Razin and colleagues (1984a).











The proliferation of cell line DA-1 (generously provided by

J.N. [hle), which requires interleukin 3 for growth, served

as an assay for interleukin 3. DA-1 cells were

centrifuged, washed twice in PBS, and resuspended at 5x105

per ml in appropriate condititioned media or cell lysates.

One-tenth milliliter aliquots were pipeted in triplicate

into separate compartments of a 96-well microtiter cluster

(Linbro, Flow Laboratories, McLean, VA). The cells were

incubated (37C, five percent carbon dioxide) for sixteen

hours, at which time each well was pulsed with one

microcurie of 3H-TdR (5 Ci per mMol, Amersham Corporation,

Arlington Heights, IL) in ten microliters of EM. Following

six hours additional incubation, the ceils were collected

on Whatman glass microfiber filter strips (Whatman Paper

Ltd., Maidstone, U.K.) in water by multiple automated

sample harvester (MASH, Otto Hiller Company, Madison, WI).

The filter strips were air dried. 3H-TdR which was

incorporated into filter-immobilized DNA was counted in

toluene-based scintillation fluid in a liquid scintillation

spectrometer.



Results



Histamine Content of Transformed Placental Cells

Initial light microscopic examination of some of the

transformed cell lines revealed large mononuclear cells











with basophilic granules which stained metachromatically

with acidic toluidine blue (H.R. Katz, personal

communication). Since the cells fit the working definition

of mast cells by these criteria, we began our

characterizations by assaying for intracellular histamine.

All but two of the placental cell lines analyzed contained

histamine as detected by a sensitive, isotopic-enzymatic

microassay (Table 11-2). The quantity of histamine detected

in the cells (5 to greater than 500 nanograms per million

cells) was similar to that found in cultured mast cells

derived from mouse spleen (450 to 500 nanograms per million

cells; Razin et al., 1981a), bone marrow (80 to 150

nanograms per million cells; Razin et al., 1982a), and

fetal liver (200 to 1400 nanograms per million cells; Nabel

et al., 1981), and mucosal mast cells (160 to 2000

nanograms per million cells; Befus et al., 1982b;

Bienenstock et al., 1982), but one order of magnitude less

than that found in serosal mast cells (15 micrograms per

million cells; Bienenstock et al., 1982). Histamine was

not detected in lymphoid or myelomonocytic cell controls,

nor was it detected in the mastocytoma P815, which

reportedly has variants which are devoid of mast cell

granules (Mori et al., 1979). Histamine biosynthesis was

confirmed by chromatographic identification (Galli et al.,

1976) of [3H]-histamine in extracts of [3H]-histidine-

labeled cells (data not shown).












Table 11-2. Histamine Content of Embryonic Tumor Cell Lines
and Control Tumor Cell Lines

CELL LINES HISTAMINE
(ng/104 cells)a


PLACENTAL CELL LINES

9P1

10P2

10P6

10P8

10P12

11PO-1

11P62

CONTROL CELL LINES

WEHI-3

RLd11

18-81

FLEI-4

P815


>5.00

2.70

<0.05

0.05

0.06

<0.05

1.60


<0.05

<0.05

<0.05

<0.05

<0.05


a: Histamine content was determined as
Materials and Methods.


described in











Analysis of Hematopoietic Lineage Markers

In view of the morphological and biochemical

similarity of the placental cell lines to mucosal and

culture-derived mast cells, we sought to further confirm

the relationship by analysis of the cell surface

differentiation antigens with an antibody which

discriminated between serosal and culture-derived mast

cells (Katz et al., 1983). This and other cell surface

antigens were analyzed by a simple and sensitive S. aureus-

antibody sandwich method (Uchanska-Ziegler et al., 1982).

Figure II-1A shows the binding reaction of the cell line

10P12 with bacteria coated with anti-cultured mast cell

antibody B23.1, while Figure II-IB shows that the same cell

line, incubated with bacteria coated with unimmmunized rat

immunoglobulin, resulted in no bacteria bound to the mouse

cell surfaces.

Cell surfaces of the remaining A-MuLV placental

transformants and of a number of control tumor cell lines

were probed with monoclonal antibody B23.1 as well as a

panel of other monoclonal and conventional antibodies

reported to be specific for hematopoietic differentiation

markers (Table II-l). Table IT-3 summarizes the results of

the cell surface analyses. Every virus-transformed

embryonic cell line expressed the B23.1 epitope, which is























*J


b^


Figure II-i.


Detection of Cell Surface Determinants on Abelson
Murine Leukemia Virus-Transformed Embryonic Cells.


Killed Staphylococcus aureus bacteria coated with anti-mast
cell/monocyte B23.1 antibodies (A) and normal rat serum
antibodies (B) were reacted with cells from line 10P12 as
indicated in Materials and Methods.
















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--I ai


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(n 3
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also found on cells of the monocyte lineage (Leblanc et

al.. 1982) as well as on culture-derived mast ceils. This

marker was also detected on control mastocytoma (P815) and

myelomonocytic leukemia cells (WEHI-3).

Two of the cell lines expressed three related

antigenic determinants of the 200 to 220 kilodalton surface

glycoprotein family which is found on mouse lymphoid cells.

The cell lines 11P0-1 and 10P12, as well as all of the

control lymphoid and adult-derived mastocytomas which we

examined, expressed the antigens defined by the monoclonal

antibodies 14.8 and RA3-2C2. These epitopes are expressed

on B cells and their surface immunoglobulin-negative

precursors. The progenitor cells react with the monoclonal

antibody RA3-3A1 as well. The latter antibody, however,

did not recognize a 220 kilodalton glycoprotein on tumor

cells of the T lineage which was detected by 14.8 and RA3-

2C2. The selective reactivity of RA3-3A1 has been

confirmed previously (Coffman and Weissman, 1981b). It is

interesting to note, however, the novel expression of this

previously characterized B cell differentiation antigen on

mastocytoma P815 as well as our embryo-derived mast cell

lines.

Placental and control tumor cell lines were also

analyzed for the expression of other primitive lymphoid

markers. The neutral glycolipid asialo-GMl, which is

expressed on the early embryonic, Thy-l-negative thymocytes











(Habu et al., 1980) and for the expression of receptors for

the Dolichos biflorus agglutinin, which is expressed on

early fetal thymocytes and on some thymic leukemia cells

(Muramatsu et al., 1980). The monoclonal antibody M6

(Kasai et al., 1983) reportedly recognizes cells bearing

receptors for Dolichos biflorus agglutinin and probably

binds to the receptor itself. Table 11-3 shows that only

one of the embryonic cell lines, 11P0-1, expressed the

early thymocyte differentiation marker recognized by anti-

FT-1. The same ceil line also bears surface asialo-GMl, as

do two other embryonic cell lines, 9P1 and 10P6. It is

also interesting to note that two A-MuLV-induced adult

mouse mastocytomas, CB6ABMC4 and BALABMC20 were both

positive for FT-1, and that the latter cell line also

expressed the neutral glycolipid asialo-GMl. These

observations will be discussed later in this chapter.

Despite the presence of markers specific for early T

lineage cells on several embryonic cell lines, mature T

lineage markers were only detected on three control cell

lines. Lack of reactivity with monoclonal antibody

T24/31.7 (Dennert et al., 1980) indicated that none of the

cell lines expressed the Thy-l differentiation marker.

Also conspicuously absent from A-MuLV-transformed embryonic

cell lines was the antigen recognized by the monoclonal

antibody 5H1, which is expressed on Abelson murine leukemia

virus transformation-sensitive targets from mouse bone











marrow as well as on thymocytes, fetal liver red blood

cells, and pre-B cells (Shinefeld et al., 1980; E. Siden

and L. Shinefeld, unpublished observations).

Analysis of Receptors for IgE and IgG

High affinity membrane receptors for the Fc regions of

immunoglobulins were detected with a sensitive, isotype-

specific assay developed in this laboratory (Siden and

Siegel, 1986). Coated bacteria were prepared by

derivatizing the bacterial surfaces with the hapten

trinitrophenol (TNP) and then reacting the modified

bacteria with mouse monoclonal anti-TNP antibodies of the

IgE or IgG classes. Experimental and control cell lines

were incubated with the immunoglobulin-coated bacteria at

37C for one hour and processed for cytocentrifugation and

staining. Typical positive and negative reactions are

illustrated in Figure 11-2. Observations are summarized in

Table 11-4. IgE receptors were detected on all but two of

the embryonic cell lines analyzed, while none of the cell

lines expressed high affinity receptors for IgG. Receptors

for IgG were detected, however, on the myelomonocytic

leukemia cell line WEHI-3 (Table 11-4) and on the

macrophage-like tumor cell line P388D1 (data not shown).

The results of the bacterial assay for IgG receptors were

duplicated by the method of Schrader (1981), which used

xenogeneic (rabbit) 7S antibodies immobilized on sheep red

















V




U


Figure 11-2.


Detection of Surface Receptors for IgE on A
MuLV-Transformed Embryonic Cells.


Killed Staphylococcus aureus bacteria haptenated with
trinitrophenol (TNP) were incubated with cells from line
10P8 which had previously been incubated with monoclonal
mouse IgE (A) and IgG (B) as described in Materials and Methods.


0 0 -i












Table 11-4.



CELL LINE


Analysis of Surface Membrane Receptors for IgE
and IgG on A-MuLV-Transformed Embryonic
Cell Lines and on Control Tumor Cell Lines

MEMBRANE RECEPTORS FOR


Mouse IgEa


Mouse IgGa


Rabbit IgGb


10P8

10P12

11PO-1

11P62-4


CONTROL

WEHI-3

RL 11

18-81

FLEI-4

P815

CB6ABMC4

BALABMC20


NDc


a: Membrane receptors for allogeneic (mouse) IgE and IgG were
detected by the TNP-bacteria/anti-TNP method (see Materials
and Methods).
b: Membrane receptors for xenogeneic (rabbit) IgG were detected
by the rabbit anti-sheep RBC/SRBC method of Schrader (1981).
c: ND indicates analysis not performed.


PLACENTAL

9P1

10P2


10P6











blood cells. Rosettes of sheep red blood cells on IgG Fc

receptor-positive cells were detected by phase microscopy

after staining with crystal violet.

Metachromatic Granules in Transformed Cell Lines

Initial examination of several of the embryonic cell

lines, which provided the first evidence for their

relationship to mast cells, was followed by a more detailed

study of the remaining cells. Cells were fixed in Mota's

lead subacetate and stained in acidic toluidine blue to

detect granules rich in basophilic glycosaminoglycan. As

seen in Table 11-5, the majority of the original (BALB/c x

BI0.BR)F1 embryonic cell lines did not possess

metachromatic granules, although two cell lines with high

histamine content (9P1 and 11P62) did express the

characteristic. Additionally, four of six embryonic cell

lines derived from a different paternal background, and all

long-term culture-derived mast cells (see Chapter III),

stained metachromatically with toluidine blue. Many of the

A-MuLV-transformed embryonic cell lines synthesize and

secrete chondroitin-4,6-disulfate proteoglycan (D. Levitt,

R. Porter, and E. Siden, manuscript in preparation), in

support of our observations.

Analysis of A-MuLV Provirus Integration

Although the embryonic tumor cell lines were derived

from cells infected with Abelson murine Leukemia virus, we

sought to confirm the presence of A-MuLV-specific sequences












Table 11-5. Metachromatic Granules in A-MuLV-Transformed
Embryonic Cell Lines and Control Tumor Cell Lines.


Cell Line

placental

9P1
10P2
10P6
10P8
10P12
11PO-1
11P62
O10PC1
1I1PCI4
11PC19
11PC20
11PC32
12PC1


control

WEHI-3
18-81
P815
CB6ABMC4
BALABMC20


Metachromatic
Granulesa


a. Cytocentrifuged smears of cells were stained with
toluidine blue as indicated in Materials and Methods.
Cells with metachromatic granules (+) and without
metachromatic granules (-) were scored.











in cellular DNA by identifying the proviral genome. DNA

isolated from four of the embryonic cell lines was probed

with the Abelson virus-specific recombinant plasmid

pAB3Sub3. The cellular DNA was first digested with

endonuclease BamHI, which recognizes no restriction sites

within the virus-specific sequences of the plasmid. Thus,

each integrated A-MuLV provirus detected was represented by

a single band on the autoradiographed blot. All but one of

the cell lines so analyzed showed two copies of the A-MuLV

provirus integrated into high molecular weight DNA (Figure

11-3); cell line 11P62 showed only one copy. In addition to

integrated viral sequences, the probe also detected two

germ line fragments of the endogenous c-abl gene, which

contains cellular sequences and BamHI sites not present in

the plasmid. The v-abl and c-abl patterns of the control

cell line 160N54, from which the infecting virus was

isolated, are also shown.

Two of the embryonic cell lines, 10P12 and 11PO-1,

were injected into mice to determine whether the cells were

tumorigenic. Both cell lines caused tumors in syngeneic

mice inoculated within two weeks of birth. DNA was

prepared from tumors isolated from the mice (output DNA).

Restriction analysis of the input (original cell line) and

output DNA was performed to determine whether the lesions

were due to cell line proliferation or to subsequent

infection of host cells by shed virus. Tumor cells derived




Full Text
CHARACTERIZATION OF ABELSON MURINE LEUKEMIA VIRUS-
TRANSFORMED MIDGESTATION EMBRYONIC CELLS
AND THEIR NORMAL HOMOLOGUES
By
MICHAEL L. SIEGEL
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1986


73
Mice
BALB/cAn mice used in these studies were bred in our
colony. B10.BR/SgSn and CBA/J mice were purchased from The
Jackson Laboratory (Bar Harbor, ME). For studies involving
timed pregnancies, females were placed in the cages housing
one or two males at ratios of one to three females per
maLe. Females were observed for vaginal plugs following
overnight cohabitation and the date of detection was noted
as day zero of gestation.
Analysis of A-MuLv Proviral Integration
High molecular weight DNA was extracted from tumor
cells by the method of Steffan et al. (1979). Tumor cells
were harvested from liquid culture by centrifugation and
washed in cold PBS. The cells were resuspended at ten
million per milliliter in TES (10 mM Tris, 5 mM EDTA, 100 mM
sodium chloride, pH 7.5) and added dropwise to an equal
volume of lysis buffer consisting of TES with one percent
w/v SDS (Sigma Chemical Co.) and 0.04 percent Proteinase K
(Fisher Scientific Co.). The lysates were digested
overnight at 37C with gentle mixing. DNA was extracted
from the lysate, twice for thirty minutes with an equal
volume of glass-distilled phenol and twice again with
chloroform containing 4 percent isoamyl alcohol (Fisher
Scientific Co.). The extracted DNA was precipitated in 2.5
volumes of 95 percent ethanol or isopropanol, dissolved in
T10E1 buffer (10 mM Tris, 1 mM EDTA, pH 7.5), and dialyzed


31
and serosal tissues, while those of lymphoid origin (lymph
node and thoracic duct) resembled mucosal mast cells, being
smaller in size with sparser, but larger granules than the
former cells. Pure "mucosal mast cells" (in fact, culture-
derived mast cells of lymphoid origin) could be grown on
selected feeder layers which were free of serosal type
precursors. The "mucosal mast cells" persisted, however,
only in the presence of T cell-derived factors. In
contrast, the mast cells derived from embryonic feeder
layers continued to persist, albeit without further
expansion, for six months or longer in the absence of
exogenous factors.
The same group (Davidson et al., 1983) later reported
that lymph node ceils derived from unimmunized, horse
serum-immunized, and helminth-infected mice, grown in the
presence of conditioned media (from antigen-stimulated
mesenteric lymph node cells) but in the absence of
irradiated embryonic mouse fibroblast feeder layers,
proliferated (as large, vacuolated cells) but failed to
develop granules. When the undifferentiated, culture-
derived cells were transferred to fibroblast monolayers,
however, the cells developed metachromatic granules
containing histamine within seven days. Intimate contact
between the two populations of viable cells was apparently
essential to granule maturation, as neither fibroblast
conditioned media, fibroblast homogenates, glutaraldehyde-


63
significant observations. First, unlike previously
reported Abelson virus-transformed embryonic cells, the
placental cell lines isolated in our laboratory exhibit
many characteristics of culture-derived and mucosal mast
cells including differentiation antigens, high affinity
receptors for IgE, and metachromatic granules containing
histamine and sulfated proteoglycans. Second, we describe
the development of a simple, sensitive, nonisotopic,
nonfluorometric method to detect membrane receptors for
immunoglobulins. Third, all of the cell lines analyzed had
at least one, and sometimes more than one integrated A-MuLV
provirus. Finally, the cell lines proliferated independent
of exogenous mast cell growth factor, and no growth factor
could be detected in either cell lysates or the
supernatants of exponentially growing cultures maintained
at high cell density.
Materials and Methods
Cell Lines
Cloned A-MuLV-transormed embryonic cell lines were
established in our laboratory as previously reported
(Siegel et al., 1985). All had been adapted to growth in
liquid culture and had been successfully maintained in 10P
(RPMI 1640 (GIBCO, Grand Island, NY) with 5x10^ M 2-


146
midgestation placenta and nonplacental embryonic tissues
(NPET) are a rich reservoir of mast cell precursors,
containing proportionately at least as many such cells as
adult bone marrow. Third, in characterizing the cells
in mast cell colonies, we have found that they
express a variant of the Ly5 differentiation antigen
previously reported to be specific for B lymphocytes.
Finally, we describe preliminary experiments which
differentiate mast cell precursors from other bone marrow
elements on the basis of surface membrane determinants.
These findings provide significant new information about
mast cell differentiation and ontogeny. Based upon our
findings, we discuss the role of mast cells and their
precursors in terms of murine embryonic hematopoiesis and
the immunobiology of the maternal-fetal interface.
Materials and Methods
Procedures for the husbandry of mice, detection of
cell surface determinants and Fc receptors, and cytological
staining were performed as described in Chapter II.
The procedure for the preparation of WEHI-3 conditioned media
(W3CM) was performed as described in Chapter III.
Cultivation of Mast Cell Precursors in Semisolid Media
Mast cell precursors were cultivated from fresh


20
1963; Ginsburg and Lagunoff, 1967) and the rat (Ishizaka et
al. 1976) and Enerback's atypical mast cells of the lamina
propria spurred erroneous theories that the mucosal mast
cell was derived from the thymus (Burnet, 1965, 1975,
1977). Based upon observations of mucosal mastocytosis
following experimental infection of rats and mice with the
nematodes Nippostrongylus brasiliensis and Tnchinella
spiralis, a lymphoid origin of these cells was also
proposed by other investigators (Rose et al., 1976; Befus
and Bienenstock, 1979; Mayrhofer, 1979a,1979b; Nawa and
Miller, 1979).
The derivation of mucosal mast cells was ultimately
resolved by Crowle (1982), who reconstituted mucosal mast
cell-deficient mice with cells derived from a variety of
hematopoietic tissues. Crowle observed that W/Wv mice
could be reconstituted by bone marrow and spleen cells but
not by thymocytes or thymus grafts, while athvmic mice
could be reconstituted by thymus grafts, thymocytes or
splenocytes. Crowle proposed that the W/Wv mice were
defective in mucosal mast cell precursors which were
present in bone marrow (and spleen) of normal mice, while
athymic mice possessed the precursor population and needed
a thymus-related component to effect differentiation.
Crowle concluded that mucosal mast cells were derived from
bone marrow and required a thymic influence for
accumulation in mucosal surfaces. These relationships


116
described for bmadhCM. Cells from the healthiest, most
confluent monolayers (seeded at 1x10^ per ml) were washed
twice with cold PBS and overlayered with 2 ml of 0.3
percent agar containing 10P/LPS or 50% W3CM.
Exponentially growing cultures of WEHI-3
myelomonocytic cells at 4x10-5 per Were centrifuged for
ten minutes at 200xg and resuspended at 1x10^ per ml in
fresh 10P/LPS. Individual compartments of a twelve-well
tissue culture cluster (Costar) received 1x10^, 0.5x10^ or
O.lxlO^ cells; all volumes were normalized to one ml with
10P/LPS. Cells were cultured for two days under these
conditions, at which time the conditioned media were
harvested and processed as previously described. Only
those wells seeded with 0.1x10^ cells had healthy adherent
layers and were selected for coculture experiments. The
latter cells were washed twice with cold PBS prior to
overlayering with 1 ml 0.3 percent agar in either 50% W3CM
or 10P/LPS.
Mast cells derived from (BALB/cAN x CBA/J)F1 placenta
and BALB/cAN bone marrow were selected and enriched to a
homogeneous population as previously described. At day 37
of culture, the cells were prepared for the experiment by
centrifugation at 200xg for ten minutes at room
temperature. The cell pellet was resuspended at a
concentration of 2x10^ per ml in 100% W3CM and one ml of
the suspension was pipeted into each well of the six-well


1983b). Similar cells have been cultured from rat bone
marrow (Haig et al., 1982, 1983), peripheral blood (Zucker-
Franklin et al., 1981; Czarnetzki et al., 1983), and thymus
(Ishizaka et al., 1976, 1977) as well as human fetal liver
(Razin et al., 1981b), umbilical cord blood (Ogawa et al.,
1983), and adult peripheral blood (Denburg et al., 1983;
Czarnetzki et al., 1984).
The development of m vitro methods for mast cell
culture provided additional means of detecting embryonic
mast cell precursors. As previously noted, Ginsburg (1963)
observed occasional mast cell outgrowth in cultures of day
eighteen embryonic mouse skin. Similar experiments with
the rat model demonstrated that embryonic rat thymus,
isolated between eighteen and twenty days post coitum and
cocultured with adult rat thymocytes or thymocyte
conditioned media, contained cells capable of
differentiating into mast cells (Ishizaka et al., 1976). A
third group (Nabel et al., 1981; Galli et al., 1982a) was
able to culture murine mast cells derived from day thirteen
fetal liver suspensions cultured in lymphocyte conditioned
media. Using the adherent cell system, Ginsburg and
colleagues (1982) were also able to demonstrate that mast
cells could be derived in culture from disaggregated mouse
embryos between ten and thirteen days of gestation. It was
thus apparent that the precursors of culture-derived mast
cells were present in the mouse embryo at ten days post


Table III-l. Continued
Mast Cell Treatment Percent Cells Expressing
bone marrow-derived mast cellse
cocultured with:
RA-3A1
Epitope
B23.1
Epitope
IgE
Receptors
Metachromatic
Granules
50% W3CM only
Adherent bone marrow cells underlayer
with bmadh CM in agar
<1
99
56
100
+50% W3CM + 50% bmadh CM
Adherent bone marrow cell underlayer
<1
99
50
93
with 50% W3CM in agar
Agar underlayer
<1
100
52
96
with bmadh CM
No underlayer
2
93
ND
+
+ bmadh CM
<1
95
60
98
a. Long term (four weeks and older) mast cell cultures were cocultured with adherent cells
in agar underlayers, with cell-free agar underlayers and with no underlayers. In some
experiments, media other than 50% W3CM was used in formulating the agar (these are
prefixed by "with"). In some experiments, the liquid media in which the mast cells were
grown was supplemented with adherent cell conditioned media (these are prefixed by "+").
For details see Materials and Methods.
b. Following coculture under the indicated conditions,mast cells were analyzed as described
in Materials and Methods. One hundred to two hundred cells were assessed microscopically
for each characteristic and the number of characteristic-positive cells was calculated
as a percentage of the entire population. In some instances, insufficient cell numbers
precluded assay (ND); in one instance, less than one hundred cells were counted, and no
percentage was determined of the marker-positive cells (+).
c. Mast cell cultures were derived from embryonic placenta of (BALB/c x CBA)F1 concepti
isolated at 11 days post coitum as described in Materials and Methods.
d. Abbreviations used: W3CM (WEHI-3 conditioned media), LPS (bacterial lipopolysaccharide),
10P/LPS (standard medium 10P supplemented with LPS, 20 micrograms per ml), P388CM
(conditioned media of cell line P388), W3/LPSCM (conditioned media of cell line WEHI-3
grown in media supplemented with LPS), bm (bone marrow), bmadh CM (conditioned media of
adherent bone marrow cells).
e. Mast cells cultures were derived from BALB/cAN bone marrow as described in Materials and
Methods.
134


136
are present in significant numbers in both types of tissues
derived ten and eleven days post coitum. Previously,
Ginsburg and colleagues (1982) reported that mast cells
occassionally arose in control cultures of day ten to day
thirteen adherent embryonic cells which were generally used
for feeder layers in cultures of mouse thymus-derived mast
cells. The authors, however, did not provide detailed
description of the phenotype of such cells.
Numerous authors have reported that mast cells derived
from a variety of adult tissues can be maintained in
culture for prolonged periods of time in the presence of
required growth factors. Although no attempts were made to
establish permanent mast cell lines like those previously
reported (Nabel et al., 1981; Nagao et al., 1981; Razin et
al., 1981a; Schrader et al., 1981; Tertian et al., 1981), we
were able to maintain bone marrow-derived mast cells in
vitro for more than seven weeks without appreciable loss of
viability (determined by trypan blue exclusion). In
contrast, mast cells derived from placenta seldom survived
more than five weeks before catastrophic decline in viable
cells. Similar results were reported by Ginsburg and
colleagues (1982) for culture of adult lymphoid tissue-
derived mast cells on embryonic feeder layers. Those
authors, however, ascribed the decline in cell number to
loss of growth factor, a variable which we have controlled
throughout culture. We propose that the embryo-derived


6
to A-MuLV transformation are also sensitive to the
proliferative effects of interleukin 3 (reviewed in Iscove
and Roitsch, 1985; Rennick et al., 1985). Seminal studies
on the culture of mast cells revealed that cells of this
lineage would proliferate in the presence of media
conditioned by lectin- or antigen-stimulated T lymphocytes
or by the myelomonocytic leukemia cell line WEHI-3B
(Hasthorpe, 1980; Nabel et al., 1981; Nagao et al., 1981;
Schrader, 1981; Tertian et al., 1981). More recently,
interleukin 3, the proliferative factor in the conditioned
media, was purified to homogeneity (Ihle et al., 1983;
Razin et al., 1984a). Subsequent studies performed with
the glycoprotein product of cloned interleukin 3 gene have
substantiated the proliferative activity of the factor
(Yokota et al., 1984; Rennick et al., 1985).
Mast Cell Function, Origin, Ontogeny, and Heterogeneity
Contemporary knowledge and interest in the mast cell
has its roots in the midnineteenth century. The earliest
description of these cells is found in the work of von
Recklinghausen (1863), who observed and illustrated the
mast cell in the unstained mesentery of the frog. Credit
for the discovery of the mast cell, however, is generally
assigned to Paul Ehrlich, a young physician-scientist who
was then interested in the differential staining affinities
of certain tissue cells and their organelles. Ehrlich
(1877) first described mast cell-like cells in several


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
CHARACTERIZATION OF ABELSON MURINE LEUKEMIA VIRUS-
TRANSFORMED MIDGESTATION EMBRYONIC CELLS
AND THEIR NORMAL HOMOLOGUES
By
Michael L. Siegel
May 1986
Chairman: Edward J. Siden
Major Department: Immunology and Medical Microbiology
The embryonic origin and ontogeny of mast cells is
poorly understood, despite a growing body of literature
relevant to that area of study. We have systematically
investigated the development of mast cells in the embryonic
mouse, beginning our studies with the observation of mast
cell characteristics of midgestation embryonic placental
cells transformed with the defective retrovirus, Abelson
murine leukemia virus. Unlike previously reported Abelson
virus-transformed cells, the placental cell lines exhibited
many of the characteristics of culture-derived mast cells,
including differentiation antigens, high affinity receptors
for IgE, and metachromatic granules containing histamine
and sulfated proteoglycans. Some of the cell lines also
expressed the B220 marker previously reported to be
specific for cells of the B lymphoid lineage. We also
viii


164
Tokyo, Japan) and were picked under sterile conditions by
gentle aspiration with a Pasteur pipet. The colonies were
expelled from the pipet into a well of a 96-well tissue
culture cluster (Costar, Broadway, MA) and the cells were
disaggregated by gentle trituration with 0.1 ml of 50%
W3CM. Some cells were analyzed immediately, while others
were incubated in a humidified atmosphere of five percent
carbon dioxide in air for one to two days prior to
analysis. Approximately half of the cells were prepared
for toluidine blue staining by cytocentrifugation and air
drying. Following fixation in Mota's fixative (Yam et al.,
1971), or acidic ethanol (Mota's fixative without lead
subacetate), the slides were stained with 0.1 percent w/v
toluidine blue, ph 0.5. Most (at least 90%) of the cells
from each colony contained metachromatic granules
indicative of mast cells (Figure IV-IF, Table IV-2).
Similarly identified and picked cells were also
analyzed for surface markers as previously described. The
cells had receptors for IgE but not for IgG, further
confirming their identity as mast cells. Most of the cells
also expressed the surface determinant recognized by
monoclonal antibody B23.1. As previously noted, the latter
marker is expressed by both mast cells and mononuclear
phagocytes.
The expression of the B220 lymphoid marker recognized
by the monoclonal antibody RA3-3A1 on A-MuLV-transformed


7
species as elements which stained atypically red-violet
with the blue basic analine dye dahlia. The term mast cell
is derived from Ehrlich's nomenclature (Mastzellen), which
he assigned to granular cells which were abundant in well-
nourished tissues of frogs (Ehrlich, 1879). In the same
work, Ehrlich first used the term "Metachromasie," or
metachromasia, to describe the anomalous staining of these
connective tissue cells. Aside from their role as
histochemical curiosities, however, few references to mast
cell derivation, functions, and heterogeneity were
published in the ensuing fifty years (Selye, 1965).
Mature mast cells have been attributed with a number
of physiologic functions, many of which have been reviewed
in the recent literature (Austen, 1984; Shanahan et al.,
1984; Katz et al., 1985a; Lagunoff, 1985). The most widely
known mast cell function is the anaphylactic response,
which was first described by Selye in 1937 (cited in Selye,
1965). The response was originally experimentally induced
in rats by intraperitoneal injection of egg white and
produced hyperemia and edema of the lips, ears, paws, and
genitalia which were aggravated by adrenalectomy and
ablated by stressors like formaldehyde, which induce
adrenocortical hyperplasia. The release of histamine
during anaphylactoid inflammation was hypothesized at this
time, although release of the mediator was not
experimentally associated with mast cells until 1954


53
(480 to 1600 nanograms per million cells) were cultured
from human umbilical cord blood grown in phytohemagglutinin
A-stimulated human T cell conditioned media (Ogawa et al.,
1983). Horton and O'Brien (1983) reported the culture of
granulated cells with centrally placed, round or indented
nuclei from human bone marrow harvested from a patient with
systemic mastocytosis. The culture-derived mast cells
showed no growth advantage with a number of conditioned
media, but required an adherent, bone marrow-derived feeder
layer to persist.
Other In Vitro-Derived Metachromatic Cells
As noted previously, some of the confusion in the
terminology applied to culture-derived mast cells has been
generated by the appearance of characteristics of one or
more of the in vivo basophilic cell correlates. Thus,
although basophils in mice are lacking (Lagunoff and Chi,
1980) or extremely rare (Dvorak et al., 1982), a report of
cloned, basophil-like cell lines with IgE receptors has
appeared in the literature (Galli et al., 1982a). The cell
line in question, derived from mouse splenocytes cultured
in concanavalin A-stimulated splenocyte conditioned media,
lacked histamine and had both natural killer cell
differentiation markers and natural killer activity.
In a series of studies on the rat, Czarnetzki and
colleagues described the growth of connective tissue-like
mast cells in vitro. In the earliest study (Czarnetzki et


2
stochastic processes may play a role in the differentiation
of hematopoietic cells (Nakahata et al., 1982a; Suda et
al., 1984).
The differentiation pathways of hematopoietic cells
and the molecular events involved in normal hematopoiesis
are best understood and defined by delineating discrete
cellular intermediates. Observation and identification is
frequently hampered by hematopoietic tissue heterogeneity,
short life span, and low frequency of cells of interest.
These difficulties are overcome in part by virus-induced
transformation of such cells, resulting in relatively
homogeneous populations of adequate size and frequently
unlimited growth potential. Although transformed
homologues of normal cells are frozen at a particular stage
of development by the action of the transforming gene
product, virus infection may also induce phenotypic changes
which are unparalleled in the course of normal
differentiation. To avoid this pitfall, it is therefore
prudent to confirm that the characteristics of tumor cell
models of early blood progenitors mimic their naturally
occurring counterparts.
Abelson murine leukemia virus (A-MuLV) is a
replication-defective retrovirus which arose by
recombination of portions of the genome of the replication-
competent, thymotropic Moloney murine leukemia virus and a
cellular gene, c-abl (Shields et al., 1979; Goff et al.,


95
proliferation assay, we were unable to detect interleukin 3
in the supernatants or cell lysates of several of the
embyonic cell lines, even when the samples were
concentrated (Table II-6). Appropriate WEHI-3 conditioned
media and cell lysate controls, however, revealed the assay
was functional.
Discussion
Despite over one hundred years of systematic study,
little is known of the processes of differentiation and
maturation of the pharmacologically-active secretory cell
termed the mast cell (Ehrlich. 1877). In the broadest
sense, mast cells have been associated with a population of
mononuclear cells containing biogenic amines and sulfated
proteoglycans which are stored in cytoplasmic basophilic
granules (Selye, 1965; Metcalfe et al., 1981). The
histochemical signature of these cells is the anomalous,
metachromatic staining of the granules in the presence of
analine dyes (Ehrlich, 1879). Mast cells also have high
affinity membrane receptors for IgE, through which, by
specific immune reactions, the cells elaborate their
biogenic effectors (Austen, 1984).
During the course of the last twenty-five years, a
number of reports have established the existence of two


41
of mast cells based upon a number of criteria. First, the
number of culture-derived mast cell precursors in
particular tissues was determined quantitatively. Prior to
the development of mast cell culture techniques which
utilized sources of interleukin 3 for mast cell
proliferation, Pluznik and Sachs (1965) enumerated mast
cell clones in soft agar with feeder layers. The authors
reported approximately thirty mast cell colony forming
units per million spleen cells seeded, while the frequency
of culture-derived mast cell precursors in embryos, thymus,
and lymph nodes were five per million, three per hundred
million, and less than one per fifty million cells,
respectively. Schrader and colleagues (1981) cloned mast
cell precursors from mouse bone marrow in soft agar with
WEHI-3 conditioned media and found thirty to two-hundred
progenitors per million Thy 1-negative ceils. These
results were contradicted by a more recent report (Sredni
et al., 1983) which found 600 to 700 bone marrow
precursors, 400 to 500 spleen precursors, 25 to 30 thymus
precursors, and 100 to 200 Lymph node precursors per
million seeded cells. The latter results, however, were
generated in a system using concanavalin A-activated
splenocvte conditioned media as a source of interleukin 3
and different strains of mice, thereby making comparison
difficult. Interestingly, the latter authors also observed
that athymic nude mice had as many mast ceil precursors as


152
IgE receptor selection
Media from high density cultures of hybridoma IgELa2
(American Type Culture Collection, Rockville, MD) were
cleared by centrifugation and preserved with sodium azide
as previously described. The processed supernatants were
dialyzed for two days against PBS (three changes of 200
volumes) at 4C to remove the sodium azide, filter
sterilized and stored at 4C in a polypropylene tube.
Sheep red blood cells in Alsever's solution (see
above) were washed four times by centrifugation with PBS/
one percent w/v glucose. Haptenating reagent was prepared
by dissolution of trinitrobenzene sulfonic acid (TNBS,
Sigma Chemical Co.) in 0.28M cacodylate buffer, pH 6.9 at
18.75 mg TNBS per ml and sterile filtration (Mishell and
Shiigi, 1980). The sterile TNP-SRBC were prepared by
dropwise addition of one volume of the flicked cell pellet
into seven volumes of haptenating reagent with constant,
gentle mixing. The reagents were pipeted back into the
tube previously emptied of the SRBC and then incubated on a
rotator for thirty minutes at room temperature. Following
centrifugation and removal of the supernatant, the TNP-SRBC
were washed in PBS/one percent w/v glucose/one percent v/v
heat inactivated fetal bovine serum four to six times,
until the supernatants were colorless, resupended to 5
percent by volume and stored at 4C.


157
of several distinguishable cell types (Figure IV-1C, -ID,
-IE) were not enumerated in these studies. In addition to
the colonial populations, long-term agar cultures also
contained a large number of adherent cells. Although the
identity of mast cells was not verified for each colony
counted, staining of cells (picked from agar) with
toluidine blue indicated that more than ninety percent of
the colonies enumerated contained cells with metachromatic
granules indicative of mast cells (Figure IV-IF).
Cells for culture were prepared from embryonic tissues
of the two FI crosses (BALB/cAN x CBA/J and BALB/cAN x
B10.BR/SgSn) used in the Abelson murine leukemia virus
infection experiments and from BALB/cAN homozygous embryos.
As seen in Figure IV-2, the number of mast cell precursors
in embryonic tissues is greatest in midgestation, peaking
at eleven or twelve days post coitum and following the same
distribution in both placenta and nonplacental embryonic
tissues. This tendency was most evident in the
heterozygous tissues, notably (BALB/cAN x CBA/J)F1, in
which the number of mast cell colonies per one million
inoculating cells increase twenty- to thirty-fold between
days eight and twelve post coitum (Figure IV-2A). The
increase in mast cell precursors during midgestation was
less pronounced, but still significant, in the cells
derived from (BALB/cAN x BIO.BR/SgSn)Fl embryonic tissues,
peaking between days ten and twelve with a ten- to twenty-


26
mast cells in several species, including the mouse, will be
further discussed in a later section of this review.
Mastocytomas
The study of mastocytomas has contributed to the
understanding of mast cell growth, differentiation, and
function, and has provided the bridge between complex in
vivo investigations and better-defined, clonal population
analyses of in vitro cultures. Spontaneous mastocytoma,
while common in such species as dogs (Cobb et al., 1975;
Yoffee et al., 1984, 1985), is a more infrequent condition
in other species, notably rats and mice (Lagunoff, 1985).
Efrati and colleagues (1957) described human mastocytoma
cells as large, lymphocyte-like elements similar to the
early mast cell progenitors described in lower mammals
(Maximow, 1906) .
The link between m vivo and in vitro mast cell
studies was established in 1959 when Schindler and colleagues
(1959) reported the successful adaptation of the
methylcholanthrene-induced, murine mastocytoma P815 (Dunn
and Potter, 1957) to growth in culture. The latter
investigators had isolated the neoplasm from a disseminated
disease with foci in the spleen and subcutaneous tissue and
had subsequently adapted it to a highly transplantable
ascites form from which a number of observations were made.
Variations in cell morphology, granule size and density,
and nuclear morphology were noted in the descriptive study.


122
culture by the end of the third week. The latter
determinant, however, was expressed at significant levels
(25 to 25 percent) on freshly dissociated placental cells
of (BALB/c x BI0.BR)F1 origin, but not on syngeneic embryo
(NPET) cells or on cells derived from (BALB/c x CBA/J)
concepti (Figure III-2).
The expression of high affinity receptors for IgG and
the lymphoid B-220 determinant recognized by the antibody
RA3-3A1 remained relatively insignificant during the course
of culture. Expression of markers on mast cells derived
from days ten and eleven placenta and NPET were similar;
furthermore, there were no qualitative differences between
mast cells isolated from (BALB/c x B10.BR)F1 and (BALB/c x
CBA)F1 embryonic tissues.
After four weeks in culture, no adherent cells were
observed in the dishes and essentially all of the cells
contained metachromatic granules as assessed by toluidine
blue staining. In addition to expression of the surface
determinant recognized by monoclonal antibody B23.1,
membrane receptors for IgE (but not IgG), and metachromatic
granules, mast cells derived from midgestation murine
placenta also expressed the paternal Class I
histocompatability antigen (k haplotype), confirming that
the precursors to the cells were embryonic, rather than


91
in cellular DNA by identifying the proviral genome. DNA
isolated from four of the embryonic cell lines was probed
with the Abelson virus-specific recombinant plasmid
pAB3Sub3. The cellular DNA was first digested with
endonuclease BamHI, which recognizes no restriction sites
within the virus-specific sequences of the plasmid. Thus,
each integrated A-MuLV provirus detected was represented by
a single band on the autoradiographed blot. All but one of
the cell lines so analyzed showed two copies of the A-MuLV
provirus integrated into high molecular weight DNA (Figure
II-3); cell line 11P62 showed only one copy. In addition to
integrated viral sequences, the probe also detected two
germ line fragments of the endogenous c-abl gene, which
contains cellular sequences and BamHI sites not present in
the plasmid. The v-abl and c-abl patterns of the control
cell line 160N54, from which the infecting virus was
isolated, are also shown.
Two of the embryonic cell lines, 10P12 and 11P0-1,
were injected into mice to determine whether the cells were
tumorigenic. Both cell lines caused tumors in syngeneic
mice inoculated within two weeks of birth. DNA was
prepared from tumors isolated from the mice (output DNA).
Restriction analysis of the input (original cell line) and
output DNA was performed to determine whether the lesions
were due to cell line proliferation or to subsequent
infection of host cells by shed virus. Tumor cells derived


209
Razin, E. R.L. Stevens, K.F. Austen, J.P. Caulfield, A.
Hein, F.-T. Liu, M. Clabby, G. Nabel, H. Cantor, and S.
Friedman. 1984b. Cloned mast cells derived from
immunized lymph node cells and from foetal liver cells
exhibit characteristics of bone marrow-derived mast
cells containing chondroitin sulfate E proteoglycan.
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Bindegewebskorperchen. Virchow Arch. Pathol. Anat. 28:
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Thymus dependence of specific IgE production and
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Mice. Fisher, New York.
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Esterhuizen, and R.G. Newcombe. 1981. Effect of
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39-46.
Remacle-Bonnet, M.M., R.J. Ranee, and R.C. Depieds. 1983.
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Multiple activities of interleukin 3. J. Immunol. 134:
910-914.
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2719-2726.


145
Guy-Grand et al., 1984) as well as clonal cultures in
semisolid media (Schrader et al., 1981; Zucker-Franklin et
al., 1981; Nakahata et al., 1982b). All of these methods
exploit the ability of mast cell precursors to proliferate
and differentiate in the presence of a factor now termed
interleukin 3 (Ihle et al., 1981) which has been isolated
from media conditioned by lectin- or antigen-stimulated T
lymphocytes as well as several permanent cell lines
(reviewed by Clark-Lewis et al., 1985; Ihle, 1985; Iscove
and Roitsch, 1985; Schrader et al., 1985; Yung and Moore,
1985). Under such conditions, mast cell precursors have
been found in a variety of tissues including adult bone
marrow, spleen, thymus, lymph nodes, gastric and intestinal
mucosa, blood, neonatal cord blood, and fetal liver
(reviewed by Katz et al, 1985a; Austen, 1984; Jarrett and
Haig, 1984).
In previous chapters we have characterized the mast
like, A-MuLV-transformed murine cell lines derived from
midgestation embryonic placenta (Chapter II) and the
subsequent detection of precursors of mast cells from
similar uninfected tissues (Chapter III). We now report the
isolation and quantitation of mast cell precursors from
embryonic tissues of mid- and late gestation. A number of
significant and novel findings are described. First, we
have described mast cell precursors at the earliest
reported time in embryonic mouse development. Second, the


196
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Number of Cells Per Milliliter
126
Days In Culture Post Infection
Figure III-4. Population Dynamics of Bone Marrow-Derived Mast Cells
Infected with A-MuLV
Long-term (at least 28 days) bone marrow-derived mast cells were
infected with A-MuLV or sham-infected and subsequently grown in
2.5 ml cultures with or without W3CM.


175
We propose that mast cell precursors in the
midgestation placenta either perish (due to deprivation of
interleukin 3 in their microenvironment or programmed
annihilation) or migrate around day thirteen to a new
microenvironment in the fetal liver, mucosal sites, or
perhaps the thymus. The fetal liver is the major
hematopoietic organ of late gestation (Metcalf and Moore,
1971). In support of the model of mast cell precursor
annihilation in the third trimester placenta is a recent
report of "waves" of pluripotent hematopoietic stem cells
(CFU-S) which respond to interleukin 3 (Spivak et al.,
1985). Like mast cells, CFU-S require interleukin 3 for
survival as well as proliferation in vitro; in the absence
of interleukin 3, CFU-S concentration decreased
precipitously. Even in the presence of interleukin 3, the
number of proliferating cells exhibited cyclic
fluctuations, which may be an inherent property of all
hematopoietic cells (King-Smith and Morley, 1970). If such
processes were in force in vivo as well as in vitro, we
would propose the loss of interleukin 3 responsiveness in
placenta-associated mast cell precursors beginning at day
thirteen (following at least three days of extensive
proliferation) and the concurrent increase in
responsiveness of fetal liver-associated mast cell
precursors (Kitamura et al., 1981) represent these waves.


96
Table II-6. Interleukin 3 Content of Conditioned Media and Cell
Lysates of A-MuLV-Transformed Embryonic Cells and
Control Tumor Cellsa.
Sample
Stimulation
¡ Conditioned Media^
IL3
Standard
lb
Indexc
1.000+/
-0.000
i
i
! 50% W3CM
IL3
Standard
2
0.836
0.048
! lx W3CM (50% SAS ppt)
¡ lx W3CM (80% SAS ppt)
IL3
Standard
3
0.569
0.064
1.8x W3CM (Amicon ret)
¡ 0.9x W3CM (Amicon ret)
IL3
Standard
4
0.319
0.063
¡ lx W3CM (Amicon filtrate)
1
1
IL3
Standard
5
0.146
0.020
¡ 100% 10P12-2 CM
¡ 10% 10P12-2 CM
IL3
Standard
6
0.069
0.006
¡ 5.5x 10P12-2 CM (80% SAS ppt)
¡ 1.4x 10P12-2 CM (80% SAS ppt)
IL3
Standard
7
0.042
0.005
8.3x 10P12-2 CM (Amicon ret)
¡ lx 10P12-2 CM (Amicon ret)
IL3
Standard
8
0.017
0.004
! lx 10P12-2 CM (Amicon fit)
i
i
IL3
Standard
9
0.010
0.003
| 100% 11PC19 CM
¡ 50% 11PC19 CM
i
Blank
0.009
0.002
1
1
1


199
Ihle, J.N.. 1985. Biochemical and biological properties
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2435.
Ihle, J.N., J. Keller, S. Oroszlan, L.E. Henderson, T.D.
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Schrader, E. Palaszynski, M. Dy, and B. Lebel. 1983.
Biologic properties of homogeneous interleukin 3. I.
Demonstration of WEHI-3 growth factor activity, mast
cell growth factor activity, P cell stimulating factor
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histamine-producing cell-stimulating factor activity.
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20 alpha-hydroxysteroid dehydrogenase in splenic
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C. Song and A. Schimpl (eds.). Cellular and Molecular
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Ishizaka, T., T. Adaci, T. Chang, and K. Ishizaka. 1977.
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217.
Ishizaka, T., H. Okudaira, L.E. Mauser, and K. Ishizaka.
1976. Development of rat mast cells in vitro. I.
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in vivo and in vitro. Immunol. Today 5: 115-119.


142
with adherent cell monolayers which grew in the presence
or absence of WEHI-3 conditioned media. Early reports of
mouse mast cell culture noted the absolute requirement of
such monolayers for mast cell differentiation and
proliferation (Ginsburg, 1963; Ginsburg and Sachs, 1963;
Ginsburg and Lagunoff, 1967). Ishizaka and colleagues
(1976) observed that rat thymus-derived mast cells grew
more rapidly in the presence of a feeder layer, while
Davidson and colleagues (1983) reported that fibroblasts
were required for mast cell granule synthesis.
In conclusion, we have demonstrated the presence of
mast cell precursors in the murine placenta and
nonplacental embryonic tissues during days ten and eleven
of gestation. The mast cells generated in liquid culture
are phenotypically similar to some Abelson virus-
transformed cell lines derived from the same tissue as well
as cultured mast cells derived from other tissues. We have
described the evolution of homogeneous mast cell cultures
from heterogeneous uninfected tissues in terms of
histochemical and surface marker expression. Finally, we
have demonstrated that while A-MuLV can infect mature mast
cells and abrogate their requirement for interleukin 3, the
virus does not induce the ectopic expression of the Ly 5
differentiation antigen which is expressed on some of the
Abelson virus-transformed mast-like cell lines.


154
avidin-SRBC and TNP-SRBC, as appropriate. Further
processing was identical to that noted above, with
hypotonic shock used to deplete the reactants of red blood
cells.
Results
Frequency of Mast Cell Precursors in Midgestational
Embryonic Tissues
Following cultivation of disaggregated placental- or
nonplacental embryonic tissue (NPET)-derived cells in 0.3
percent w/v agar, colonies were observed at the time of the
first feeding (one week in culture) and at each subsequent
weekly feeding. No attempt was made to quantitate the
number of mast cell colonies prior to three weeks in
culture because previous experience with liquid culture-
derived mast cells indicated that early cultures were
heterogeneous in morphology and cell surface phenotype
(CHAPTER III). Reports of other cell lineages in similar
semisolid cultures of less than three weeks duration
confirmed our observations (Nakahata et al., 1982b; Pharr
et al., 1984). All observations were therefore made
between twenty-one and twenty-eight days of culture. Mast
cell colonies, which predominate in long-term cultures,
were identified by distinctive colony and cell morphology
(Figure IV-1A, -IB). Non-mast cell colonies, consisting


168
suspension. Cytocentrifuged samples of the preparations
showed somewhat lower numbers of rosetted cells, presumably
due to mechanical shearing and lysis of SRBC during the
processing. The latter data, however, are similar to those
generated by the S. aureus-antibody sandwich technique.
The data for the sorting of BALB/cAN bone marrow are
summarized in Table IV-4. Several significant
observations are evident. First, if bone marrow cells
(unfractionated) were incubated on ice but not subjected to
antibody treatment, rosetting, gradient separation, lysis
and numerous centrifugations in the time-consuming (eight
to twelve hour) procedure, the number of mast cell colonies
was reduced approximately forty percent compared to
previous, unsorted experiments. This decrement was
presumably due to the extended processing time in the
absence of growth factor, which was two to three times that
required to do the initial experiments. Second, the
manipulation of cells by incubation with antibody and sheep
red blood cells, separation on a density gradient medium,
incubation with ammonium chloride or hypotonic salts
solutions, and numerous centrifugations resulted in no
significant further depletion of mast cell precursors, as
evident from the similar number of colonies in sorted and
unsorted marrow populations. Finally, the method of lysis
of SRBC did not significantly alter the results of the
assay. This last observation is surprising in light of the


LIST OF FIGURES
Page
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
II-l Detection of Cell Surface Determinants
on Abelson Murine Leukemia Virus-
Transformed Embryonic Cells 81
II-2 Detection of Surface Receptors for IgE
on A-MuLV-Transformed Embryonic Cells 87
II-3 Virus-Transformed Cells Contain Abelson
Murine Leukemia Virus-Specific DNA
Sequences 92
II-4 Tumors Isolated from Mice Injected with
Cell Lines 10P12 and 11P0 Contain
A-MuLV-Specific DNA Sequences 94
III-l Progression of Hematopoietic Lineage
Markers in Long-Term Mast Cell Cultures
Derived from Embryonic Tissues 120
III-2 Metachromatic Granules of Long-Term,
Culture-Derived Embryonic Mast Cells 121
III-3 Progression of Hematopoietic Lineage
Markers In Long-Term Mast Cell Cultures
Derived from Adult Bone Marrow 124
III-4 Population Dynamics of Bone Marrow-
Derived Mast Cells Infected with
A-MuLV 126
III-5 Expression of Ly5 Antigen on A-MuLV-
Infected Mast Cells 128
III-6 Mixed Population of RA3-3Al-Positive
Lymphoid Cells and RA3-3A1-Negative
Cultured Mast Cells in Long-Term Bone
Marrow Cultures 130
III-7 Abelson Murine Leukemia Virus-Infected
Mast Cells Express v-abl Gene
Product 131
IV-1 Colonies in Long-Term Agar Cultures of
Embryonic Cells in Conditioned Media 156
IV-2 Frequency of Mast Cell Precursors in
Midgestation Embryonic Tissues 158
IV-3 Frequency of Placental Mast Cell
Precursors in the Third Trimester of
Gestation 160
Vll


13
and wild type bone marrow cells into the primary,
irradiated recipients and screening the colonies for cells
with only one type of granule), the authors were able to
conclude that the colony forming unit of the spleen (Till
and McCulloch, 1961) was the ultimate progenitor cell for
mast cells in the spleen, stomach, caecum, and skin as well
as for peripheral blood granulocytes and erythrocytes.
These results were corroborated by Sonada and colleagues
(1983), who demonstrated that late (twelve days post
injection) spleen colonies, which included both erythroid
and myeloid elements, contained mast ceil precursors which
differentiated in secondary recipient skin.
The ontogeny of embryonic mast cells in mice is
meagerly defined in the literature. As previously
mentioned, iji vivo mast cell precursors are present,
despite the absence of more mature forms, in the mouse
fetal Liver thirteen and more days post coitum (Kitamura
et ai., 1979c). Embryonic mast cell ontogeny was better
defined in the rat. Csaba and Kapa (1960) demonstrated the
presence of mast cells, which incorporated exogenous
heparin, in the thymus, spleen, lymph nodes, myocardium,
and kidney of day sixteen to seventeen rat embryos. By
studying sections through rat embryos between fifteen and
twenty-three days of gestation, Combs and colleagues (1965)
placed mast cells into four stages of differentiation based
upon their staining characteristics with alcian blue and


130
Figure III-6. Mixed population of RA3-3A1-Positive Lymphoid
Cells and RA3-3A1-Negative Cultured Mast
Cells in Long-Term Bone Marrow Cultures.
Bone marrow cells were cultivated for one week and infected
with A-MuLV (A) or sham infected (B) as described in
Materials and Methods. After three additional weeks of
culture, cells were probed with aureus-RA3-3A1 as
previously described.


Table III-l. Effect of Cocultivation of Culture-Derived Mast Cells
with Adherent Cells and Their Conditioned Media.
Mast Cell Treatment3 Percent Cells Expressing^
RA3-3A1
B23.1
IgE
Metachromat
Epitope
Epitope
Receptors
Granules
day 11 Placenta-derived mast
cellsc cocultured with:
50% W3CMd only
8
95
55
72
LPS-induced P388 cell underlayer
with 10P/LPS in agar
4
93
52
70
with 50% W3CM in agar
<1
99
51
66
Agar only underlayer
with 10P/LPS in agar
2
98
54
69
with 50% W3CM in agar
<1
92
49
68
No underlayer
+50% W3CM + 50% 10P/LPS
3
99
47
69
+50% W3CM + 50% P388CM
4
97
45
67
50% W3CM only
2
96
55
72
LPS-induced WEHI-3 underlayer
with 10P/LPS in agar
<1
94
ND
ND
with 50%W3CM in agar
3
90
46
73
Agar only underlayer
with 10P/LPS in agar
2
92
45
80
with 50% W3CM in agar
<1
89
42
71
No underlayer
+50% W3CM + 50% 10P/LPS
<1
94
47
72
+50% W3CM + 50% W3/LPSCM
2
96
48
74
50% W3CM
2
100
52
67
Adherent bm cell underlaver
with 50% W3CM
8
97
48
53
No underlayer
+50% W3CM + 50% bmadh CM
5
98
49
67
133


103
pre-B cells (Boss et al., 1979: Siden et al., 1979; Alt et
al. 1981) as well as interleukin 3-dependent mast cells
(Pierce et al., 1985; Chapter III of this work) and early
myeloid lineage cells (Rapp et al., 1985; Cook et al.,
1985). All of these cells are sensitive to the
proliferative effects of interleukin 3 (Iscove and Roitsch,
1985; Palacios et al., 1984). Our observations, however,
are that A-MuLV-infected mast cells do not synthesize
detectable interleukin 3 (Table II-6) nor do they contain
interleukin 3-specific messenger RNA (E. Siden, personal
communication); the former observation has recently been
corroborated by others studying Abelson virus-infected mast
cells (Pierce et al., 1985) and myeloid cells from more
mature stages of development (Cook et al., 1985).
We are presently uncertain of the apparent
nonautocrine mechanism of interleukin 3 independence in
Abelson virus-infected cells, although several
possibilities, including interaction of the v-abl gene
product with the interleukin 3 receptor (modifying the
activity of the receptor), substitution by the viral gene
product for an interleukin 3 receptor intracellular
function, or short-circuiting of an interleukin 3-induced
proliferation pathway (Pierce et al., 1985) are potentially
satisfying. Farrar and colleagues (1985) recently reported
that interleukin 3 stimulates the transient redistribution
of protein kinase C from the cytosol to the plasma membrane


11
cells exhibited evidence of thymic origin or dependence,
participated in immunologic reactions, were similarly
activated by lectins and immune stimuli, and were
relatively amitotic, speculated that the mast cells were
"post-mitotic" T lineage cells.
Other hematopoietic cell types have been postulated to
be mast cell by precursors in vivo and in vitro cultivation
techniques. Desaga and colleagues (1971) performed
repeated peritoneal lavages on rats to deplete them of
mature serosal mast cells. Mast cell-deficient
peritoneal exudate cells, cytochemically identified as
monocytes at the beginning of in vitro growth, developed
into metachromatic, granulated mast cells within two days
of harvest. Similarly, Czarnetzki and Behrendt (1981)
reported that peritoneal exudate cells from mast cell
depleted rats (injected with sterile water
intraperitoneally) resembled mononuclear phagocytes both
morphologically and cytochemically before and shortly after
culture in L-cell conditioned media. The in vitro-
propagated population, however, differentiated into mast
cells which were identified morphologically and which
contained granules with histamine and alpha-naphthol
acetate esterase.
The hematopoietic origin of some, and perhaps all,
mast cells was defined in great detail by Kitamura and
colleagues in a series of reports beginning less than ten


181
on the virus transformation-sensitive target cells which
may then differentiate into interleukin 3- responsive
culture-derived mast cell precursors.
The studies described in this chapter may also
contribute to the understanding of the immunobiology of the
maternal-fetal interface. The allogeneically foreign
conceptus first presents immunogenic antigens from the
cytotrophoblast to its mother between days nine and eleven
of gestation (Roe and Bell, 1982). The spectrum of
maternal-fetal immunological reactions have been
extensively reviewed in the literature (Bell and
Billington, 1983; Chaouat et al., 1983; Lala et al., 1983).
The mother, recognizing the fetal graft as nonself,
responds with both humoral and cell-mediated arms of the
immune system. The placenta, which has been described as
an immunological barrier and an immunoadsorbent (Wegmann et
al., 1979), also has been reported to play an active
immunoregulatory role (Chaouat et al., 1980; Remacle-Bonnet
et al., 1983; Chaouat and Kolb, 1985), although the
cellular source(s) of immunomodulatory factors is not
defined. Histamine, which is reported to play an important
role in blastocyst implantation (Dey et al., 1979; Dey and
Johnson, 1980a, 1980b; Nalbandov, 1971), has also been
shown to inhibit cytotoxic T lymphocyte effector functions
(Plaut et al., 1973; Schwartz et al., 1980; Chaouat and
Kolb, 1985), to inhibit the production of macrophage


CHAPTER II
ABELSON MURINE LEUKEMIA VIRUS-INFECTED CELLS FROM
MIDGESTATION PLACENTA EXHIBIT MAST CELL AND LYMPHOID
CHARACTERISTICS
Introduction
Abelson murine leukemia virus (A-MuLV) is a
replication-defective transforming retrovirus which was
isolated from a tumor in a steroid-treated BALB/c mouse
inoculated with the Moloney murine leukemia virus (Abelson
and Rabstein, 1970). Molecular analysis of the A-MuLV
genome has revealed that the virus arose by recombination
between the thymotropic Moloney virus genome and a cellular
gene termed c-abl (Goff et al., 1980; Shields et al.,
1979). The recombinant virus can infect and immortalize
hematopoietic cells jni vivo and ini vitro, and can transform
certain fibroblast cell lines in vitro (Scher and Siegler,
1975). The virus has demonstrated the ability to transform
in vivo mature cells of the B lineage (Potter et al., 1973;
Premkumar et al., 1975) as well as those of the T (Cook,
1982), myelomonocytic (Raschke et al., 1978; Ralph et al.,
1976), and mast cell (Risser et al., 1978; Mendoza and
Metzger, 1976) lineages. In contrast, in vitro infection
of adult or embryonic primary hematopoietic tissues
followed by clonal selection in semisolid media results in
the immortalization of cells exhibiting characteristics of
the earliest stages of B lymphoid differentiation
(Rosenberg et al., 1975; Rosenberg and Baltimore, 1976b;
61


200
Johnson, G.R., and D. Metcalf. 1977. Pure and mixed
erythroid colony formation in vitro stimulated by spleen
conditioned medium with no detectable erythropoietin.
Proc. Natl. Acad. Sci. USA 74: 3879.
Kahlson, G., and E. Rosengren. 1968. New approaches to the
physiology of histamine. Physiol. Rev. 48: 155-196.
Kasai, M., M. Iwamori, Y. Nagai, K. Okumura, and T. Tada.
1980. A glycolipid on the surface of mouse natural
killer cells. Eur. J. Immunol. 10: 175-180.
Kasai, M., T. Takashi, T. Takahashi, and T. Tokunaga. 1983.
A new differentiation antigen (FT-1) shared with fetal
thymocytes and leukemia cells in the mouse. J. Exp. Med.
159: 971-980.
Katz, H.R., P.A. LeBlanc, and S. Russell. 1983. Two classes
of mast cells delineated by monoclonal antibodies. Proc.
Natl. Acad. Sci. USA 80: 5916-5918.
Katz, H.R., R.L.. Stevens, and K.F. Austen. 1985a.
Heterogeneity of mammalian mast cells differentiated in
vivo and in vitro. J. Allergy Clin. Immunol. 76: 250-259.
Katz, H.R., G.A. Schwarting, P.A. LeBlanc, K.F. Austen, and
R.L. Stevens. 1985b. Identification of the neutral
glycosphingolipids of murine mast cells: Expression of
Forrsman glycolipid by the serosal but not the bone
marrow-derived subclass. J. Immunol. 134: 2617-2623.
Kay, H.D., G.D. Bonnard, W.H. West, and R.B. Herberman. 1977.
A functional comparison of human Fc-bearing lymphocytes
active in natural cytotoxicity and antibody-dependent
cellular cytotoxicity. J. Immunol. 118: 2058-2066.
Kennedy, T.G. 1977. Evidence for a role for prostaglandin
in the initiation of blastocyst implantation in the rat.
Biol Reprod. 16: 286-291.
Kiernan, J.A. 1972. The involvement of mast cells in
vasodilatation due to axon reflexes in injured skin.
Q. J. Exp. Physiol. 57: 311-317.
Kincade, P.W., G. Lee, C.J. Paige, and M.P. Scheid. 1981a.
Cellular interactions affecting the maturation of murine
B-lvmphocyte precursors in vitro. J. Immunol. 127:
255-260.
Kincade, P.W., G. Lee, T. Watanabe, L. Sun, and M.P. Scheid.
1981b. Antigens displayed on murine B lymphocyte
precursors. J. Immunol. 127: 2262-2268.


67
two hours at 37C, each reaction was stopped with 0.1 ml of
1 M sodium hydroxide and saturated with solid sodium
chloride. The contents of each tube were extracted with
0.2.5 ml chloroform in the presence of 10 microliters of
unlabeled SAMe (16 micrograms per ml)and the phases were
separated by brief centrifugation. The aqueous phase was
carefully removed with a pasteur pipet and the organic
phase was re-extracted with an equal volume of 1 M sodium
hydroxide and unlabeled SAMe. Aliquots (150 microliters)
of the organic phase were added to toluene-based
scintillation fluid (10 ml) and counted on a Beckman liquid
scintillation counter (Beckman Instruments, Palo Alto, CA).
All samples and standards were prepared in duplicate or
triplicate. Histamine content was determined by comparison
of tritiated counts in dilutions of tumor lysates to those
in histamine standard solutions and were expressed as
nanograms (ng) per 10^ cells.
Cytological Staining
Staining procedures were performed on air dried
cytocentrifuge (Shandon Scientific Company, Ltd., London,
U.K.) preparations on 25x75 mm precleaned glass slides.
Slides were fixed in methanol for one to two minutes prior
to staining with either Wright's Giemsa (Schalm et al.,
1975) or May-Gruenwald Giemsa (Thompson and Hunt, 1966).
Metachromatic cell granules were identified by staining for
five minutes with 0.1% w/v toluidine blue in 30% v/v


23
precursors in BALB/c mice bearing the myelomonocytic
leukemia WEHI-3, a constitutive producer of the mast ceil
growth factor interleukin 3, which is identical to the mast
cell growth factor produced by activated T lymphocytes
(Yung et al., 1981).
A variety of other distinguishing characteristics have
been ascribed to the cells alternately called mucosal mast
cells, atypical mast cells, and histaminocytes (Code,
1977). Early studies of mucosal mast cells were performed
on tissue sections or on heterogeneous populations of cells
isolated from the gut mucosa. The previously cited
methodologies of Enerback (1966a, 1966b) were later
optimized by the inclusion of techniques which further
stabilized the granules (neutral formalin fixation) and
enzymatically (.with trypsin) stripped stain-retarding
proteins from glutaraldehyde-treated preparations (Wingren
and Enerback, 1983). The recent development of methods for
isolating such cells from the small intestine, which
exploited the mucosal mastocytosis induced by parasitic
infection, was reported by Befus and colleagues (1982a),
and made it possible to analyze mucosal mast cells in the
absence of extraneous elements and to confirm some previous
observations. In contrast to peritoneal mast cells,
isolated mucosal mast cells are smaller and have a shorter
lifespan. Mucosal mast cells have fewer granules which
contain nonheparin, lower sulfated proteoglycans (as yet


86
marrow as well as on thymocytes, fetal liver red blood
cells, and pre-B cells (Shinefeld et al., 1980; E. Siden
and L. Shinefeld, unpublished observations).
Analysis of Receptors for IgE and IgG
High affinity membrane receptors for the Fc regions of
immunoglobulins were detected with a sensitive, isotype-
specific assay developed in this laboratory (Siden and
Siegel, 1986). Coated bacteria were prepared by
derivatizing the bacterial surfaces with the hapten
trinitrophenol (TNP) and then reacting the modified
bacteria with mouse monoclonal anti-TNP antibodies of the
IgE or IgG classes. Experimental and control cell lines
were incubated with the immunoglobulin-coated bacteria at
37C for one hour and processed for cytocentrifugation and
staining. Typical positive and negative reactions are
illustrated in Figure II-2. Observations are summarized in
Table II-4. IgE receptors were detected on all but two of
the embryonic cell lines analyzed, while none of the cell
lines expressed high affinity receptors for IgG. Receptors
for IgG were detected, however, on the myelomonocytic
leukemia cell line WEHI-3 (Table II-4) and on the
macrophage-like tumor cell line P388D1 (data not shown).
The results of the bacterial assay for IgG receptors were
duplicated by the method of Schrader (1981), which used
xenogeneic (rabbit) 7S antibodies immobilized on sheep red


149
course of one to one and one-half hours) to the stirring
liquid to a final concentration of 9g per 50 ml. The
slurry was poured into large polyethylene tubes and
centrifuged at room temperature in a J-21C centrifuge with
a JA-20 rotor (Beckman, Palo Alto, CA) for fifteen minutes
at 5000 RPM. The supernatant was discarded and the pellet
was dissolved in a small volume of phosphate-buffered
saline (PBS) and dialyzed extensively (two changes of 400
to 1000 volumes) against 0.1M sodium bicarbonate (Fisher
Scientific Co.) at 4C. The protein concentration of the
dialysate was estimated by absorbance at 280 nm in a SP8-
100 ultraviolet spectrophotometer (Pye Unicam, Ltd.,
Cambridge, UK) prior to further processing. N-
hydroxysuccinyl biotin (NHS-biotin, Sigma Chemical Co.) was
dissolved in dimethylsulfoxide (Sigma Chemical Co.) at 1 mg
per ml. The sodium bicarbonate dialysate and NHS-biotin
were mixed vigorously at 200 micrograms of derivatized
biotin per milligram of protein, then incubated for four
hours at room temperature on a rotator. The modified
antibody preparation was dialyzed extensively against PBS
at 4C and was filtered through a 0.2 micron disposable
nitrocellulose filter (Gelman Sciences, Inc., Ann Arbor,
MI) into a sterile polypropylene tube (Fisher Scientific
Co.) prior to storage at 4C.
Sheep red blood cells (SRBC, from blood freshly drawn
at the J. Hillis Miller Health Center Animal Resources


182
inhibitory factor (Rocklin, 1976) and to have other
immunomodulatory effects (Askenase et al., 1981) as well as
promoting tissue growth and repair (Kahlson and Rosengren,
1968). Prostaglandins and leukotrienes, related mast cell
products, may play similar immunomodulatory roles (Lala et
al., 1983). Although our studies of freshly dissociated
tissues have indicated that mast cells are, at most, a
minor fraction of the total conceptus between days ten and
twelve post coitum, a small but significant population of
cells at the maternal-fetal interface could indeed
contribute to the maintenance of the fetal graft.


66
Analysis of Histamine Content
Histamine content was determined by a modification of
the enzymatic-isotopic microassay of Taylor et al. (1980).
Histamine methyltransferase (HMT) reagent was prepared from
BALB/cAN mouse brain which was homogenized in iced 5mM
sodium phosphate buffer, pH7.9 (10 ml per gram of brain)
with an iced, sintered glass homogenizer. The crude
homogenate was transferred to an Oak Ridge type tube and
cleared centrifugally at 50,000xg for ten minutes at 4C.
Histamine standard solutions and tumor cell lysates were
prepared in PIPES-BSA which contained 25mM PIPES, pH 7.4
(Sigma Chemical Co., St. Louis, MO), 0.4 mM magnesium
chloride, 5 mM potassium chloride, 120 mM sodium chloride,
1 mM calcium chloride, 5.6 nm glucose (Fisher Scientific
Co., Fairlawn, NJ), 0.1 percent BSA (Sigma); standard
solutions and cells were boiled for ten minutes and cleared
for five minutes in a microfuge (Brinkmann Instruments,
Westbury, NY). Immediately before starting the reaction,
HMT-SAMe was formulated: 500 microliters of HMT reagent
was mixed with 10 microliters (5 microcuries) S-[methyl-
^H]-adenosylmethionine (76.1 Ci per mMol; New England
Nuclear, Boston, MA) and 5 microliters unlabeled S-
adenosylmethionine (SAMe, Sigma Chemical Co.; 16 micrograms
per ml). Each reaction mixture consisted of 10 microliters
of HMT-SAMe and 20 microliters of histamine standard
solution or tumor cell lysate. Following incubation for


1.24
Figure III-3. Progression of Hematopoietic Lineage Markers in
Long-Term Mast CeLl Cultures Derived From Adult
Rone Marrow
Cultures were maintained and analyzed as described in Materials and
Methods. Data shown are for BALB/c bone marrow-derived mast cell
cultures characterized for expression of determinants recognized by
monoclonal antibodies RA3-3A1 (A), B23.HB), receptors for IgE (E)
and IgG (G), and cells with metachromatic granules (M).


32
fixed fibroblasts, nor separation of fibroblasts from
"large lymphocyte" mast cell precursors by a membrane could
effect the change.
Despite the initial success in culturing mast cells
from lymphoid tissue cocultivated with adherent cell feeder
layers, reports in the literature of the technique's use
were limited to those of the previously cited groups. Two
factors probably contributed to the limited use of adherent
cell monolayers in mast cell culture. First, the system
was quite complex, requiring considerable time and
extensive subculture (or irradiation) of feeder layers to
eliminate the contribution of connective tissue type mast
cell precursors. Secondly, and perhaps more significantly,
the development of culture-derived mast cells in media
conditioned by activated lymphocytes by at least three
independent groups provided the opportunity to maintain
mast cells in the absence of a continuous monolayer of
feeder cells. As previously noted, however, even in the
absence of fibroblast feeder layers, islands of adherent
cells are observed in early cultures of lymphoid and
hematopoietic tissue-derived mast cells (confirmed in our
studies; see Chapters ill and IV). Since the adherent
cells are present in such cultures before the selection and
enrichment of mast cells, it is, at this juncture,
plausible to speculate that the adherent cells may assume a
transient, maturational role in mast cell differentiation.


212
Schrader, J.W., R. Scollay, and F. Battye. 1983b.
Intramucosal lymphocytes of the gut: Lyt 2 and Thy 1
phenotype of the granulated cells and evidence for the
presence of both T cells and mast cell precursors. J.
Immunol. 130: 558-564.
Schwartz, A., P.W. Askenase, and R.K. Gershon. 1980.
Histamine inhibition of the in vitro induction of
cytotoxic T-cell responses. Immunopharmacology 2:
179-190.
Sefton, B.M., T. Hunter, E.H. Ball, and S.J. Singer. 1981a.
Vinculin: A cytoskeletal target of the transforming
gene of Rous sarcoma virus. Cell 24: 165-174.
Sefton, B.M., T. Hunter, and W.C. Raschke. 1981b. Evidence
that the Abelson virus protein functions in vivo as a
protein kinase which phosphorylates tyrosine. Proc.
Natl. Acad. Sci. USA 78: 1552-1556.
Selye, H. 1965. The Mast Cells. Butterworth, Washington,
D.C.
Serunian, L.A., and N. Rosenberg. 1986. Abelson virus
potentiates long-term growth of mature B lymphocytes.
Mol. Cell. Biol. 6: 183-194.
Shanahan, F., J.A. Denburg, J. Bienenstock, and A.D. Befus.
1984. Mast cell heterogeneity. Can. J. Physiol.
Pharmacol. 62: 734-737.
Shelesnyak, M.C. 1960. Nidation of the fertilized ovum.
Endeavor 19: 81-86.
Shields, A., S. Goff, M. Paskind, G. Otto, and D. Baltimore.
1979. Structure of the Abelson murine leukemia virus
genome. Cell 18: 955-962.
Shinefeld, L.A., V.L. Sato, and N. E. Rosenberg. 1980.
Monoclonal rat anti-mouse brain antibody detects Abelson
murine leukemia virus target cells in mouse bone marrow.
Cell 20: 11-17.
Siden, E.J., D. Baltimore, D. Clark, and N.E. Rosenberg.
1979. Immunoglobulin synthesis by lymphoid cells
transformed in vitro by Abelson murine leukemia
virus. Cell 16: 389-396.
Siden, E.J. and M.L. Siegel. 1986. A simple method for the
simultaneous detection of Fc receptors and
differentiation markers on normal and neoplastic murine
hematopoietic cells. J. Immunol. Methods, in press.


194
Daeron, M., A.R. Sterk, F. Hirata, and T. Ishizaka. 1982.
Biochemical analysis of glucocorticoid-induced
inhibition of IgE-mediated histamine release from mouse
mast cells. J. Immunol. 129: 1212-1218.
Davidson, S., A. Mansour, R. Gallily, M. Smolarski, M.
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differentiation depends on T cells and granule synthesis
on fibroblasts. Immunology 48: 439-452.
Denburg, J.A., A.D. Befus, and J. Bienenstock. 1980a.
Growth and differentiation in vitro of mast cells
from mesenteric lymph nodes of Nippostrongylus
brasiliensis-infected rats. Immunology 41: 195-202.
Denburg, J.A., M. Davison, and J. Bienenstock. 1980b.
Basophil production. J. Clin. Invest. 65: 390-399.
Denburg, J.A., M. Richardson, S. Telizyn, and J. Bienenstock.
1983. Basophil/mast cell precursors in human peripheral
blood. Blood 61: 775-780.
Dennert, G., R. Hyman, J. Lesley, and I.S. Trowbridge. 1980.
Effects of cytotoxic monoclonal antibody specific for
T200 glycoprotein on functional lymphoid cell
populations. Cell. Immunol. 53: 350-364.
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mastzellvorstufen bei der ratte. Z. Zellforsch 121:
292-300.
Dey, S.K. 1981. Role of histamine in implantation:
Inhibition of histidine decarboxylase induces delayed
implantation in the rabbit. Biol. Reprod. 24: 867-869.
Dey, S.K., and D.C. Johnson. 1980a. Histamine formation by
mouse preimplantation embryos. J. Reprod. Fert. 60:
457-460.
Dey, S.K., and D.C. Johnson. 1980b. Reevaluation of
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Selective growth of natural cytotoxic but not natural
killer effector cells in interleukin 3. Nature 306:
788-791.


58
populated both mucosal and connective tissue-serosal
compartments. Nakano and colleagues (1985) injected
culture-derived mast cells and partially purified (30 to 40
percent) peritoneal mast cells into W/Wv mice by various
routes. At high levels of inoculum (10^ to 10^ cells),
intravenously- or intraperitoneally-injected cultured mast
cells populated the spleen and stomach (mucosa and muscle),
while at lower levels of inoculum (10- to lO^cells), only
intraperitoneallv injected cells were able to populate the
same anatomical sites. Both cultured and peritoneal mast
cells were relatively inefficient at populating the skin,
however, possibly due to the presence of mast cells, or
their precursors, which were already present in the skin
(Kitamura et al., 1977). An interesting note to the Nakano
studies was the evolution of mucosal or serosal mast cell
characteristics from injected cells (regardless of origin)
depending on the anatomical site of subsequent lodging.
Thus, the granules of mast cells isolated from the
peritoneal cavity, spleen, skin, and gastric muscularis
propria of reconstituted animals stained preferentially
with safranin (which has an affinity for highly sulfated
mucopolysaccharides like heparin) and the fluorescent dve
berberine sulfate (which also binds to heparin), and were
ultrastructurally homogeneous in size and electron density,
while mast cells identified in the glandular stomach mucosa
stained preferentially with alcian blue and were unstained


186
cytochemical analysis performed at the microscopic level,
with surface determinant characterization performed on
mature culture-derived mast cells. In contrast, our
investigations documented the sequential selection and
enrichment of cells expressing both membrane receptors for
IgE and the culture-derived mast cell determinant
recognized by the monoclonal antibody B23.1 (Katz et al.,
1983), as well as metachromatic granules, from a
heterogeneous population of cells relatively impoverished
of the markers. We feel that our observations complement
those of Ginsburg (1963) and later investigators and add a
further level of analytical sophistication to their
historical contributions.
Two features of the Abelson virus-transformed mast
cell-like lines, interleukin 3 independence and expression
of the B lineage variant of the Ly 5 differentiation
marker, were notably absent from cultured mast cells
derived from the same tissues. Culture-derived mast cells
were infected with Abelson virus to investigate the role of
that agent in the induction of those characteristics. The
virus-infected cells exhibited factor-independent growth,
but were still devoid of the lymphoid marker. Observation
of nonadherent cells expressing the same lymphoid
determinant in cultures of unselected (adherent and
nonadherent) uninfected cells grown in the presence of
interleukin 3 prompted us to investigate the role of


170
remaining TNP-SRBC in the cultures of IgE-sorted bone
marrow cells; these cells apparently had no effect on the
proliferation of mast cell precursors in agar.
Sorting of bone marrow with B23.1-biotin and avidin-
modified SRBC resulted in the segregation of all of the
mast cell precursors to the marker-positive fraction.
Ninety to one hundred percent of the precursors were
recovered from the rosetted pellet when compared to
unsorted cells. Dilutions of sorted cells cultured in agar
showed linearity of inoculum versus colony number,
indicating that the assay was a reliable measure of
precursor frequency. If cells from each fraction were
combined in proportion to the number which partitioned with
the rosettes (pellets) or free cells (supernatants), the
frequency of colonies enumerated was the same as that in
unfractionated bone marrow. This last observation
indicated that the minute B23.1 marker-negative population
contributes neither stimulatory nor inhibitory factors
required for the efficient seeding of mast cell precursors.
When bone marrow cells were sorted with biotinylated-
RA3-3A1 antibody and avidin-modified SRBC, all of the mast
cell precursors were found in the marker-negative
(supernatant) fraction. As seen with the B23.1 sorting,
essentially all (ninety percent) of the mast cell
precursors were recovered after RA3-3A1 sorting, and the
plot of the cells seeded versus mast cell colonies was


35
coitem, several days before mast cells per se were
observable in the embryo (Kitamura et al., 1979c).
Interleukin 3 From Lymphocyte-Conditioned Media And Other
Sources
The analysis of culture-derived mast ceils and their
precursors evolved from studies of hematopoietic cell
growth factors produced by lymphocytes. Reports of mast
cell-supporting factors (which, for the sake of convention,
we will commonly call interleukin 3) in the supernatants of
mitogen-stimulated spienocytes began to surface at the
beginning of the present decade (Burgess et al., 1980;
Hasthorpe, 1980). Since that time, a number of other
investigators have utilized media conditioned by a variety
of means to support the differentiation and growth of
culture-derived mast cells. Such media have thus been
derived from spienocytes activated by concanavalin A
(Clark-Lewis and Schrader, 1981; Tertian et al., 1981; Yung
et al., 1981; Schrader et al., 1981; Nakahata et al.,
1982b; Yung and Moore, 1982; Sredni et al., 1983), by
pokeweed mitogen (Hasthorpe, 1980; Nakahata et al., 1982b,
Wedling et al., 1983, 1984; Pharr et al., 1984), by
phytohemagglutinin A (Ogawa et al., 1983), by bacterial
lipopolysaccharide (Nakahata et al., 1982b), and by mixed
lymphocyte reactions augmented by lectin (Razin et al.,
1981a, 1982a, 1982c). Conditioned media with analogous
activity have been elicited from concanavalin A-stimulated


178
was optimized and proven effective on previously
characterized mouse cell lines (Table IV-3). The time-
consuming technique in the loss of approximately forty
percent of the mast cell precursors, presumably due to the
extended processing time in the absence of interleukin 3
and not numerous manipulations. By combining the sorting
procedure with the cultivation of cells in semisolid agar
media, we were able to effectively deplete all of the mast
cell precursors with B23.1 antibody; in contrast, neither
IgE nor RA3-3A1 significantly depleted bone marrow of mast
cell precursors. These preliminary results, however, were
questionable in light of previous experience with the same
antibodies in the system used to detect surface markers on
liquid culture cell populations (see Chapter III). Under
the latter conditions, fresh bone marrow contained twenty
two percent RA3-3A1 reactive cells and six percent B23.1
positive cells. In the sorting procedure, bone marrow
contained three to twenty-four percent RA3-3A1 positive
cells and ninety-six to ninety-nine percent B23.1 positive
cells. The reason for this discrepancy is unknown, since
all experimental controls gave predicted values. Despite
the intertechnique inconsistencies, we are confident that
few or no mast cell precursors express receptors for IgE or
the determinant recognized by monoclonal antibody RA3-3A1,
while it would appear that most, if not all, may express
the determinant recognized by B23.1. Further studies, with


215
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627 .


ACKNOWLEDGEMENTS
This dissertation and ray accomplishments over the past
several years are the result of a team effort. I wish to
acknowledge the contributions of all of my "teammates," and
hope that I slight none by inadvertent oversight.
First, I would like to thank my friend, mentor, and
supervisory chairperson, Edward J. Siden. His profound
powers of perception and his ability to assimilate new
observations into an existing body of knowledge have set
ideals which I shall take with me. His friendship and
camaraderie will always be of value to me.
Second, I wish to acknowledge and thank my supervisory
committee, J. Bert Flanegan, Carlo Moscovici, Steve Russell,
and Roy Weiner, who collectively guided me through my
dissertation research, sometimes despite my reluctance and
protestations.
My family has been a constant source of support,
cheer, and inspiration. My wife Jeremie has coped with
every crisis I brought home and has survived more mood
fluctuations in the last few years than most people endure
in a lifetime. David and Rebecca have also endured the
journey with few complaints and good humor, and it is to
them I owe the preservation of my humor. I must also thank
my father, Seymour Siegel, who inspired me to strive to be
the best at whatever I tried (even if I was a garbage man),
11


184
In the course of characterizing the Abelson virus-
transformed embryonic cell lines, we developed a sensitive,
nonisotopic, nonfluorometric assay for membrane receptors
for cytophillic immunoglobulin. The method utilizes
hapten-specific monoclonal antibodies and homologous
hapten-derivatized bacteria which form rosettes with cells
bearing the appropriate receptors. The marker positive
cells are easily identified by light microscopy of
cytocentrifuged, fixed, and stained preparations. The
technique has the advantage over previously described
isotopic (Mendoza and Metzger, 1976) and nonisotopic
(Schrader, 1981) methods of allowing morphological
characterization of the reactive cells. This methodology
is applicable to the characterization of other
hematopoietic lineages and can be modified to identify two
specificities on the same cell (Siden and Siegel, 1986).
The identification of mast cell characteristics in
lines derived by the transformation of midgestation
enbryonic placenta by Abelson murine leukemia virus led us
to analyze homologous, untransformed embryonic tissues for
mast cells and their precursors. Similar to the
observations of Kitamura and colleagues (1979c) on mouse
fetal liver as early as day thirteen post coitum, we
detected no mast cells in embryonic tissues at days ten and
eleven of gestation. We were, however, able to culture
mast cells from those tissues, thus providing the novel


36
mesenteric lymph node cells of parasitized animals
(McMenamin et al., 1985). Phytohemagglutinin A- or
concanavalin A-stimulated human blood lymphocytes also
produced activities which supported the growth of human
basophilic cells and a growth factor-dependent mouse cell
line (Tadokoro et al., 1983; Stadler et al., 1985).
The phenotype of the murine lymphoid cell which
produces interleukin 3 was deduced from the activities of
conditioned media of a number of related T cell clones. In
contrast to Lyt 1+2+, Lyt 1-2+, and Lyt 1-2- cells, which
did not support culture-derived mast cell growth, the
supernatants of Lyt 1+2- T cell clones, corresponding to
the inducer T lymphocyte subset, supported the
proliferation of such cells (Nabel et al., 1981). The
observations of Nabel and colleagues were subsequently
confirmed by other investigators (Fung et al., 1984; Yokota
et al., 1984), and by the observation that rat mesenteric
lymph node cells expressing differentiation markers of
helper T lymphocytes (0X19+, W3/25+, 0X8-) were responsible
for the production of a factor with analogous activity to
mouse interleukin 3 (McMenamin et al., 1985).
A number of permanent cell lines also produce factors
which support the growth of culture-derived mast cells.
The best known of these cell lines is the myelomonocytic
WEHI-3 line, which constitutively produces high levels of
interleukin 3 (Nagao et al., 1981; Schrader et al., 1981;


93
from the input cells would have the same sites of provirus
integration, while in vivo virus infection from cells
shedding virus would probably result in the observation of
different sites, due to the random nature of A-MuLV
integration. Both scenarios were observed (Figure II-4).
Cells recovered from lymph nodes of mice which were
injected with 11P0-1, a virus-producing cell line (E.
Siden, personal communication), had viral sequences
integrated into cellular restriction fragments distinct
from the input DNA pattern. The cell line 10P12, however,
which sheds no detectable virus (E. Siden, personal
communication), was reisolated from the liver and had A-
MuLV-specific sequences integrated into cellular
restriction fragments similar in size to the input pattern.
Analysis of Interleukin 3 Production by Embryonic Cell
Lines
The phenotypic similarity of the A-MuLV-transformed
embryonic cell lines and culture-derived mast cells was
sharply contrasted by the growth factor requirements of the
respective populations. The former cells required no
growth factors beyond those provided by medium 10P
(containing fetal bovine serum), while cultured mast cells
required an exogenous source of interleukin 3,
conventionally provided in WEHI-3-conditioned media. We
therefore sought to determine whether the cell lines
produced their own growth factor. Using a sensitive


117
clusters used in the adherent bone marrow and P388D1
experiments; alternatively, 0.5 ml of suspension was
pipeted into each well of the twelve-well cluster used in
the WEHI-3 experiments. Mast cells were cocultured with
adherent monolayers prepared as previously described and
with control underlayers of 0.3 percent agar in appropriate
media but without adherent cells. All suspensions were
made 50% W3CM by addition of an equal volume of EM.
The effects of adherent cell-derived factors were
investigated by making the mast cell suspensions 50% W3CM
by addition of an equal volume of bmadhCM, P388/LPSCM or
W3/LPSCM. Control cultures of mast cells were similarly
made by addition of equal volumes of EM or 10P/LPS as
appropriate. Cultures were fed weekly for three weeks by
addition of an equal volume of homologous media and
analyzed for surface markers as previously described.
Results
Progression of Lineage Markers in Mast Cell Cultures in
Liquid Media
Previous studies have dealt with the expression of
surface and cytochemical markers in the cells alternatively
called cultured mast cells and P cells, but only after the
populations had become homogeneous by successive selection


119
Figure III-l. Progression of Hematopoietic Lineage Markers in Long-Term
Mast Cell Cultures Derived From Embryonic Tissues
Cultures were maintained and analyzed as described in Materials
and Methods. Cultures were derived from nonplacental embryonic
tissue (NPET, panels A, C, E) and from placenta (P, panels B, D, F)
isolated from (BALB/c x B10.BR)F1 (panels A, B, C, D) and (BALB/c
x CBA)F1 (panels E,F) concepti at 10(panels A,B) and ll(panels C,
D,E,F) days post coitum. Percent cells expressing determinants
recognized by monoclonal antibodies RA3-3A1 (A) and B23.1(B),
receptors for IgE(E) and IgG(G), and cells with metachromatic
granules (M) are indicated.


192
Clark-Lewis, I., R.M. Crapper, K. Leslie, S. Schrader, and
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A. Schimpl (eds.). Cellular and Molecular Biology of
Lymphokines. Academic Press, Orlando, FL.
Clark-Lewis, I., S.B.H. Kent, and J.W. Schrader. 1984.
Purification to apparent homogeneity of a factor
stimulating the growth of multiple lineages of
hemopoietic cells. J. Biol. Chem. 259: 7488-7494.
Clark-Lewis, I.. and J.W. Schrader. 1981. P cell-
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new T cell-derived factor. J. Immunol. 127: 1941-1947.
Clark-Lewis, I., J.W. Schrader, Y.-Y. Wu, and A.W. Harris.
1982. A B lymphoma cell line produces growth factors
for hemopoietic, lymphoid, and mast cells. Cell
Immuno1. 69: 196-200.
Cobb, C.M., H. Birkedal-Hansen, and F.R. Denys. 1975.
Ultrastructural characteristics of mast cells from a
canine mastocytoma maintained in vitro. J. Oral
Pathol. 4: 244-256.
Code, C.F. 1977. Reflections on histamine gastric
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Coffman, R.L., and I.L. Weissman. 1981a. A monoclonal
antibody that recognizes B cells and B cell precursors
in mice. J. Exp. Med. 153: 269-279.
Coffman, R.L., and I.L. Weissman. 1981b. B220: A B cell-
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Differentiation and proliferation of embryonic mast
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independent proliferation in vitro and tumorigenicity
in vivo are associated in basophil/mast-cell lines and
their somatic hybrids. Differentiation 28:291-295.
Cook, W.D. 1982. Rapid thymomas induced by Abelson murine
leukemia virus. Proc. Natl. Acad. Sci. USA 79: 2917-
2921.


218
Zucker-Franklin, D. 1980. Ultrastructural evidence for the
common origin of human mast cells and basophils. Blood
56: 534-540.
Zucker-Franklin, D., G. Grusky, N. Hirayama, and E. Schnipper.
1981. The presence of mast cell precursors in rat
peripheral blood. Blood 58: 544-551.


17 b
cell precursors increases between days eight and twelve.
In the most dramatic case studied, this augmentation
represented a twenty- to thirty-fold increase in mast cell
colonies for (BALB/cAN x CBA/J)F1 embryos (reaching levels
similar to those detected in adult bone marrow), while
smaller increases were observed for (BALB/cAN x B10.BR)F1
and homozygous BALB/cAN embryos. The discrepancies between
heterozygous and homozygous embryonic mast cell precursor
frequencies were not observed in adult bone marrow, leading
us to speculate that allogeneic differences may be
responsible for mast cell precursor proliferation at this
critical period of fetal development.
A third significant observation of this study is that
the number of mast cell precursors in the murine embryonic
placenta drops rapidly from its peak at twelve days to the
lowest levels noted in that tissue during the course of the
third trimester of pregnancy. Our observation that day
eleven embryonic tissue contained fewer mast cell
precursors than those of day thirteen were supported by
similar reports in the literature (Ginsburg et al., 1982).
Although precursor frequency in the embryo proper was not
investigated during the same period of time, we have noted
that Kitamura et al. (1979c) reported the isolation of mast
cell precursors in the fetal liver of day thirteen and day
fourteen mouse embryos; the former observation was
substantiated by Nabel et al. (1981).


190
Baltimore, D., N. Rosenberg, and O.N. Witte. 1979.
Transformation of immature lymphoid cells by Abelson
murine leukemia virus. Immunol. Rev. 48: 3-22.
Bazill, G.W., M. Haynes, J. Garland, and T.M. Dexter. 1983.
Characterization and partial purification of a
haemopoietic cell growth factor in WEHI-3 cell
conditioned medium. Biochem. J. 210: 747-759.
Befus, A.D., and J. Bienenstock. 1979. Immunologically
mediated intestinal mastocytosis in Nippostrongylus
brasiliens is-infected rats. Immunology 38: 95-101.
Befus, A.D., F.L. Pearce, J. Gauldie, P. Horsewood, and J.
Bienenstock. 1982a. Mucosal mast cells. I. Isolation
and functional characteristics of rat intestinal mast
cells. J. Immunol. 128: 2475-2480.
Befus, A.D., F.L. Pearce, G. Goodacre, and J. Bienenstock.
1982b. Unique functional characteristics of mucosal
mast cells. Adv. Exp. Med. Biol. 149: 521-527.
Bell, S.C., and W.D. Billington. 1983. Anti-fetal allo-
antibody in the pregnant female. Immunol. Rev. 75:
5-30.
Benditt, E.P., S. Bader, M. Arase, C. Corley, and K.B. Lam.
1954. Relationship of mast cells and histamine to
mechanism of edema production by ovomucoid in rats.
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Bienenstock, J., A.D. Befus, F. Pearce, J. Denburg, and R.
Goodacre. 1982. Mast cell heterogeneity: Derivation
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lymphocytes, natural killer cells, and mast cells. Ann.
N.Y. Acad. Sci. 409: 164-170.


81
Figure II-l. Detection of Cell Surface Determinants on Abelson
Murine Leukemia Virus-Transformed Embryonic Cells.
Killed Staphylococcus aureus bacteria coated with anti-mast
cell/monocyte B23.1 antibodies (A) and normal rat serum
antibodies (B) were reacted with cells from line 10P12 as
indicated in Materials and Methods.


29
be considered models of their in vivo homologues until
definitive evidence permits us to conclude that the two
populations are completely identical. We will therefore
continue to use the term "culture-derived" mast cells, and
similarly distinctive terms, to maintain the tenor of this
caveat throughout the following discussion.
Adherent Feeder Layer Studies
The earliest reports of culture-derived mast cells by
Ginsburg (Ginsburg,1963; Ginsburg and Sachs, 1963) involved
a complex system of mouse thymocytes cultured on feeder
layers of mouse embryonic fibroblasts. It was apparent to
the authors that the monolayer was essential for
proliferation of mast cells. Culture of thymocytes in the
absence of the feeder layer failed to produce mast cells.
On the other hand, culture of embryonic skin fibroblasts
from eighteen day fetuses, without additional thymocytes,
infrequently resulted in mast cell outgrowth. The tissue
source of the mast cells, therefore, was disputable, and
quite possibly both thymocytes and embryonic feeder layers
contributed progenitors to the mast cell culture. The
issue was better defined several years later when the same
group reported that irradiated embryonic fibroblast
monolayers, which could no longer produce mast cells when


202
Lagunoff, D., and Y.C. Chi. 1980. Cell biology of mast cells
and basophils. In G. Weissman (ed.), Cell Biology of
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and V. Colavincenzo. 1983. Immunobiology of the fetal-
maternal interface. Immunol. Rev. 75: 87-116.
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activity of natural cytotoxic cells is augmented by
interleukin 2 and interleukin 3. J. Exp. Med. 157:
1070-1075.
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mononuclear phagocyte antigenic heterogeneity detected
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219-231.
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homeostasis and inflammation by leukotrienes and other
mast cell-dependent compounds. Nature 293: 103-108.
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224: 271-274.
Maximow, A. 1906. Uber die zellformen des lockeren
Bindegewebes. Arch. f. Mikr. Anat. 67: 680-757.
Mayrhofer, G. 1979a. The nature of the thymus dependency of
mucosal mast cells. I. An adaptive secondary response
to challenge with Nippostrongylus brasiliensis.
Cell. Immunol. 47: 304-311.


21
were further reinforced by the same investigator (Crowle
and Reed, 1984). Reconstitution of athymic mice was
ablated by pretreatment of wild type mouse bone marrow
cells or splenocytes with anti-Thy 1 and complement, while
similar treatment of beige bone marrow or spleen cells
still resulted in the detection of some mast cells, albeit
fewer, in the mucosa of thymus-intact W/Wv mice.
The thymic dependence of mucosal mast cells, in
contrast to the thymus-independent growth and development
of serosal mast cells, has been documented bv a number of
other investigators. Prior to the reports of Crowle
(1982,1984), Ruitenberg and Elgersma (1976) observed that
nude mice infected with Trichinella spiralis experienced no
intestinal mucosal mast cell response unless reconstituted
by thymus or parasite-immune thoracic duct cell grafts,
concluding that thymus-derived T-lineage cells were required
for mucosal mastocytosis. These results were substantiated
by a number of other researchers (Olson and Levy, 1976;
Mayrhofer and Bazin, 1981; Reed et al., 1982). Similar
studies were performed in the rat. Mayrhofer (1979a) noted
that the number of mucosal mast cells in Nippostrongylus
brasiliens is-infected rats increased in a pattern similar to
primary and secondary immune responses. Adult thymectomy or
chronic thoracic duct drainage several months prior to
nematode challenge (to deplete mature T cells) resulted in
significantly depressed intestinal mastocytosis, while


8 9 10 II 12 8 9 10 II 12 8 9 10 II 12
DAYS OF GESTATION
Figure IV-2. Frequency ot Mast Cell Precursors in Midgestation Embryonic Tissues
Bars represent weighted means ot 2 to 11 samples; error bars represent weighted
standard deviations. Tissues analyzed: TC: cotal conceptus; P: placenta;
E: nonplacental embryonic tissues.
158


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87
B
Figure. II-2. Detection of Surface Receptors for IgE on A-
MuLV-Transformed Embryonic Cells.
Killed Staphylococcus aureus bacteria haptenated with
trinitrophenol (TNP) were incubated with cells from line
10P8 which had previously been incubated with monoclonal
mouse IgE (A) and IgG (B) as described in Materials and Methods.


193
Cook, W.D., D. Metcalf, N.A. Nicola, A.W. Burgess, and F.
Walker. 1985. Malignant transformation of a growth
factor-dependent myeloid cell line by Abelson virus
without evidence of an autocrine mechanism. Cell 41:
677-683.
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of retroviruses induce phosphorylation of tyrosine
present in similar cellular proteins. Mol. Cell. Biol.
1: 394-407.
Crapper, R.M., and J.W. Schrader. 1983. Frequency of mast
cell precursors in normal tissues determined by an in
vitro assay: Antigen induces parallel increases in the
frequency of P cell precursors and mast cells. J.
Immunol. 131: 923-928.
Crowle, P.K. 1982. Bone marrow origin of mucosal mast
cells. Fed. Proc. 41: 581.
Crowle, P.K., and N.D. Reed. 1984. Bone marrow origin of
mucosal mast cells. Int. Archs. Allergy Appl. Immunol.
73: 242-247.
Csaba, G., and E. Kapa. 1960. Uptake of heparin by cells.
Nature 187: 711-713.
Czarnetzki, B.M., and H. Behrendt. 1981. Studies on the
in vitro development of rat peritoneal mast cells.
Immunobiology 159: 256-268.
Czarnetzki, B.M., C.G. Figdor, G. Kolde, T. Vroom, R.
Aalberse and J.E. de Vries. 1984. Development of human
connective tissue mast cells from purified blood
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vitro studies on the development of rat peritoneal mast
cells. Immunobiology 156: 470-476.
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vitro generation of mast cell-like cells from human
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1982. Evidence that the tissue mast cells derive from
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Immuno1. 67: 44-48.


77
The proliferation of cell line DA-1 (generously provided by
J.N. Ihle), which requires interleukin 3 for growth, served
as an assay for interleukin 3. DA-1 cells were
centrifuged, washed twice in PBS, and resuspended at 5x10^
per ml in appropriate condititioned media or cell lysates.
One-tenth milliliter aliquots were pipeted in triplicate
into separate compartments of a 96-well microtiter cluster
(Linbro, Flow Laboratories, McLean, VA). The cells were
incubated (37C, five percent carbon dioxide) for sixteen
hours, at which time each well was pulsed with one
microcurie of ^H-TdR (5 Ci per mMol, Amersham Corporation,
Arlington Heights, IL) in ten microliters of EM. Following
six hours additional incubation, the cells were collected
on Whatman glass microfiber filter strips (Whatman Paper
Ltd., Maidstone, U.K.) in water by multiple automated
sample harvester (MASH, Otto Hiller Company, Madison, WI).
The filter strips were air dried. ^H-TdR which was
incorporated into filter-immobilized DNA was counted in
toluene-based scintillation fluid in a liquid scintillation
spectrometer.
Results
Histamine Content of Transformed Placental Cells
Initial light microscopiic examination of some of the
transformed cell lines revealed large mononuclear cells


125
We were therefore interested in determining whether A-MuLV
infection of culture-derived mast cells could induce the
expression of the lymphoid B-220 antigen.
Initial experiments were designed to infect bone
marrow-derived mast cells throughout the course of their
maturation and to analyze the surface marker distribution
of the population. Cells infected one, two, and three
weeks after initial isolation and culture were considered
"transitional" in that the target population was
heterogeneous with respect to the surface and cytological
markers assayed; similarly, cells four weeks or later in
the culture period were relatively homogeneous (Figure III-
3) .
Following A-MuLV infection, mast cell cultures
experienced a decrease in population size attributed to
virus-related killing of some infected cells (Figure III-4).
The effects were more dramatic in virus-infected cultures
which were grown in the absence of growth factor and were
attributed to the inability of factor-dependent cells to
proliferate in EM alone, as demonstrated by the short half-
life of uninfected mast cells which were grown in parallel
cultures without W3CM (Figure III-4). The period of
attrition peaked within the first four days of culture
post-infection and was followed by an equally dramatic
period of proliferation of surviving cells; parallel


25
degranulation inhibitory effects of Doxantrazole (Befus et
al., 1982b; Pearce et al., 1982, 1985).
Basophils
Cells with membrane receptors for IgE are not limited
to the mast cell lineage. Both lymphocytes (Gonzales-
Molina and Spiegelberg, 1978) and macrophages (Melewicz et
al. 1982) have been reported to bind IgE; the affinity of
the membrane receptors of the non-mast cells, however, was
ten to one-hundred times Less than that of mast cells
(Ogawa et al., 1983). The best known of the mast-like
cells, however, are the basophils (basophilic
granulocytes), polymorphonuclear leukocytes which are
present in the blood of several mammalian species (Lagunoff
and Chi, 1980). Like mast cells, basophils contain
metachromatic granules and express surface membrane
receptors for IgE. Histamine is released by antigenic
challenge of IgE-bearing basophils (Lagunoff and Chi,
1980). The granules of basophils of several species have
been reported to contain chondroitin sulfate proteoglycan
(Olsson et al., 1970; Orenstein et al., 1978; Metcalfe et
al., 1980b) similar in its degree of sulfation to the
proteoglycan of mucosal mast cells (Tas and Berndsen,
1977). Basophilic granulocytes, however, are apparently
absent from mouse peripheral blood (Lagunoff and Chi,
1980). The relationship of these cells to culture-derived


88
Table II-4. Analysis of Surface Membrane Receptors for IgE
and IgG on A-MuLV-Transformed Embryonic
Cell Lines and on Control Tumor Cell Lines
CELL LINE MEMBRANE RECEPTORS FOR
Mouse IgEa Mouse IgGa Rabbit IgG*3
PLACENTAL
9P1 +
10P2 +
1P6
10P8 +
10P12 +
11P0-1
11P62-4 +
CONTROL
WEHI-3 + +
RL 11 - NDC
18-81
FLEI-4 - ND
P815 - ND
CB6ABMC4 ND
BALABMC20 + ND
a: Membrane receptors for allogeneic (mouse) IgE and IgG were
detected by the TNP-bacteria/anti-TNP method (see Materials
and Methods).
b: Membrane receptors for xenogeneic (rabbit) IgG were detected
by the rabbit anti-sheep RBC/SRBC method of Schrader (1981).
c: ND indicates analysis not performed.


79
Table II-2. Histamine Content of Embryonic
and Control Tumor Cell Lines
Tumor Cell Lines
CELL LINES
HISTAMINE
(ng/lO^ cells)3
PLACENTAL CELL LINES
9P1
>5.00
10P2
2.70
10P6
<0.05
10P8
0.05
10P12
0.06
11P0-1
<0.05
11P62
1.60
CONTROL CELL LINES
WEHI-3
<0.05
RLdl 1
<0.05
18-81
<0.05
FLEI-4
<0.05
P815
<0.05
a: Histamine content was determined as described in
Materials and Methods.


160
DAYS OF GESTATION
Figure 1V-3. Frequency of Placental Mast Cell Precursors in the
Third Trimester of Gestation.
Mast cells were cultured from (BALB/cAN x CBA/J)F1 placenta as
indicated in Materials and Methods. Bars represent weighted
means (Bahm,1972) of 5 to 13 samples; error bars represent
standard deviations.


developed a simple, sensitive, nonfluorometric, nonisotopic
assay to detect membrane receptors for immunoglobulins.
The observation of the mast-like, Abelson virus-
transformed cell lines led us to investigate the presence
of mast cell precursors in normal midgestation embryonic
tissues. We found embryonic precursors to mast cells in
homologous, noninfected tissues and conducted a detailed,
systematic analysis of the differentiation of mast cells in
liquid cultures over the course of several weeks of
selection and enrichment. We also studied the effects of
Abelson virus infection and adherent cell cytokines on
lymphoid differentiation antigens in mast cell cultures.
Mast cell precursors in embryonic tissues of mid- and
late gestation were quantitated by a clonal assay. We
described the embryologically earliest reported mast cell
precursors in the mouse and report that the mouse embryo is
a rich reservoir of such precursors, containing
proportionately at least as many such cells as adult bone
marrow. We have observed that mast cells which
differentiate in agar culture, like some of the Abelson
virus-transformed cell lines, express the B220 determinant.
We have also described preliminary experiments in which we
selected mast cell precursors in bone marrow on the basis
of surface membrane determinants.
ix


204
Metcalf, D., and M.A.S. Moore. 1971. Embryonic aspects of
haemopoiesis. In D. Metcalf and M.A.S. Moore (eds.),
Haemopoietic Cells. North-Holland Publishing Company,
Amsterdam.
Metcalfe, D.D., M. Kaliner, and M.A. Donlon. 1981. The
mast cell. CRC Crit. Rev. Immunol. 3: 23-74.
Metcalfe, D.D., R.A. Lewis, J.E. Silbert, R.D. Rosenberg,
S.I. Wasserman, and K.F. Austen. 1979. J. Clin.
Invest. 64: 1537-1543.
Metcalfe, D.D., S.I. Wasserman, and K.F. Austen. 1980b.
Isolation and characterization of sulfated
mucopolysaccharides from rat leukaemic (RBL-1)
basophils. Biochem. J. 185: 367-372.
Michell, B. 1984. Oncogenic intelligence: Oncogenes and
inositol lipids. Nature 308: 770.
Michels, W.A. 1963. The mast cells. Ann. N.Y. Acad. Sci.
103: 235-372.
Mishell, B.B., and S.M. Shiigi (eds.). 1980. Preparation and
testing of reagents. In Selected Methods in Cellular
Immunology. W.H. Freeman and Co., San Francisco, CA.
Moore, M.A.S., and D. Metcalf. 1970. Ontogeny of the
haematopoietic system: Yolk sac origin of jji vivo
and in vitro colony forming cells in the developing
mouse embryo. Brit. J. Haematol. 18: 279-296.
Mori, Y., H. Akedo, K. Tanaka, Y. Tanigaki, and M. Okada.
1979. Effect of sodium butyrate on the granulopoiesis
of mastocytoma cells. Exp. Cell Res. 118: 15-22.
Morse, H.C. Ill, W.F. Davidson, R.A. Yetter, E.D. Murphy,
J.B. Roths, and R.L. Coffman. 1982. Abnormalities
induced by the mutant gene lpr: Expansion of a unique
lymphocyte subset. Proc. Natl. Acad. Sci. USA 129:
2612-2615.
Muller, R., D.J. Slamon, J.M. Tremblay, M.J. Cline, and I.M.
Verma. 1982. Differential expression of cellular
oncogenes during pre- and postnatal development of the
mouse. Nature 299: 640-644.
Muramatsu, T., H. Muramatsu, M. Kasai, S. Habu, and K.
Okumura. 1980. Receptors for Dolichus bifloris
agglutinin: New cell surface markers on a spontaneous
leukemia. Biochem. Biophys. Res. Commun. 96: 1547-1553.


5
virus was similarly capable of abrogating the interleukin
3-dependence of both myeloid (Greenberger et al., 1979;
Cook et al., 1985) and mast (Pierce et ai., 1985; Chapter
III, this dissertation) lineages. Taken with the
observation of erythropoietin-independent evthroid cells
(Waneck and Rosenberg, 1981) and interleukin 3-independent
mast cells (Pierce et al., 1985; Siegel et al., 1985) from
A-MuLV-infected primary cell populations, the data suggest
that A-MuLV may alter the requirements of these cells for
growth factors while permitting the cells to differentiate.
Although the precise role of the v-abl oncogene in
maintaining factor-independent proliferation is not yet
known, it is interesting to note that the cellular
homologue, c-abl, is transcribed at its highest level in
the developing embryo at the same time that the number of
A-MuLV targets reaches its peak. At least two retroviral
oncogene products have recently been shown to have
analogous structures in normal cells. The epidermal growth
factor receptor exhibits striking similarity to the erb B
oncogene product of avian erythroblastosis virus (Downward
et al., 1984), while the v-sis oncogene of simian sarcoma
virus encodes a protein structurally and immunologically
related to platelet-derived growth factor (Doolittle et
al., 1983; Robbins et al., 1983; Waterfield et al., 1983).
Although the function of c-abl has not been established, it
is known that all of the hematopoietic lineages sensitive


109
The spleens were transferred to fresh medium and residual
fat and membranes were removed. The trimmed spleens were
minced with sterile scissors onto nylon screens and
dissociated through the screens by massaging the tissue
with the rubber end of a plunger of a disposable 3cc
syringe. Disaggregated cells were washed through the
screen into the underlying centrifuge tube with ten to
twenty milliliters of EM.
Embryonic tissues
Embryonic tissues were isolated at the indicated days
of gestation after vaginal plugs were observed on day 0.
Pregnant mice were killed by cervical dislocation and the
gravid uteri were surgically removed and placed in a petri
dish containing EM. After two to five minutes (to allow
the tissues to bleed into the media) each uterus was
transferred to a second dish of EM and the residual
mesentery and fat were trimmed off. The uteri were
transferred to third dish of EM and concepti were dissected
away from the maternal tissues and placed in a fresh dish
of EM. Concepti of ten or more days of age were teased
into placental and nonplacental embryonic tissue (NPET)
components. The ectoplacental cones were dissected free of
attached membranes and placed in a fresh dish of EM.
Concepti of eight and nine days were processed without
further dissection. All tissues were dissociated through
nylon screens as previously described.


18
of neuropeptides and endorphins, thymus dependence, and
life span. These characteristics have been surveyed in
detail in reviews previously cited. We will therefore only
briefly survey the literature which is cogent to the
ultimate topic of this discussion, the in vitro, culture-
derived mast cell.
Connective Tissue Mast Cells
Although typical mast cells have been isolated from a
variety of connective tissue sources throughout the rodent
body, the most frequently studied member of this subset is
that which is isolated, free of extraneous tissues, from
the serosal surfaces of the peritoneal cavity. As
previously discussed, by injecting bone marrow into mast
cell-deficient hosts, Kitamura and colleagues (1977) were
able to demonstrate the relationship of hematopoietic
precursors to the serosal mast cells. The ultimate
precursor cell in the bone marrow was shown to be the
colony-forming unit of the spleen (Kitamura et al., .1981;
Sonoda et al., 1983). From the bone marrow, mast cell
precursors migrate through the blood (Kitamura et al.,
1979a; Zucker-Franklin et al., 1981; Sonoda et al., 1983)
and subsequently proliferate and differentiate in
connective tissue (Hatanaka et al., 1979; Kitamura et al.,
1979b,1979d). Connective tissue mast cells in rodents have
been found to proliferate and differentiate independently of
thymic influences. Thus, the athymic nude mouse has mast


155
Figure IV-1. Colonies in Long-Term Agar Cultures of Embryonic
Cells in Conditioned Media.
Colonies of (BALB/c x CBA)F1 placental cells (day 12)
were photographed at constant magnification after four
weeks of growth in 0.3% agar with 50% W3CM.
(A) Small to medium mast cell colony;
(B) large mast cell colony;
(C) adherent, non-mast cells;
(D) diffuse non-mast cells;
(E) small granulocyte colony;
(F) mast cells were picked from the colony in A and
stained with toluidine blue to visualize metachromatic
granules.


173
a clonal agar assay (Schrader et al., 1981). We modified
the assay, however, to be consistent with our previous
liquid culture experiments (Chapter III), by substituting
WEHI-3 condidtioned media (Razin et al., 1984a) for the Con
A- spleen-conditioned media of those authors. We verified
the reliability of our assay by quantitation of bone marrow
and spleen-derived mast cell precursors and found that our
data were consistent with those previously published.
The results of our analyses of mast cell precursors in
midgestation and late gestation embryonic tissues present
several significant observations. First, we have described
the earliest mast cell precursors reported in the mouse,
being first detected at eight days post coitum. Previous
to this report, the earliest reported mast cell precursors
were derived from disaggregated day ten or eleven embryos
which had been cultured in the presence of T cell-derived
growth factors (Ginsburg et al, 1982). In the mouse,
embryonic mast cell precursors have also been identified in
the fetal liver as early as day thirteen post coitum
(Kitamura et al., 1979c; Nabel et al., 1981). Similar
observations have been reported in the embryonic rat
(Ishizaka et al., 1976) and in the embryonic human (Razin
et al., 1981b).
A second significant observation concerns the
frequency of embryonic mast cell precursors as gestation
progresses. As seen in Figure IV-2, the number of mast


138
(Tertian et al., 1981) that most of the cells in long-term
cultures of mast cells have IgE receptors. This
discrepancy may be the result of differences in receptor
assay technique or may result from other experimental
variances such as cell cycling which could temporally
affect the expression of membrane receptors. We have
observed on one A-MuLV-transformed embryonic cell line that
the rosetting assay used by Tertian and colleagues, detects
more cells with membrane receptors for IgE than the S.
aureus assay (data not shown).
The progression of surface markers on cultured
embryonic cells (Figure III-l) follows a pattern similar to
those observed for bone marrow-derived mast cells (Figure
III-3). Freshly disaggregated placenta contains a
population of cells which have receptors for normal rat IgG
and are recognized by monoclonal antibody B23.1, but few or
no such cells bind IgE or have metachromatic granules.
Based on previous reports (Katz et al., 1983), these cells
are probably related to the monocyte-mononuclear phagocyte
lineage and may be the cells responsible for the binding
and degradation of anti-paternal antibodies (Raghupathy et
al., 1984). Fewer cells expressing the B23.1 epitope are
found in the nonplacental embryonic tissues of (BALB/c x
B10.BR)F1 concepti. The development of metachromatic
granules and IgE receptors in cultures of cells derived
from embryonic tissues was delayed (perhaps because the


167
Table
IV-3. Sorting of
Control Cells
by Rosetting
Percent Rosetted Cells
Cell
Rosetting Phase of Separation Crystal
Cyto-
Line
Agents Medium Analyzed
Violet
Centrifuge
Method3
Method^
10P8
IgE/TNP-SRBC
supernatant
7
ND
pellet
96
82
PBS/TNP-SRBC
supernatant
0
ND
pellet
0
0
11P62
B23.1-Biotin
supernatant
11
ND
/Avidin-SRBC
pellet
98
0
PBS/Avidin-
supernatant
0
ND
SRBC
pellet
0
0
18-81
RA3-3A1-
supernatant
8
ND
Biotin/
Avidin-SRBC
pellet
100
95
PBS/Avidin-
supernatant
0
ND
SRBC
pellet
0
0
a. Following sorting of cells, an aliquot was made 0.2%
crystal violet and the number of rosetted and
unrosetted cells per 10-t ml hemocytometer field
were counted on an inverted phase microscope.
b. Following sorting of cells, an aliquot was cyto-
centrifuged and stained with Wright's Giemsa.
Coverslips were affixed with Permount and at least
one hundred cells were microscopically analyzed for
association with three or more sheep red blood cells.


17
mucosal mast cell granules. Mucosal mast cells also
required higher concentrations of thiazine dyes, like
toluidine blue, and azure A dyes, as well as prolonged
staining times, when compared to serosal mast cells, while
the granules of the former cells had higher affinity to
copper phthalocyanine dyes such as Astra blue at ph 0.3.
Enerback therefore concluded that the mucosal mast and
connective tissue mast cells differed not only
morphologically, but biochemically as well, and offered
that the mucosal mast cells contained less highly sulfated
mucopolysaccharides than the dermal cells. Finally,
Enerback noted that in rats systemicallv exposed to the
histamine releasing agent 48/80, serosal mast cells in the
mesentery, tongue, and skin were degranuiated and therefore
undetectable, while mast cells in the duodenal mucosa were
unaffected and perhaps greater in number. While being
unable to explain the latter observation, Enerback was able
to conclude that mucosal mast cells differed from their
serosal counterparts functionally as well.
The differences between mucosal and serosal mast
cells, first noted by Maximow and then Enerback, have
since been appended by the observations of numerous
investigators and extend beyond those mentioned to surface
markers, histamine content, IgE receptors and
internalization of bound IgE, proteoglycan composition,
proteases, sensitivity to histamine secretagogues, effects


72
partially purified mouse IgG anti-TNP (generous gift of M.
Rittenberg, Oregon Health Center University) were cleared
by centrifugation at 12,000xg for 15 minutes at 4C. The
latter was diluted in Dulbecco's modified Eagle's minimal
essential medium (GIBCO) with 10% v/v heat-inactivated
fetal bovine serum, and both contained 0.1% w/v sodium
azide. Cells to be analyzed were suspended in H10BNF1 at
1x10^ per ml, aliquoted at 0.1 ml per well of a 96-well PVC
cluster (Dynatech, Inc., Alexandria, VA) and pelleted by
centrifugation (2 minutes, lOOxg, 4C). The cluster was
"flicked" and briefly vortexed (five to ten momentary
touches) and the cells were resuspended in 0.1 ml of
cleared IgE or IgG anti-TNP. Clusters were covered with
plastic wrap and placed in a 37C, 5% carbon dioxide
incubator for one hour. Following incubation, the treated
cells were washed twice with H10BNF1 by centrifugation.
The cell pellets were dispersed by vortexing and 5
microliters of 10% w/v TNP-S_^ aureus or TNP-E. col i were
added to each well. The covered microtiter clusters were
placed on ice for thirty minutes and the contents of the
wells were washed six times as previously described. The
pellets were resuspended in 0.1 ml of H10BNF1. Ten to
thirty microliter samples of each well were
cytocentrifuged, stained with May-Gruenwald Giemsa and
observed by bright field microscopy.


172
Discussion
The development of mast cells from embryonic tissues
in liquid cultures led us to quantitate the number of mast
cell precursors in those tissues. The frequency of such
precursors has historically been analyzed by several
methods. Nakahata et al. (1982b) developed a culture assay
for mouse mast cell colonies in methylcellulose. The assay
was adapted from previous work (Parmley et al., 1976) which
enumerated multipotent hematopoietic progenitors, but
selected for mast cell growth with lectin-stimulated
conditioned media. Subsequent use of the technique by
others in the same laboratory (Pharr et al., 1984; Suda et
al., 1985) has established the reliabilty of this method.
Concurrent to the development of the methlcellulose assay,
Schrader et al. (1981) and Zucker-Franklin et al. (1981)
reported the development of a similar clonal assay using
agar-based semisolid media with results similar to those
obtained with methylcellulose. In addition to the
semisolid medium techniques, limiting dilution analysis in
liquid culture has been used to quantitate the cultured
mast cell precursor frequency in a variety of tissues
(Crapper and Schrader, 1983; Guy-Grand et al, 1984).
Based on experience, supplies, and the similar results
obtained in independent studies (above), we chose to
analyze the frequency of mast cells in embryonic tissues by


>
Figure II-4. Tumors Isolated from Mice Injected with Cell Lines
10P12 and 11P0 Contain A-MuLV-Specific DNA Sequences.
DNA was isolated from embryonic cell lines and tumor tissue,
restricted with endonuclease Bam HI, blotted onto nitrocellulose,
and probed with nick-translated pAB3Sub3 as described in
Materials and Methods. Autoradiograph of probed blots is shown.
Lanes contain DNA from input cell line 11P0 (A), cultured lymph
node tumor cells from animals which were injected with 11P0 (B,C),
input cell line 10P12 (D), cultured liver tumor cells from
animals which were injected with 10P12 (E,F). Arrows mark
c-abl-containing fragments.


208
Rapp, U.R., J.L. Cleveland, K. Brightman, A. Scott, and J.N.
Ihle. 1985. Abrogation of IL-3 and IL-2 dependence by
recombinant murine retroviruses expressing v-myc
oncogenes. Nature 317: 434-438.
Raschke, W.C., S. Baird, P. Ralph, and I. Nakoinz. 1978.
Functional macrophage cell lines transformed by Abelson
leukemia virus. Cell 15: 261-267.
Razin, E., C. Cordon-Cardo, and R. A. Good. 1981a. Growth
of a pure population of mouse mast cells in vitro
with conditioned medium derived from concanavalin A-
stimulated splenocytes. Proc. Natl. Acad Sci. USA 78:
2559-2561.
Razin, E., C. Cordon-Cardo, C.R. Minick, and R.A. Good.
1982a. Studies of the exocytosis of cultured mast cells
derived from mouse bone marrow. Exp. Hematol. 10: 524-
532.
Razin, E., J.N. Ihle, D. Seldin, J.-M. Mencia-Huerta, H.R.
Katz, P.A. LeBlanc, A. Hein, J.P. Caulfield, K. F.
Austen, and R. L. Stevens. 1984a. Interleukin 3: A
differentiation and growth factor for the mouse mast
cell that contains chondroitin sulfate E proteoglycan.
J. Immunol. 132: 1479-1486.
Razin, E., J.M. Mencia-Huerta, R.A. Lewis, E.J. Corey, and
K.F. Austen. 1982b. Generation of leukotriene C4 from
a subclass of mast cells differentiated in vitro
from mouse bone marrow. Proc. Natl. Acad. Sci. USA 79:
4665-4667.
Razin, E., J.-M. Mencia-Huerta, R.L. Stevens, R.A. Lewis,
F.-T. Liu, E.J. Corey, and K.F. Austen. 1983. IgE-
mediated release of leukotriene C4, chondroitin sulfate
E proteoglycan, beta-hexosaminidase, and histamine from
cultured bone marrow-derived mouse mast cells. J. Exp.
Med. 157: 189-201.
Razin, E., A.B.Rifkind, C. Cordon-Cardo, and R.A. Good.
1981b. Selective growth of a population of human
basophils ini vitro. Proc. Natl. Acad. Sci. USA 78:
5793-5796.
Razin, E., R.L. Stevens, F. Akiyama, K. Schmid, and K.F.
Austen. 1982c. Culture from mouse bone marrow of a
subclass of mast cells possessing a distinct chondroitin
sulfate proteoglycan with glycosaminoglycans rich in N-
acetylgalactosamine-4,6-disulfate. J. Biol. Chem. 257:
7229-7236.


65
c. YAC-1, a Moloney leukemia virus-induced
lymphoma obtained from R. Weiner (University
of Florida);
3. Monocyte-macrophage
a. WEHI-3, a myelomonocytic leukemia obtained
from M. Norcross (University of Florida);
b. P388D1, a methylcholanthrene-induced monocytic
tumor obtained from American Type Culture
Collection (Rockville, MD);
4. Basophil-mast cell
a. P815, a methylcholanthrene-induced mastocytoma
obtained from S. Noga (University of Florida);
b. CB6ABMC4, an A-MuLV-induced mastocytoma
obtained from M. Potter (National Cancer
Institute, Bethesda, MD);
c. BALABMC20, an A-MuLV-induced mastocytoma
obtained from M. Potter.
All of the above cell lines were maintained in 10P with
twice-weekly passage.
Cell line DA-1, which was used in the analysis of
interleukin 3 production, was generously supplied by J.N.
Ihle (National Cancer Institute, Frederick, MD) and was
maintained in a one-to-one mixture of WEHI-3 conditioned
media and enriched media (EM; Razin et al., 1984a).


97
Table II-6. Extended
Stimulation
¡ Cell Lysatese
Stimulation
Index
1
1
1
Index
1.384
WEHI-3 (5.2xl05 C.E.)
0.496
0.061
(2.6xl05 C.E.)
0.321
0.588
(1.3xl05 C.E.)
0.174
1.252
¡ (0.6xl05 C.E.)
0.082
1.242
1
1
0.007
1
1
1
0.007
10P12-2 (7.3xl05 C.E.)
0.005
0.007
(3.6xl05 C.E.)
0.005
0.013
(1.8xl05 C.E.)
0.004
0.008
! (0.9x10s C.E.)
0.004
0.006
1
1
0.011
1
1
0.009
1
1
1
0.007
10P12-2 (7.3xl05 C.E.):
0.009
Standard 1 (1:1)
1
0.764
a. Interleukin 3 content was assayed by a proliferation assay
as described in Materials and Methods.
b. Homogeneous interleukin 3 standard was generously supplied by
Dr. J.N. Ihle as a concentrate in RPMI 1640 and used to his
specifications. Standard 1 was formulated by fifty-fold
dilution of the concentrate. Standards 2 through 9 were
made by serial two-fold dilutions from standard 1. All
dilutions were made in EM, which also served as the blank.
c. Stimulation Index was calculated from raw counts retained on
filters by the formula: Stimulation Index= Mean Sample cpm/
Mean Standard 1 cpm, where mean Standard 1 cpm was 46236
(standard deviation, 16129). Stimulation indices for standards
(expressed as means +/- 1 standard deviation) were compiled from
the results of five experiments of three replicates each, except
for Standard 7 which was compiled from three experiments of three
replicates. All other stimulation indices were compiled from a
minimum of three determinations.
d. Conditioned media were prepared as indicated in Materials and
Methods. Abbreviations: CM (conditioned media), SAS (saturated
ammonium sulfate), Amicon ret (retenate of 10 Kd cutoff Amicon
stirred cell filter), Amicon fit (filtrate of 10 Kd cutoff Amicon
filter).
e. Cell lysates were prepared as indicated in Materials and Methods
from the number of cells noted parenthetically (C.E. is cell
equivalents).


CHAPTER IV
ISOLATION, ENUMERATION, AND CHARACTERIZATION OF IN VITRO
MAST CELL PRECURSORS DERIVED FROM MIDGESTATION EMBRYONIC
PLACENTA
Introduction
The understanding of the relationships between the
various components of the hematopoietic system has greatly
benefited from the development of clonal assay systems
during the last quarter century. From the seminal work of
Till and McCulloch (1961), the concept of a pluripotent
hematopoietic stem cell (CFU-S), capable of clonally
reconstituting the spleen and bone marrow of lethally
irradiated mice, led to the paradigm that all of the
cellular elements of blood were related by a single
progenitor. The rapid evolution of in vitro clonal culture
techniques has further contributed to our understanding of
normal and pathogenic hematopoiesis. Techniques are
presently available for quantitation of pluripotent
hematopoietic precursors from a variety of sources (Johnson
and Metcalf, 1977; Hara and Ogawa, 1978; Fauser and
Messner, 1979). In addition, methods for isolation,
differentiation, and enumeration of committed, multipotent
hematopoietic precursors have been described for a number
of lineages.
The in vitro quantitation of mast cell precursors in a
variety of tissues has thus been accomplished by limiting
dilution in liquid culture (Crapper and Schrader, 1983;
144


BIOGRAPHICAL SKETCH
Michael L. Siegel was born on June 15, 1949, in New
York City, the younger of two sons of Frances and Seymour
Siegel and the brother of Victor (who sometimes claimed to
be an only child). After a relatively uneventful childhood
and adolescence in Punta Gorda, Florida, Bronx, New York,
and Spring Valley, New York, Michael attended Cornell
University and managed to achieve a bachelor of science
degree in animal science while attending Woodstock,
protesting social injustice and the American way, and
locking in to age seventeen. Late in his senior year at
Cornell, Michael abandoned his lifelong goal of becoming a
veterinarian and decided instead to pursue an academic and
research career at the University of Florida. His new
pursuit was soon postponed, however, as financial problems
forced him to withdraw from classes.
Serendipidously, Union Carbide Corporation was looking
for someone with Michael's background (so he convinced
them), and thus began another era in his life. Michael
worked for Union Carbide for five years developing
radioimmunoassays, and then accepted an offer from Meloy
Laboratories to manage its immunoreagents production unit.
Living in Manassas, Virginia, Michael met his wife,
Jeremie, and her children, David and Rebecca. In June of
219


Ill
enrichment-selection period and thereafter. Transforming
virus stocks, prepared as previously described (Siegel et
al., 1985) by superinfection of Abelson P160 nonproducer
cell line 160N54, contained 1 to 2x10^ PFU of Moloney
murine leukemia virus (M-MuLV) and 0.5 to 1.0x10^ of A-MuLV
per ml and were stored at -70C prior to rapid thawing
immediately before use. Mast cells were pelleted at 200xg
at room temperature for ten minutes and resuspended in A-
MuLV virus stock with 4 micrograms Polybrene (Aldrich
Chemical Co., Milwaukee, WI) per ml at 0.5 to 4x10^ cells
per ml. Cells were incubated with virus for two and one-
half hours at 37C (Rosenberg and Baltimore, 1976a) in
capped 12x75 mm polypropylene culture tubes (Fisher
Scientific Co.) with gentle, end-over-end rotation.
Following the adsorption period, the suspension was diluted
to a final cell concentration of 1x10^ per ml with EM or
W3CM (final concentration of 50% W3CM, plus virus stock and
EM) and cultured in a humidified atmosphere of five percent
carbon dioxide in air. Cells in culture were counted at
two to five day intervals and fed weekly by centrifuging
the cells and resuspending them at 1 to 2x10^ per ml in 50%
W3CM or EM, as appropriate. Disaggregated cells from fetal
livers dissected from day 18 embryos were infected with A-
MuLV and cultured as above.


90
Table II-5. Metachromatic Granules in A-MuLV-Transformed
Embryonic Cell Lines and Control Tumor Cell Lines.
Metachromatic
Cell Line Granules3
placental
9P1 +
10P2
10P6
10P8
10P12
11P0-1
11P62 +
10PC1 +
11PC14 +
11PC19
11PC20
11PC32 +
12PC1 +
control
WEHI-3
18-81
P815
CB6ABMC4
BALABMC20 +
a: Cytocentrifuged smears of cells were stained with
toluidine blue as indicated in Materials and Methods.
Cells with metachromatic granules (+) and without
metachromatic granules (-) were scored.


101
activity (M. Jadus, personal communication). The
expression of B lineage and T lineage markers, as seen on
11P0-1, may be characteristic of one stage of mast cell
differentiation. Alternatively, the expression of B, T,
monocyte, and cultured mast cell markers on the Abelson
virus-transformed embryonic cell lines may be analogous to
that of a yet undefined in vivo multipotent proliferative
stem cell which is present in the midgestation placenta.
The present studies have not further pursued this matter.
We have characterized cell lines, generated by
transformation of embryonic cells by Abelson murine
leukemia virus, which phenotypically resemble culture-
derived mast cells. However, analysis of nontransformed
cells from freshly disaggregated embryonic tissues
indicated that few, if any, mast cells are present in the
midgestational conceptus (Chapter III). Infection may
therefore have transformed the cells prior to stem cell
commitment to the mast cell lineage or before committed
mast cell precursors can be identified. Undifferentiated,
multipotent hematopoietic stem cells, which are responsible
for colonization of other fetal tissues, have been
described in the mouse embryonic yolk sac blood islands at
this stage of development (Moore and Metcalf, 1970).
Abelson murine leukemia virus infection of primary
hematopoietic cells followed by culture in semisolid media
has previously been shown to generate permanent cell lines


64
mercaptoethanol (Sigma Chemical Co., St. Louis, MO)) and 10
percent heat-inactivated fetal bovine serum (Sterile
Systems, Logan, UT)) for at least two years with twice
weekly passages prior to analysis. The nomenclature used
to designate each cell line included the number of days of
gestation (detection of vaginal plug on day 0) followed by
a suffix ("P" for placenta of (BALB/cAN x BIO.BR/SgSn)Fl
origin, "PC" for placenta of (BALB/cAN x CBA/J) origin, and
a clone number. Cell line 10P12, for example, was the
twelfth clone isolated from 10 day placental cells derived
from matings of BALB/cAN females and B10.BR males.
Mouse tumor cell lines used as experimental controls in
the studies were chosen to represent the major
hematopoietic lineages:
1. B 1ymphoid
a. FLEI-4, an pre-B cell line derived by E.J.
Siden by infection of day 15 BALB/c fetal
liver with A-MuLV strain P120;
b. 18-81 (Siden et al., 1979), a pre-B cell line
induced by infection of bone marrow cells with
A-MuLV strain P120;
2. T lymphoid
a. RLd 11, a radiation-induced leukemia
obtained from N. Rosenberg;
b. B2-4-4, a Moloney virus-induced leukemia
obtained from N. Rosenberg;


75
lambda DNA were generally cut away and discarded. The gel
was then exposed to shortwave ultraviolet light for an
additional ten minutes to break the DNA and thereby
facilitate transfer. DNA in the gel was denatured with 0.5
M sodium hydroxide, 0.6 M sodium chloride for one hour,
neutralized in 1 M Tris, pH 7.4, 1.5 M sodium chloride (two
changes of 150 to 200 milliliters for 30 to 45 minutes
each), and transferred to nitrocellulose (Schleicher and
Scheull, Keene, NH) by the method of Southern (1975).
Probes for A-MuLV-related sequences were prepared from
the virus-specific recombinant plasmid pAB3Sub3 (Goff et
al., 1980) by nick translation (Rigby et al., 1977) to a
specific activity of 10 dpm per microgram. The 2p_
labeled sequences were hybridized to the nitrocellulose-
immobilized DNA for twenty hours at 68C by the method of
Wahl (1979). The nitrocellulose blot was then washed
extensively under stringent conditions (0.015 M sodium
chloride, 0.0015 M sodium citrate at 68C). The probed
blots were autoradiographed on XAR-5 film (Eastman-Kodak,
Rochester, NY) with two calcium-tungstate-phosphor
intensifying screens (Cronex Lightning Plus, E.I.DuPont de
Nemours and Co., Wilmington, DE) for two to five days.
Conditioned Media
Embryonic tumor cells were cultured under conditions
similar to those used to generate WEHI-3 conditioned media
(W3CM) (Razin et al., 1984a). Cells from log phase


92
ABODE
r
Figure II-3. Virus-Transformed Cells Contain Abelson Murine
Leukemia Virus-Specific DNA Sequences.
DNA isolated from embryonic cell lines was restricted,
electrophoresed, blotted, and probed with v-abl recombinant
plasmid pAB3Sub3, and the filter was autoradiographed as
described in Materials and Methods. Lanes contain DNA from
control cell line 160N54 (A), 10P8 (B), 10P12 (C), 11P0-1
(D), 11P62 (E). Arrows mark c-abl-containing fragments.


112
Immunoprecipitation of Viral Proteins
Viral proteins in A-MuLV-infected cells were
identified by immunoprecipitation as previously described
(Siden et al., 1979). Virus-infected culture-derived mast
cells and control cells were pelleted at 200xg for ten
minutes at room temperature and washed once with balanced
salts solution (BSS). The cells (1.5 to 2x10^) were
resuspended at 2x10^ per ml in labeling media consisting of
RPMI 1640 media without methionine (Flow Laboratories,
Rockville, MD), 2 mM glutamine, lx RPMI vitamins (GIBCO),
100 units penicillin and 100 micrograms streptomycin per ml
(GIBCO), 10 mM HEPES (Sigma Chemical Company), pH 7.35,
0.05 mM 2-mercaptoethanol (Sigma Chemical Company), and 78
microcuries of 35s_met]1onne (H26 Ci per mMol, New England
Nuclear, Boston, MA). Cells were incubated in labeling
media for two hours at 37C with gentle rocking. The
labeled cells were centrifuged for five minutes at 400xg,
washed once with BSS and lysed in 1 ml of phosphate lysis
buffer (PLB, 10 mM sodium phosphate (Fisher Scientific
Co.), pH 7.5, 100 mM sodium chloride (Fisher Scientific
Co.), 1 percent v/v Triton X-100 (Fisher Scientific Co.),
0.5 percent w/v sodium deoxycholate (Fisher Scientific
Co.), and 0.1 percent w/v sodium dodecyl sulfate (SDS, BDH
Chemicals, Ltd., Poole, U.K.)) which was supplemented with
0.098 percent w/v bovine serum albumin (Sigma Chemical
Co.), 1 mM EDTA (Fisher Scientific Co.), 1 mM TAME (Sigma


leukemia cells, peritoneal mast cells, or chondrocytes.
Chondroitin sulfate proteoglycan E was shown in this study
to be chemically distinct from heparin by a number of
criteria including sensitivity to enzymatic degradation and
molecular weight (chondroitin sulfate proteoglycan E has an
estimated molecular weight of 200 kilodaltons, in contrast
to heparin, which has a molecular weight of 750
kilodaltons). These results have been confirmed in the
literature (Razin et al., 1983; Sredni et al., 1983).
Mouse culture-derived mast cell granules also contain a
number of other in vivo mast cell-associated biological
mediators, including serotonin (5-hydroxytryptamine) and
dopamine (Tertian et al., 1981).
Arachidonic acid metabolites, the prostaglandins and
leukotrienes, are important biological mediators
asssociated with metachromatic cells. IgE-dependent
activation of mouse culture-derived mast cells results in
the synthesis and release of leukotriene C4 (Razin et al.,
1982b, 1983), a component of the slow releasing substance
of anaphylaxis (Austin, 1984). These studies also showed
that bone marrow-derived cultured mast cells generated
approximately twenty-five times more leukotriene C4 than
prostaglandin D2 upon activation by calcium ionophore
A23187 or IgE receptor-mediated pathways. In contrast, rat


128
CO
LlI
o
L
>
H
CO
O
Q.
WEEKS IN CULTURE PRIOR TO INFECTION
Figure III-5. Expression of Lv5 Antigen on A-MuLV-Infected
Mast Cells
Bone marrow cells were cultured under conditions favorable for
selection and enrichment of mast cells and infected with A-MuLV
at the times noted above. Uninfected cells were assayed at the
time of infection (unshaded bars) and parallel cultures of
infected (shaded bars) and sham-infected (stippled bars) were
assayed twenty-one days post infection for the presence of Lv5
with monoclonal antibody RA3-3A1 as described in Materials
and Methods.


191
Boss, M., M. Greaves, and N. Teich. 1979. Abelson virus-
transformed haematopoietic cell lines with pre-B-cell
characteristics. Nature 278: 551-553.
Brandon, J.M., and M.C. Bibby. 1979. A study of changes
in uterine mast cells during early pregnancy in the rat.
Biol. Reprod. 20: 977-980.
Burgess, A.W., D. Metcalf, S.H.M. Russell, and N.A. Nicola.
1980. Granulocyte/macrophage-, megakaryocyte-,
eosinophil-, and erythroid-colony stimulating factors
produced by mouse spleen cells. Biochem J. 185: 301-314.
Burnet, F.M. 1965. Mast cells in the thymus of NZB mice.
J. Path. Bact. 89: 271-284.
Burnet, F.M. 1975. Possible identification of mast cells
as specialised post-mitotic cells. Med. Hypoth. 1: 3-5.
Burnet, F.M. 1977. The probable relationship of some or
all mast cells to the T-cell system. Cell Immunol. 30:
358-360.
Burton, A.L. 1963. Studies on living normal mast cells.
Ann. N.Y. Acad. Sci. 103: 245-263.
Capron, M., J. Rousseaux, C. Mazingue, H. Bazin, and A.
Capron. 1978. Rat mast cell-eosinophil interaction in
antibody-dependent eosinophil cytotoxicity to
Schistosoma mansoni schistosomula. J. Immunol. 121:
2518-2525.
Caulfield, J.P., R.A. Lewis, A. Hein, and K.F. Austen. 1980.
Secretion in dissociated human pulmonary mast cells.
J. Cell. Biol. 85: 299-311.
Chaouat, G., S. Chaffaux, M. Duchet-Suchaux, and G.A. Voisin.
1980. Immunoactive products of the mouse placenta. I.
Immunosuppressive effects of crude and water-soluble
extracts. J. Reprod. Immunol. 2: 127-139.
Chaouat, G., and J.-P. Kolb. 1985. Immunoactive products
of the placenta. IV. Impairment by placental cells
and their products of CTL function at effector stage.
J. Immunol. 135: 215-222.
Chaouat, G., J.P. Kolb, and T.G. Wegmann. 1983. The murine
placenta as an immunological barrier between the mother
and the fetus. Immunol. Rev. 75: 31-60.


100
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
0
120
C.dll(BALB/c x B 10. BR)F I NPET
B B B B
D.dIKBALB/c x B 10. BR)FI P
E. d II (BALB/c x CBA) FI NPET
F.d INBALB/c x CBA) F I P
1234567 1234567
TIME IN CULTURE (WEEKS)


45
Characteristics of Culture-Derived Mast Cells
The development of refined analytical methods, first
applied to other hematopoietic cells, aided in the
characterization of culture-derived mast cells. Yung and
colleagues (1983) analyzed bone marrow cells by
centrifugation techniques and reported that interleukin 3-
responsive cells could be isolated by nature of their
median buoyant density (1.033) from interleukin 2-
responsive ceils (1.075). The authors also noted that the
buoyant densities of long-term bone marrow-derived cultured
mast cells (1.062 to 1.095 g/ml) were similar to those
determined by Pretlow and Cassidy (1970), who analyzed
heterogeneous populations of freshly isolated peritoneal
mast cells and reported that immature mast cells have a
median buoyant density of 1.087. Interleukin 3-
responsive cells were separated by sedimentation velocity
analysis from the in vitro precursors of macrophages (CFU-
M) and the pluripotent colony-forming cell of the spleen
(CFU-S), but not the bipotent precursor of granulocytes and
macrophages (CFU-GM).
Culture-derived mast cells and their precursors have
been characterized for the expression of a broad variety of
hematopoietic differentiation markers in attempts to assign
them to a particular lineage. Bone marrow and intestinal
precursors to culture-derived mast cells were observed to
lack the T lineage antigens Thy 1, Lyt 1 and Lyt 2


214
Steffen, D., S. Bird, W.P.Rowe, and R.A. Weinberg. 1979.
Identification of DNA fragments carrying ecotropic
proviruses of AKR mice. Proc. Natl. Acad. Sci. USA 76:
4554-4558.
Sterry, W., and B.M. Czarnetzki. 1982. In vitro
differentiation of rat peritoneal macrophages into mast
cells: An enzymecytochemical study. Blut 44: 211-220.
Stevens, R.L., and K.F. Austen. 1981. Proteoglycans of
the mast cell. In E.L. Becker, A.S. Simon, and K.F.
Austen (eds.). Biochemistry of the Acute Allergic
Reaction: Fourth International Symposium (Kroc
Foundation Series, Vol. 14). Alan R. Liss, New York.
Stevens, R.L., and K.F. Austen. 1982. Effect of para-
nitrophenyl-beta-D-xyloside on proteoglycan and
glycosaminoglycan biosynthesis in rat serosal mast cell
cultures. J. Biol Chem. 257: 253-259.
Suda, T., J. Suda, and M. Ogawa. 1984. Disparate
differentiation in mouse hemopoietic colonies derived
from paired progenitors. Proc. Natl. Acad. Sci. USA 81:
2520-2524.
Suda, T., J. Suda, S.S. Spicer, and M. Ogawa. 1985.
Proliferation and differentiation in culture of mast
cell progenitors derived from mast cell deficient mice
of the genotype W/Wv. J. Cell. Physiol. 122: 187-192.
Tadokoro, K., B.M. Stadler, and A.L. de Week. 1983. Factor
dependent in vitro growth of human normal bone marrow-
derived basophil like cells. J. Exp. Med. 158: 857-871
Tas, J., and R.G. Berndsen. 1977. Does heparin occur in
mucosal mast cells of the rat small intestine? J.
Histochem. Cytochem. 25: 1058-1062.
Taylor, K.M., S. Krilis, and B.A. Baldo. 1980. An
enzymatic-isotopic microassay for measuring ailergic
release of histamine from blood and mast cells in
vitro. Int. Archs. Allergy Appl. Immun. 61: 19.
Tertian, G., Y.-P. Yung, D. Guy-Grand, and M.A.S. Moore.
1981. Long-term in vitro culture of murine mast
cells. I. Description of a growth factor-dependent
culture technique. J. Immunol. 127: 788-794.
Tertian, G., Y.-P. Yung, and M.A.S. Moore. 1980. Induction
and long-term maintenance of Thy-1 positive lymphocytes:
Derivation from continuous marrow cultures. J. Supramol
Struct. Cell. Biol. 13: 533-539.


30
cultured alone, could support the growth of culture-derived
mast cells from cocultured thymocytes (Ginsburg and
Lagunoff, 1967).
Thus, the culture-derived mast cell era was ushered in
with the observation that at least some mast cell
progenitors were present in lymphoid tissue and that
adherent cells were required for jin vitro mast cell
differentiation and growth. In seminal attempts to clone
mast cell precursors in soft agar, Pluznik and Sachs (1965)
reported that embryonic feeder layers were again required
for outgrowth of mast cells from disaggregated splenocytes.
Ishizaka and colleagues (1977), investigating rat thymus-
derived mast cells in a system modeled after that of
Ginsburg, observed that clonal expansion was more prolific
in the presence of a feeder layer, although mast cells were,
indeed, isolated from cultures containing only thymocytes.
In the latter case, however, the mast cells were observed
to be associated with islands of fibroblast-like adherent
cells which arose in the thymocyte cultures.
The origin of mast cells in feeder layer cultures was
better resolved, almost twenty years after its initial
identification, by the definition of two morphologically
distinct populations of mast cells in mixed cultures of
adult lymphoid and embryonic feeder layer cells (Ginsburg
et al., 1982). Mast cells derived from the feeder layer
were morphologically similar to those found in connective


150
facilities and diluted with an equal volume of sterile
Alsever's solution (Mishell and Shiigi, 1980)) were washed
four times by centrifugation (ten minutes, 400xg, room
temperature) with sterile 0.15 M saline. The pellet of the
final wash was resuspended by flicking the tube and an
equal volume of filter-sterilized 0.5 mg per ml egg avidin
(Sigma Chemical Co.) was added. Two ml of filter-
sterilized 0.1 mg per ml chromic chloride (Fisher
Scientific Co.) was added dropwise to the red blood cell-
avidin mixture with constant, low speed vortexing over two
to three minutes at room temperature in a biological
containment hood. The reactants were held an additional
five to ten minutes at room temperature and then the
avidin-modified sheep red blood cells were washed four
times by centrifugation with balanced salts solution (BSS).
The cells were resuspended to 5 percent v/v in BSS and
stored for up to one week at 4C.
Bone marrow cells were prepared from two to four month
old BALB/cAN female mice as previously described. After
counting the washed cells, 1x10^ to 3x10^ cells were
pelleted by centrifugation and washed once in cold PBS.
The pelleted cells were resuspended in cold, biotin-
modified antibody. Pilot studies, using tumor cells rich
in the surface markers (11P62-4 for B23.1, 18-81 for RA3-
3A1), indicated 0.1 ml of antibody preparation per 1x10^
cells provided optimum labeling; the same volume to cell


114
to pellet the bacteria prior to electrophoresis on a 7.0
percent SDS-polyacrylamide gel with a 5.0 percent stacking
gel. Running buffer of 0.6 percent w/v Tris, 2.9 percent
w/v glycine, 0.1 percent w/v SDS and 0.114 percent w/v
sodium thioglycollate (Sigma Chemical Co.) was used.
Following electrophoresis at 150 volts (DC), the gel was
stained for fifteen minutes with 0.25 percent w/v Coumassie
Brilliant Blue R in aqueous 45 percent (v/v) methanol, ten
percent (v/v) glacial acetic acid. The gel was destained
over night against several changes of ten percent (v/v)
acetic acid with two "Adsorptors" destaining sponges,
rehydrated with three, ten minute changes of water, and
soaked for forty minutes in 1M sodium salicylate (Fisher
Scientific Co.) in water. The gel was sandwiched between a
clean sheet of 3MM filter paper and plastic wrap prior to
drying under vacuum (BIO-RAD gel drier, Oakland, CA). The
dried, fluorographed gel was exposed to XAR-5 film
(Eastman-Kodak, Rochester, NY) at -70C for the time
indicated.
Coculture of Long Term Mast Cells with Adherent Cell
Underlayers and Adherent Cell Conditioned Media
Bone marrow cells were prepared from normal BALB/cAN
tissue isolated as previously described (see Preparation of
Cell Suspensions for In Vitro Culture of Mast Cells).
Washed cells were resuspended at 1x10^ per ml in 50% W3CM
and 2.5 ml were aliquoted in each well of a six-well tissue


159
fold increase over the levels observed at day eight (Figure
IV-2B). In cells derived from homozygous tissues,
however, the tendency toward increased numbers of mast cell
precursors as embryonic development proceeded was much more
subtle. Although the numbers of embryonic precursors were
similar to those observed in heterozygous crosses earlier
in gestation (day eight), the number of mast cell colonies
in BALB/cAN tissues increased only three- to five-fold
(Figure IV-2C).
Frequency of Mast Cell Precursors in Embryonic Tissues of
the Third Trimester of Gestation
The increase in mast cell precursors in midgestation
embryonic tissues, most dramatically demonstrated in the
(BALB/cAN x CBA/J)F1 crosses, prompted us to ask whether
the numbers were maintained in the placenta throughout the
remaining course of gestation. As seen in Figure IV-3,
the number of embryonic mast cell precursors in the
placenta decreased seven- to eight-fold between days twelve
and thirteen and then continued to fall rapidly to the
lowest levels observed in our experiments by day fifteen.
Frequency of Mast Cell Precursors in Adult Bone Marrow
The number of mast cell precursors in the bone marrow
of adult mice has been analyzed by a number of other
investigators, thus providing a methodological control and
standard to which the frequency of mast cells in embryonic
tissue could be compared. It was additionally of interest


140
Having established the presence of mast cell
precursors in tissues identical to those used for A-MuLV
transformation, we infected "differentiated, long-term
culture-derived mast cells with A-MuLV to determine whether
such cells would be induced for the expression of the RA3-
3A1 determinant. The infected cells were phenotypically
identical to uninfected cells grown in the presence of
interleukin 3 with respect to metachromatic granules and
expression of the B23.1 epitope. The B lymphoid Ly5 marker
found on two of the original A-MuLV-transformed placental
cell lines (Chapter II) was not detected on either infected
or uninfected mast cells, although it was detected on
nongranular lymphoid cells in heterogeneous early cultures
of bone marrow cells which had been infected with A-MuLV
(Figures III-5, III-6). The RA3-3Al-positive cells were
probably Abelson virus-transformed pre-B cells, the
precursors of which are absent in long-term, homogeneous
mast cell cultures.
Our observations that Abelson murine leukemia virus
can infect mature cultured mast cells and abrogate their
requirement for interleukin 3 have recently been
corroborated in the literature. Pierce and colleagues
(1985) reported that fetal liver-derived mast cells,
infected with A-MuLV and maintained in media containing
interleukin 3 for three weeks, subsequently formed colonies
at high efficiency in the absence of the growth factor.


44
rim of metachromatic cytoplasm, similar to those described
by Maximow (1906) in stained tissue sections, arose and
became dominant in cell culture. The cytoplasm of the
monoblasts continued to increase in size as the nucleus
became more distinctly indented, with a chromophobic region
in the concave aspect of the nucleus. At twelve to
thirteen days in culture, a foamy region in the cytoplasm
was seen to spread, sometimes encompassing the entire
cytoplasm. Metachromatic material first appeared in the
foamy region as faint, amorphous substance in vacuoles,
later increasing in size and staining intensity to well-
defined granules. These latter cells were described as
"young mast cells", possessing granular, metachromatic
cytoplasm, with actively mitotic, kidney-shaped, round or
oval nuclei; such cells dominated the cultures between days
twelve and twenty-two. After three weeks in culture, the
majority of the cells were mature, round mast cells with
round to oval, eccentric or centered amitotic nuclei and
abundant, metachromatic cytoplasmic granules, similar in
morphology and histochemistry to mucosal mast cells.
Similar staging of rat culture-derived mast cell precursors
and intermediates has been reported (Ishizaka et al., 1976;
Zucker-Franklin et al., 1981; Sterry and Czarnetzki, 1982;
Czarnetzki et al, 1983), corroborating iji situ observations
(Combs et al., 1965).


48
(Katz et al., 1983). Although the Forsmann glycolipid
recognized by monoclonal antibody B1.1 is undetectable on
culture-derived mast cells, the latter cells do express the
antigen precursor, globotetrasylceramide (Katz et al.,
1985b), and may therefore be deficient or defective in the
giycosyltransferase required for the synthesis of the
mature antigen.
Culture-derived mast cells have been extensively
characterized biochemically as well. The histamine content
of cultured mast cells, like that of mucosal mast cells,
has been estimated between 450 and 500 nanograms per
million cells, at least ten-fold less than the histamine
content of comparable numbers of peritoneal mast cells
(Nabel et al., 1981; Nagao et al., 1981; Razin et al.,
1981a; Galli et al., 1982b; Sredni et al., 1983; Wedling et
al., 1985). Mouse culture-derived mast cell (and mucosal
mast cell) granules stain blue when treated with alcian
blue and safranin (Ginsburg and Lagunoff, 1967), indicating
the presence of weakly sulfated mucopolysaccharides,
whereas serosal mast cell granules, containing strongly
sulfated heparin proteoglycan, stain red. Razin and
colleagues (1982c) analyzed the proteoglycan of murine
culture-derived mast cells and found they contained
glucuronic acid-N-acetyigalactosamine-4,6-disulfate, or
chondroitin sulfate proteoglycan E, a unique
glycosaminoglycan which could not be detected in basophilic


127
cultures infected with M-MuLV or sham infected (incubated
with fresh DME with 4 micrograms Polybrene per ml) did not
proliferate if deprived of growth factors.
The expression of the lymphoid B-220 antigen was
analyzed as a function of primary culture age. Initially,
populations of cells which had been in culture for one,
two, three, or four weeks prior to infection were probed
twenty-one days post infection. The data from these
experiments are summarized in Figure III-5. Populations of
cells infected at early time points during the development
of mast cell cultures displayed similar proportions of
cells expressing the B-220 determinant as uninfected
populations, while at late times the cells retain the B-
220-negative phenotype of the cells in liquid culture
(Figure III-3). The cell culture systems, however, were
different by one important criterion. In the standard
procedure used to propagate culture-derived mast cells,
only nonadherent cells are passaged each week, thereby
selecting against the adherent population. In the virus-
and sham-infected cell cultures, however, the nonadherent
cells were maintained in the presence of the adherent cells
present in the original culture (and their progeny). Thus,
more adherent cells were present in cultures infected at
one week than at later times. Adherent cells supported the
growth of lymphoid cells as well as culture-derived mast
cells. The RA3-3Al-positive population is probably


8
(Benditt et al., 1954). Other substances shown to be
released during the anaphylactic response include heparin
and related proteoglycans, leukotrienes, prostaglandins, 5-
hydroxytryptamine and other amines, and neutral proteases
(reviewed in detail in Austen, 1984).
Mast cells have been directly or indirectly implicated
in a number of other physiologic roles. Histamine has been
associated with modulation and regulation of the immune
system (Askenase et al., 1981) including reduction of T
ceil effector function (Plaut et al., 1973; Schwartz et
al., 1980) and decreased lymphokine production (Rocklin,
1976). Mast cells have also been implicated in delayed-
type hypersensitivity reactions (Askenase, 1977), immune
complex formation (Benveniste et al., 1972), natural
cytotoxicity (Farram and Nelson, 1980; Henderson et al.,
1981), and parasite resistance (Capron et al., 1978), as
well as elaboration of a factor akin to interleukin 1 which
potentiates inflammation and collagenase activity in
synovial cells (Van Den Hoof and Tichelar-Guttar, 1983;
Yoffee et al., 1984, 1985).
Nonimmune functions have aLso been ascribed to mast
cells. "Microenvironmental hormones" (Lewis and Austen,
1981) produced by mast cells have been implicated in tissue
growth and repair (Kahlson and Rosengren, 1968) and
thyroxine secretion by the thyroid (Melander, 1977).
Histamine was shown to be required for timely blastocyst


50
peritoneal mast cells preferentially synthesized and
released prostaglandin D2 in forty-fold excess over
leukotriene C4.
Culture-derived mast cells are ultrastructurally
distinct from serosal mast cells. Cells of the former
category thus possess granules which are more heterogeneous
in size and electron density than the latter (Ginsburg and
Luganoff, 1967; Ginsburg et al., 1978; Nabel et al., 1981;
Galli et al., 1982b; Wedling et al., 1985). Granules in
culture-derived mast cells are ovoid and are frequently
associated with small vessicles (Sredni et al., 1983) and a
well-developed Golgi apparatus (Ginsburg and Lagunoff,
1967). The substance of mouse culture-derived mast cell
granules is crystalline (Razin et al., 1982a), while that
of human origin (resembling basophils, rather than mast
cells) is more particulate (Razin et al., 1981b). Mouse
bone marrow-derived mast cells form membrane channels after
activation with IgE and anti-IgE through which granules may
reach the cell surface (Razin et al., 1982a), similar to
human lung mast cells (Caulfield et al., 1980). The
cytoplasmic membrane of culture-derived mast cells is
characterized by numerous, fine protrusions (Galli et al.,
1982b; Wedling et al, 1985) which are absent from
peritoneal mast cell membranes.
The response of culture-derived mast cells to
secretagogues is similar to that of mucosal mast cells, but


60
culture-derived mast cells could be propagated from
embryonic sources in the presence of exogenously supplied
interleukin 3. These studies (and parallel experiments on
adult bone marrow-derived mast cells) also provide the
first detailed analysis of hematopoietic marker expression
of cultures progressing from heterogeneous to homogeneous
populations of mast cells (Chapter III). We have
subsequently analyzed the frequency of mast cell precursors
in embryonic placenta, nonplacental embryonic tissues, and
adult tissues, demonstrating the earliest reported mast
cell precursors as well as a heretofore unreported rich
source of such cells, the placenta (Chapter IV). In the
same chapter, we have characterized the cell surface of
mast cells grown in semisolid agar media and have presented
encouraging preliminary results of experiments designed to
sort mast cell precursors on the oasis of differentiation
antigen expression.


106
lines suggested that we probe the cells with B23.1 (Chapter
II), an antibody which recognizes culture-derived mast
cells. These studies led to the observation of other mast
cell markers on the transformed cell lines, namely
histamine and membrane receptors for IgE.
The expression of mast cell characteristics by A-MuLV
transformants derived from murine placenta opened several
related areas for investigation. We first desired to learn
if precursors to culture-derived mast cells exist in
midgestation embryonic tissue. Second, having found
embryonic precursors to these mast cells, we wanted to know
if the markers expressed on the A-MuLV transformants were
also expressed on "normal" cells cultured in interleukin 3.
We therefore conducted a detailed analysis of the
progression of cell populations in mast cell cultures over
the course of several weeks of selection and enrichment.
Third, we studied the effects of Abelson virus infection
and adherent cell cytokines on long-term cultured mast
cells. The scope and significance of our findings are
discussed in this chapter.
Materials and Methods
Procedures for the husbandry of mice, detection of
cell surface determinants and Fc receptors, and cytological
staining were performed as described in Chapter II.


51
not serosal mast cells. As previously discussed,
leukotriene C4 is synthesized and released as a result of
activation of the IgE receptor-mediated pathway. In
addition, histamine, chondroitin sulfate proteoglycan E,
and beta-hexosaminadase are released by immune activation
(Razin et al., 1983). Like both serosal and mucosal mast
cells, culture-derived mast cells are induced to
degranulate by the calcium ionophore A23187 (Razin et al.,
1982a, 1982b; Sredni et al., 1983; Robin et al., 1985).
Culture derived mast cells, however, mimic mucosal mast
ceils in their lack of response to compound 48/80 (Sredni
et al, 1983). In contrast, mouse peritoneal mast cells are
degranulated by compound 48/80.
Many of the characteristics previously described for
mouse culture-derived mast cells have been reported in
analogous rat and human systems. Ishizaka and colleagues
(1976) cultured rat thymocytes in the presence of rat
embryonic fibroblast monolayers and observed the outgrowth
of cells with receptors for IgE and metachromatic granules.
Haig and colleagues (1982, 1983) grew rat bone marrow-
derived cultured mast cells in the presence of media
conditioned by mesenteric lymph node cells. The
investigators observed that, similar to culture-derived
murine mast cells, rat culture-derived mast cells were
smaller than peritoneal mast cells, possessed sparse
granules of heterogeneous size, and expressed surface


185
observation that mast cell precursors exist in the mouse
embryo at least five days before the first mast cells are
detectable (Kitamura et al., 1979c). Subsequent analysis
of mast cell precursors in embryonic tissues between days
eight and nineteen of gestation indicated that the
precursors to culture-derived mast cells exist in very low
numbers prior to day nine of gestation, increasing by day
twelve to levels similar to those found in adult bone
marrow. The number of embryonic placental precursors to
culture-derived mast cells then falls precipitously between
days thirteen and nineteen of gestation. The results are
particularly interesting in light of the previously noted
observation that jm vivo mast cell precursors are abundant
in the fetal liver at day thirteen (Kitamura et al.,
1979c). It is enticing to speculate that mast cell
precursors either migrate through the placenta to the fetal
liver between days twelve and thirteen, or an independent
second "wave" of precursors develops in the fetal liver at
that time; however, there is no definitive evidence that
the embryonic in vitro and in vivo mast cell precursors are
identical.
In the course of our investigations, we studied the
progression of five hematopoietic markers in mast cell
cultures of embryonic and adult tissues over the course of
four weeks. Previous reports of phenotypic changes in mast
cell cultures were limited to morphological and


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148
temperature as described above. Cultures were fed once
weekly until the colonies were enumerated and analyzed.
Surface Markers on CFU-MC: Sorting of Mast Cell
Precursors with Monoclonal Antibodies
Mast cell precursors (CFU-MC) in adult BALB/cAN bone
marrow were screened for the presence of determinants
recognized by the monoclonal antibodies B23.1 (Katz et al.,
1983) and RA3-3A1 (Coffman and Weissman, 1981b) and for
expression of receptors for IgE. Initial experiments
designed to define the optimum method for selection of
cells with the desired phenotype, when performed with tumor
cell controls, indicated that neither panning (Wysocki and
Sato, 1978; Kay et al., 1977) nor complement-mediated
cytolysis were effective. A procedure for selective
depletion of rosetted cells (D. Levitt, personal
communication), however, proved effective in achieving the
desired goal.
B23.1 and RA3-3Al-positive cell selection
Hybridoma tissue culture fluids were obtained from
high cell density cultures, cleared by centrifugation at
2000xg for fifteen minutes, filtered through 0.2 micron
membranes, and were stored at 4C after addition of 0.01 to
0.02 percent w/v sodium azide. The hybridoma supernatants
were recleared centrifugally, warmed to room temperature
and placed in a beaker with a magnetic stir bar. Sodium
sulfate (Fisher Scientific Co.) was added slowly (over the


22
thymectomy shortly before challenge was ineffective in
ablating the mast cell response (Mayrhofer, 1979b). These
last observations indicated that the thymus, per se, is not
the immediate source of mast cells or mast cell growth
factors. Similar depression of mucosal mast cell response
and poor clearance of intestinal parasites were observed in
B rats, thymectomized, irradiated animals which were
reconstituted with bone marrow of T cell-depleted (chronic
thoracic duct drainage) donors (Mayrhofer and Fisher, 1979).
Interestingly, contrary to the reports of Crowle (Crowle,
1982; Crowle and Reed, 1984), unchallenged B rats, as well
as athymic nu/nu mice, were reported to have normal numbers
of mucosal mast cells when compared to appropriate controls
(Mayrhofer and Bazin, 1981).
Three other independent lines of evidence fortified
the hypothesis that mucosal mast cells were dependent on a
T cell-derived proliferation-differentiation factor.
First, the primary mucosal mast cell response to
Nippostrongylus brasiliensis in the rat was enhanced by
adoptive transfer of immune T cells (Nawa and Miller,
1979). Second, mucosal mastocytosis was demonstrated in a
variety of other immune scenarios, including the
inflammatory reactions of ulcerative colitis, Crohn's
disease, and pulmonary fibrosis (Askenase, 1980). Third,
Guy-Grand and colleagues (1984) recently reported the
direct stimulation of intestinal mucosal mast cell


137
mast cells, in contrast to adult bone marrow-derived mast
cells, prematurely become insensitive to the proliferative
effects of interleukin 3. These cells may be impoverished
of interleukin 3 receptors, perhaps by a capping mechanism
similar to that observed in B cells, in which anti-mouse mu
chain antibodies induced the disappearance of cell surface
IgM in 14 day mouse fetal liver explant and dissociated
adult lymphoid tissue cultures (Raff et al., 1975) and
inhibited the mitogenic effect of lipopolysaccharide in the
absence of 2-mercaptoethanol (Sidman and Unanue, 1978).
Alternatively, the embryonic culture-derived mast cells may
be defective in a post-receptor molecular mechanism, the
nature of which is unknown. We have not further
investigated this interesting phenomenon, which requires
more detailed examination.
Within one week of initiation of culture, over fifty
percent of bone marrow-derived cells express the B23.1
differentiation marker. The proportion of cells
expressing this marker continues to increase until, at four
weeks, almost all cells are B23.1-positive. Similar
population dynamics were observed when the cells were
stained with acidic toluidine blue. The proportion of
cells which expressed membrane receptors for IgE increased
more gradually from background levels in fresh bone marrow
to approximately fifty percent of the cells at week four.
The latter data are in contrast to a previous report


54
al., 1979), mast cell-free peritoneal exudate cells were
harvested from rats which were previously injected with
sterile water (intraperitoneally). The peritoneal cells
were cultured in L-ceil conditioned media with sodium
butyrate. The mast cells which grew out of this
population, although initially possessing blue-staining
granules by the alcian blue-safranin technique, later had
red staining granules and released histamine in response to
compound 48/80. Despite their serosal mast cell
characteristics, these culture-derived mast cells were
similar to the classical description of mucosal mast cells
in that they contained low levels of histamine (500
nanograms per million cells) and survived for only short
periods. Similarly described cells were subsequently
isolated by the same group from rat peritoneal cells
(Czarnetzki and Behrendt, 1981), and rat mononuclear
phagocytes (Czarnetzki et al., 1981, 1982; Sterry and
Czarnetzki, 1982).
In related studies, in vitro cultivated human
peripheral blood mononuclear cells (Denburg et al., 1983)
and guinea pig bone marrow cells (Denburg et al., 1980)
developed into metachromatic cells with segmented nuclei
which were more characteristic of basophils than mast
cells. The human cells, in particular, possessed the
polymorphonuclear structure with mature chromatin, Golgi
and microtubules which are more characteristic of basophils


12
years ago. Initially, they demonstrated that irradiated,
mast cell-deficient mice could be reconstituted by
injection of bone marrow from untreated donors (Kitamura et
al., 1977). Since irradiation did not eradicate all
recipient mast cells prior to reconstitution, a donor
strain with phenotvpically distinct mast cell granules
(beige) was used to definitively demonstrate the origin of
the cells which were detected. Similar experiments, using
unirradiated, genetically mast cell-deficient W/Wv mice,
demonstrated that adult bone marrow (Kitamura et al., 1978;
Hatanaka et al., 1979), blood (Kitamura et al., 1979a) and
spleen (Kitamura et al., 1979d) were rich reservoirs of
mast cell precursors which were also found in smaller
numbers in thymus, lymph node, and Peyer's patches
(Kitamura et al., 1979d). Mast cells were also detected in
fetal liver populations despite the apparent lack of mature
mast cells in that tissue (Kitamura et al., 1979c).
The hematopoietic nature of the ini vivo mast ceil
precursor was further defined bv the same group. in
preliminary studies, Kitamura and colleagues (1981) showed
that genetically mast cell-deficient W/Wv mice could be
reconstituted with cells from individual spleen colonies of
normal (C57BL/6) mice which had been irradiated and
subsequently reconstituted with bone marrow cells. Having
assured themselves of the clonality of each donor spleen
colony (by injecting mixed phenotypically distinct beige


10
The derivation of mast cells in mammalian tissues has
been the subject of considerable investigation, and
sometimes heated debate, since the early nineteen sixties.
Because the developmentaliy earliest described mast cells
were observed in connective tissue, and a gradation of
"immature" to "mature" forms of mast cells could be
isolated from this source, several investigators proposed
that mast cells were derived directly from connective
tissue precursors (Burton, 1963; Michels, 1963). Combs and
colleagues (.1965) observed the development of mast ceils in
embryonic rats between fifteen and twenty-three days post
coitum. The cells appeared to arise in undifferentiated
mesenchymal tissue and progressed through a gradation of
intermediates to characteristic mast cells. Asboe-Hanson
(1971) further noted that mast cells in the skin appeared
to differentiate locally from mesenchymal elements.
Based on observations of profound mastocytosis
associated with immune and neoplastic lymphocyte
proliferation, a second faction proposed the hematopoietic
origin of mast cells. Accordingly, Ginsburg (1963) removed
thymuses from mice and cultured them with embryonic skin
monolayers. The mast cells so derived led the author
(Ginsburg, 1963; Ginsburg and Lagunoff, 1967) to propose
that mast cells were derived from thymocytes, an
observation subsequently confirmed in the rat (Ishizaka et
al., 1976). Burnet (1965, 1975, 1977), observing that mast


14
safranin, dyes which preferentially bind to the
glycosaminoglycan components of mast ceil granules. By
this technique, alcian blue binds to poorly sulfated
glycosaminoglycans like chondroitin sulfate, while safranin
binds to highly sulfated molecules like heparin. A
gradation of cells, beginning with large, lymphocvte-like
elements with few, alcian blue-stained granules (Stage I),
and progressing through characteristic mature mast cells
with small nuclei and large numbers of safranin-staining
granules (Stage IV), was documented. Mast cell stages were
also differentiated by nuclear characteristics (mitotic
figures), granule heparin (periodic acid-Schiff staining),
granule glycosaminoglycan synthesis (sodium sulfate uptake),
granule histamine (diazotized parabromoaniline reaction),
and granule protease (phenyiproprionyl naphthol AS
reaction) content. The first recognizable mast cells
(Stage I) were found in the head mesenchyme at fifteen days
of gestation. Mast cell numbers rapidly increased during
the sixteenth day of gestation, with Stage II mast cells
found in the connective tissue of the dorsal vertebrae.
Subcutaneous (Stage III) mast cells were identified in
tissues of embryos eighteen and nineteen days post coitum.
Mature Stage IV mast cells were first present shortly
before birth and were indistinguishable from adult
connective tissue mast cells.


80
Analysis of Hematopoietic Lineage Markers
In view of the morphological and biochemical
similarity of the placental cell lines to mucosal and
culture-derived mast cells, we sought to further confirm
the relationship by analysis of the cell surface
differentiation antigens with an antibody which
discriminated between serosal and culture-derived mast
cells (Katz et al., 1983). This and other cell surface
antigens were analyzed by a simple and sensitive aureus -
antibody sandwich method (Uchanska-Ziegler et al., 1982).
Figure I-1A shows the binding reaction of the cell line
10P12 with bacteria coated with anti-cultured mast cell
antibody B23.1, while Figure II-1B shows that the same cell
line, incubated with bacteria coated with unimmmunized rat
immunoglobulin, resulted in no bacteria bound to the mouse
cell surfaces.
Cell surfaces of the remaining A-MuLV placental
transformants and of a number of control tumor cell lines
were probed with monoclonal antibody B23.1 as well as a
panel of other monoclonal and conventional antibodies
reported to be specific for hematopoietic differentiation
markers (Table II-l). Table II-3 summarizes the results of
the cell surface analyses. Every virus-transformed
embryonic cell line expressed the B23.1 epitope, which is


28
perhaps related note, Hasthorpe (1980) reported the
isolation and adaptation to in vitro growth of a factor-
dependent mast-like cell line from splenocvtes of a DBA-2
mouse infected with Friend erythroleukemia virus. Although
electron microscopy established the presence of budding C-
type virus particles on the highly granular cells,
Hasthorpe's FMP1.1 cell line was nontumorigenic upon
intraperitoneal or subcutaneous administration to syngeneic
hosts and could not proliferate in the absence of exogenous
growth factor in vitro.
Culture-Derived Mast Cells
The growing body of knowledge concerning mast cells is
due, in part, to the development of methodologies for the
selection, enrichment, and maintenance of culture-derived
mast cells. These cells possess many of the
characteristics of their in vivo correlates, the mucosal
mast cells, as demonstrated in the following text. A note
of caution, however, must be interjected into the seemingly
logical flow between naturally occurring fn vivo mast cells
and culture-derived mast cells. Although similar by a
variety of criteria including morphology, biochemistry,
function, and growth factor dependence, culture-derived
mast cells are not "normal" in the sense that they have
been produced in anatomically foreign, although perhaps
physiologically sufficient, conditions. They may therefore


123
maternal (data not shown). The expression of Class I
markers on P cells was previously reported by Schrader
(1981).
Bone marrow
The progression of cell surface and cytochemical
markers on bone marrow-derived mast cells was similar to
that observed in embryonic-derived tissues (Figure III-3).
The number of cells expressing the B23.1 determinant and
staining metachromatically with toluidine blue rose
dramatically from basal levels in fresh tissue to
essentially all of the cells at four weeks, while the
number of cells expressing receptors for IgE rose more
gradually to approximately fifty percent of the population.
At the same time, cells expressing receptors for IgG and
the B-220 lymphoid determinant were an insignificant
portion of the total cells in culture by two weeks.
Expression of Cell Surface Antigens on Cultured Mast Cells
Infected with Abelson Murine Leukemia Virus
Long term liquid cultures of mast cells are
characterized by a relatively homogeneous population of
cells which express the determinant recognized by the
monoclonal antibody B23.1, have receptors for IgE and
possess metachromatic granules in their cytoplasm. They
are, however, devoid of the B-lymphoid marker recognized by
the monoclonal antibody RA3-3A1, which is expressed on some
of the A-MuLV transformed placental mast-like cell lines.


27
In vitro analysis (Schindler et al., 1959) demonstrated
that the cells synthesized histamine and serotonin.
Through extensive culture, some sublines of P815 lost both
granules and intracellular histamine content; in some
sublines, however, the condition was reversed by addition
of sodium butyrate to the culture medium (Mori et al.,
1979) .
Focal mastocytomas have frequently been associated
with the presence of lymphocytes (Galli and Dvorak, 1979;
Askenase, 1980), supporting the once popular hypothesis
that the latter cells were precursors to the former
(Burnett, 1965, 1975, 1977). It was also noted that some
mastocytomas, like basophils and culture-derived mast
cells, synthesize chondroitin sulfate proteoglycan rather
than the heparin (Lewis et al., 1973) found in serosal mast
cells.
Some mastocytomas have been reported during the course
of experiments analyzing Abelson murine leukemia virus-
induced lymphomagenesis (Mendoza and Metzger, 1976; Risser
et al., 1978). The mastocytomas generally arose in
peritoneal oil granulomas evoked by tetramethylpentadecane
(Pristane) in mice which were inoculated with Abelson virus
thirty to forty days after administration of the oil
(Pierce et al., 1985). The tumors were generally
transplantable into syngeneic hosts and frequently could be
adapted to growth jji vitro. In a yet unexplained, but


LIST OF TABLES
Page
Table II-l Lineage-Specific Antibodies Used in Surface
Marker Analysis 69
Table II-2 Histamine Content of Embryonic Tumor Cell
Lines and Control Tumor Cell Lines 79
Table II-3 Analysis of Lineage-Specific Surface
Determinants on A-MuLV-Transformed
Embryonic and Control Tumor Cell Lines 82
Table II-4 Analysis of Surface Membrane Receptors for
IgE and IgG on A-MuLV-Transformed Embryonic
Cell Lines and on Controi Tumor Cell Lines 88
Table II-5 Metachromatic Granules in A-MuLV-Transformed
Embryonic Cell Lines and Control Tumor Cell
Lines 90
Table II-6 Interleukin 3 Content of Conditioned Media
and Cell Lysates of A-MuLV-Transformed
Embryonic Cells and Control Tumor Cells 96
Table III-l Effect of Cocultivation of Culture-Derived
Mast Cells with Adherent Cells and Their
Conditioned Media 133
Table IV-1 Frequency of Mast Cell Precursors in Adult
Bone Marrow from Homozygous and
Heterozygous Mice 162
Table IV-2 Expression of Surface and Cytochemical
Markers on Colony-Derived Mast Cells 165
Table IV-3 Sorting of Control Cells by Rosetting 167
Table IV-4 Sorting of Bone Marrow Cells by Surface
Determinants 169
vi


89
blood cells. Rosettes of sheep red blood cells on IgG Fc
receptor-positive cells were detected by phase microscopy
after staining with crystal violet.
Metachromatic Granules in Transformed Cell Lines
Initial examination of several of the embryonic cell
lines, which provided the first evidence for their
relationship to mast cells, was followed by a more detailed
study of the remaining cells. Cells were fixed in Mota's
lead subacetate and stained in acidic toluidine blue to
detect granules rich in basophilic glycosaminoglycan. As
seen in Table II-5, the majority of the original (BALB/c x
B10.BR)F1 embryonic cell lines did not possess
metachromatic granules, although two cell lines with high
histamine content (9P1 and 11P62) did express the
characteristic. Additionally, four of six embryonic cell
lines derived from a different paternal background, and all
long-term culture-derived mast cells (see Chapter III),
stained metachromatically with toluidine blue. Many of the
A-MuLV-transformed embryonic cell lines synthesize and
secrete chondroitin-4,6-disuifate proteoglycan (D. Levitt,
R. Porter, and E. Siden, manuscript in preparation), in
support of our observations.
Analysis of A-MuLV Provirus Integration
Although the embryonic tumor cell lines were derived
from cells infected with Abelson murine Leukemia virus, we
sought to confirm the presence of A-MuLV-specific sequences


113
Chemical Co.), 1 mM PMSF (Sigma Chemical Co.), and 0.0002
percent w/v Aprotinin (Boehringer Mannheim GmbH,
Indianapolis, IN). The lysates were cleared at 4C for ten
minutes at 2,000xg and sixty minutes at 45,000 revolutions
per minute in a Ti50 rotor of a Beckman L5-50
ultracentrifuge (Beckman, Palo Alto, CA).
Lysates were analyzed for incorporated radioisotope by
spotting five microliter volumes on Whatman 3MM paper
(Fisher Scientific Co.). The filters were washed in five
percent w/v trichloroacetic acid, dried, and counted in a
Beckman liquid scintillation spectrometer with toluene-
based scintillation fluid. Volumes of labeled lysates,
normalized to equivalent numbers of counts, were
immunoprecipitated with five microliters of either goat
anti-M-MuLV or normal goat serum by incubation over night
at 4C on a rotator, followed by incubation for two to three
hours with prewashed, heat-killed S. aureus bacteria (50
microliters at 10 percent w/v) in 1.5 ml microfuge tubes.
The contents of the tubes were centrifuged at 12,000xg for
thirty seconds and the pellets were washed three times with
PLB and resuspended in fifty microliters of sample buffer
containing 62.5 mM Tris, pH 6.8, one percent w/v SDS, 50 mM
dithiothreitol (Sigma Chemical Co.), 5 mM EDTA, and
bromophenol blue (Fisher Scientific Co.).
Immunoprecipitated proteins were heated for thirty minutes
at 68C and microfuged for three minutes at room temperature


CHARACTERIZATION OF ABELSON MURINE LEUKEMIA VIRUS-
TRANSFORMED MIDGESTATION EMBRYONIC CELLS
AND THEIR NORMAL HOMOLOGUES
By
MICHAEL L. SIEGEL
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1986

ACKNOWLEDGEMENTS
This dissertation and ray accomplishments over the past
several years are the result of a team effort. I wish to
acknowledge the contributions of all of my "teammates," and
hope that I slight none by inadvertent oversight.
First, I would like to thank my friend, mentor, and
supervisory chairperson, Edward J. Siden. His profound
powers of perception and his ability to assimilate new
observations into an existing body of knowledge have set
ideals which I shall take with me. His friendship and
camaraderie will always be of value to me.
Second, I wish to acknowledge and thank my supervisory
committee, J. Bert Flanegan, Carlo Moscovici, Steve Russell,
and Roy Weiner, who collectively guided me through my
dissertation research, sometimes despite my reluctance and
protestations.
My family has been a constant source of support,
cheer, and inspiration. My wife Jeremie has coped with
every crisis I brought home and has survived more mood
fluctuations in the last few years than most people endure
in a lifetime. David and Rebecca have also endured the
journey with few complaints and good humor, and it is to
them I owe the preservation of my humor. I must also thank
my father, Seymour Siegel, who inspired me to strive to be
the best at whatever I tried (even if I was a garbage man),
11

and my mother, Frances Heifer Siegel, who showed me the
value of patience and determination. My brother, Victor,
is also acknowledged for encouraging me to read and learn.
I owe much of my recent success to the support of my
fellow graduate students. In particular, I express my
thanks to Randy Horwitz, who has helped me stay young,
sharpened my cynical wit, shared my most profane moments,
and provided me with friendship which has endured almost
six years in Gainesville.
Finally, I wish to recognize the faculty and staff
(past and present) of the Department of Immunology and
Medical Microbiology who have made my experience more
fulfilling. In particular, I wish to thank Ken Berns, who
encouraged me to return to graduate studies after a seven
year hiatus. I am also indebted to Catherine and Richard
Crandall, George Gifford, and Michael Boyle, for a
seemingly endless supply of reagents and advice. Last, but
not least, I thank Muriel Reddish, Patrice Boyd, Ellen
Boukari and their superb staffs, without whom the work
would probably have taken six additional years.
iii

TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS ii
LIST OF TABLES vi
LIST OF FIGURES vii
ABSTRACT viii
CHAPTERS
I INTRODUCTION AND REVIEW OF THE LITERATURE I
Introduction: Hematopoietic Cell Differentiation
and Tumor Models 1
Mast Cell Function, Origin, Ontogeny, and
Heterogeneity 6
Connective Tissue Mast Cells 18
Mucosal Mast Cells 19
Basophils 25
Mastocytomas 26
Culture-Derived Mast Cells 28
Other In Vitro-Derived Metachromatic Cells 53
Relationship of In Vivo- and In Vitro-Derived
Mast Cells 56
Epilogue 59
IIABELSON MURINE LEUKEMIA VIRUS-INFECTED CELLS
FROM MIDGESTATION PLACENTA EXHIBIT MAST CELL
AND LYMPHOID CHARACTERISTICS 61
Introduction 61
Materials and Methods 63
Results 77
Discussion 95
III CHARACTERIZATION OF MAST CELLS DERIVED FROM
MIDGESTATION EMBRYONIC TISSUES IN LIQUID
CULTURE 105
Introduction 105
Materials and Methods 106
Results 117
Discussion 135
IV

IV ISOLATION, ENUMERATION, AND CHARACTERIZATION OF
IN VITRO MAST CELL PRECURSORS DERIVED FROM
MIDGESTATION EMBRYONIC PLACENTA 144
Introduction 144
Materials and Methods 146
Results 154
Discussion 172
V SUMMARY AND CONCLUSIONS 183
REFERENCES 189
BIOGRAPHICAL SKETCH 219
v

LIST OF TABLES
Page
Table II-l Lineage-Specific Antibodies Used in Surface
Marker Analysis 69
Table II-2 Histamine Content of Embryonic Tumor Cell
Lines and Control Tumor Cell Lines 79
Table II-3 Analysis of Lineage-Specific Surface
Determinants on A-MuLV-Transformed
Embryonic and Control Tumor Cell Lines 82
Table II-4 Analysis of Surface Membrane Receptors for
IgE and IgG on A-MuLV-Transformed Embryonic
Cell Lines and on Controi Tumor Cell Lines 88
Table II-5 Metachromatic Granules in A-MuLV-Transformed
Embryonic Cell Lines and Control Tumor Cell
Lines 90
Table II-6 Interleukin 3 Content of Conditioned Media
and Cell Lysates of A-MuLV-Transformed
Embryonic Cells and Control Tumor Cells 96
Table III-l Effect of Cocultivation of Culture-Derived
Mast Cells with Adherent Cells and Their
Conditioned Media 133
Table IV-1 Frequency of Mast Cell Precursors in Adult
Bone Marrow from Homozygous and
Heterozygous Mice 162
Table IV-2 Expression of Surface and Cytochemical
Markers on Colony-Derived Mast Cells 165
Table IV-3 Sorting of Control Cells by Rosetting 167
Table IV-4 Sorting of Bone Marrow Cells by Surface
Determinants 169
vi

LIST OF FIGURES
Page
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
II-l Detection of Cell Surface Determinants
on Abelson Murine Leukemia Virus-
Transformed Embryonic Cells 81
II-2 Detection of Surface Receptors for IgE
on A-MuLV-Transformed Embryonic Cells 87
II-3 Virus-Transformed Cells Contain Abelson
Murine Leukemia Virus-Specific DNA
Sequences 92
II-4 Tumors Isolated from Mice Injected with
Cell Lines 10P12 and 11P0 Contain
A-MuLV-Specific DNA Sequences 94
III-l Progression of Hematopoietic Lineage
Markers in Long-Term Mast Cell Cultures
Derived from Embryonic Tissues 120
III-2 Metachromatic Granules of Long-Term,
Culture-Derived Embryonic Mast Cells 121
III-3 Progression of Hematopoietic Lineage
Markers In Long-Term Mast Cell Cultures
Derived from Adult Bone Marrow 124
III-4 Population Dynamics of Bone Marrow-
Derived Mast Cells Infected with
A-MuLV 126
III-5 Expression of Ly5 Antigen on A-MuLV-
Infected Mast Cells 128
III-6 Mixed Population of RA3-3Al-Positive
Lymphoid Cells and RA3-3A1-Negative
Cultured Mast Cells in Long-Term Bone
Marrow Cultures 130
III-7 Abelson Murine Leukemia Virus-Infected
Mast Cells Express v-abl Gene
Product 131
IV-1 Colonies in Long-Term Agar Cultures of
Embryonic Cells in Conditioned Media 156
IV-2 Frequency of Mast Cell Precursors in
Midgestation Embryonic Tissues 158
IV-3 Frequency of Placental Mast Cell
Precursors in the Third Trimester of
Gestation 160
Vll

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
CHARACTERIZATION OF ABELSON MURINE LEUKEMIA VIRUS-
TRANSFORMED MIDGESTATION EMBRYONIC CELLS
AND THEIR NORMAL HOMOLOGUES
By
Michael L. Siegel
May 1986
Chairman: Edward J. Siden
Major Department: Immunology and Medical Microbiology
The embryonic origin and ontogeny of mast cells is
poorly understood, despite a growing body of literature
relevant to that area of study. We have systematically
investigated the development of mast cells in the embryonic
mouse, beginning our studies with the observation of mast
cell characteristics of midgestation embryonic placental
cells transformed with the defective retrovirus, Abelson
murine leukemia virus. Unlike previously reported Abelson
virus-transformed cells, the placental cell lines exhibited
many of the characteristics of culture-derived mast cells,
including differentiation antigens, high affinity receptors
for IgE, and metachromatic granules containing histamine
and sulfated proteoglycans. Some of the cell lines also
expressed the B220 marker previously reported to be
specific for cells of the B lymphoid lineage. We also
viii

developed a simple, sensitive, nonfluorometric, nonisotopic
assay to detect membrane receptors for immunoglobulins.
The observation of the mast-like, Abelson virus-
transformed cell lines led us to investigate the presence
of mast cell precursors in normal midgestation embryonic
tissues. We found embryonic precursors to mast cells in
homologous, noninfected tissues and conducted a detailed,
systematic analysis of the differentiation of mast cells in
liquid cultures over the course of several weeks of
selection and enrichment. We also studied the effects of
Abelson virus infection and adherent cell cytokines on
lymphoid differentiation antigens in mast cell cultures.
Mast cell precursors in embryonic tissues of mid- and
late gestation were quantitated by a clonal assay. We
described the embryologically earliest reported mast cell
precursors in the mouse and report that the mouse embryo is
a rich reservoir of such precursors, containing
proportionately at least as many such cells as adult bone
marrow. We have observed that mast cells which
differentiate in agar culture, like some of the Abelson
virus-transformed cell lines, express the B220 determinant.
We have also described preliminary experiments in which we
selected mast cell precursors in bone marrow on the basis
of surface membrane determinants.
ix

CHAPTER I
INTRODUCTION AND
REVIEW OF THE LITERATURE
Introduction: Hematopoietic Cell Differentiation
and Tumor Models
The ontogeny of the hematopoietic system of the mouse
can be viewed as a progression of finite, genetically
programmed stages in the maturation of pluripotent stem
cells into the terminally differentiated state of each of
the various blood lineages. Pluripotent hematopoietic stem
cells, defined bv their ability to reconstitute lethaliy
irradiated recipients (Till and McCulloch, 1961), are first
detected in the murine yolk sac between eight and twelve
days of gestation (Tyan, 1968). Beginning with day ten and
throughout the remainder of gestation, ceils with the same
differentiative capacity are found in the fetal blood and
liver (Moore and Metcalf, 1970). In the adult, pluripotent
hematopoietic stem cells are found in the bone marrow (Till
and McCulloch, 1961) and spleen (Nakahata and Ogawa, 1982).
The mechanisms involved in the differentiation of
pluripotent hematopoietic stem cells into mature,
functional blood elements are, for the large part, unknown.
These pathways may involve the interaction of pluripotent
or committed progeny stem cells with other cells or
macromolecular products in their inductive environment
(Kincade et al., 1981a), resulting in the cell's commitment
to one of several genetically programmed, phenotypically
distinguishable chains of events; alternately, random
1

2
stochastic processes may play a role in the differentiation
of hematopoietic cells (Nakahata et al., 1982a; Suda et
al., 1984).
The differentiation pathways of hematopoietic cells
and the molecular events involved in normal hematopoiesis
are best understood and defined by delineating discrete
cellular intermediates. Observation and identification is
frequently hampered by hematopoietic tissue heterogeneity,
short life span, and low frequency of cells of interest.
These difficulties are overcome in part by virus-induced
transformation of such cells, resulting in relatively
homogeneous populations of adequate size and frequently
unlimited growth potential. Although transformed
homologues of normal cells are frozen at a particular stage
of development by the action of the transforming gene
product, virus infection may also induce phenotypic changes
which are unparalleled in the course of normal
differentiation. To avoid this pitfall, it is therefore
prudent to confirm that the characteristics of tumor cell
models of early blood progenitors mimic their naturally
occurring counterparts.
Abelson murine leukemia virus (A-MuLV) is a
replication-defective retrovirus which arose by
recombination of portions of the genome of the replication-
competent, thymotropic Moloney murine leukemia virus and a
cellular gene, c-abl (Shields et al., 1979; Goff et al.,

3
1980) in a prednisolone-treated BALB/cCR mouse (Abelson and
Rabstein, 1970). A-MuLV is capable of rapid transformation
of bone marrow-derived thymus-independent lymphoid cells in
vivo (Sklar et al., 1975; Premkumar et al., 1975) and in
vitro (Rosenberg et al., 1975; Baltimore et al., 1979;
Rosenberg and Baltimore, 1976a, 1980).
Although most of the in vivo transformants reportedly
have been of the B cell phenotype (Premkumar et al., 1975),
plasmacytomas have also been reported (Potter et al.,
1973). T cell lymphomas (Cook, 1982), myelomonocytic
leukemias (Raschke et al., 1978), and mast cell tumors
(Mendoza and Metzger, 1976; Risser et al., 1978; Pierce et
al., 1985) have also resulted from in vivo infections,
indicating that Abelson virus may affect the growth and
differentiation of multiple hematopoietic lineages.
Abelson murine Leukemia virus is capable of
transforming both hematopoietic and nonhematopoietic cells
in vitro. Rosenberg and Baltimore (Rosenberg et al.,
1975; Rosenberg and Baltimore, 1976a, 1976b) have developed
an jji vitro culture system for the transformation and
clonal proliferation of lymphoid cells from murine
hematopoietic tissues. Under well-defined conditions,
permanent cell lines with pre-B lymphoid characteristics
(Siden et al., 1979) have been generated. These cell lines
are believed to exhibit the earliest known differentiation
markers and immunoglobulin gene organizational structures

4
of pre-B ceils (Boss et al., 1979; Siden et al., 1979; Alt
et al., 1981, 1984). Using identical conditions for
transformation of mouse placenta and fetal liver, Waneck
and Rosenberg (1981) described colonies of cells expressing
various differentiated erythroid characteristics,including
cessation of growth. Unlike its Moloney leukemia virus
ancestor, A-MuLV transforms the NIH/3T3 fibroblast cell
line (Scher and Siegler, 1975).
Modifications of the original Rosenberg and Baltimore
in vitro transformation protocol have resulted in the
transformation of phenotypically disparate lineages.
Whitlock and colleagues (1983) and Serunian and Rosenberg
(1986) have reported the transformation of more
differentiated B lineage ceils from in vitro-derived
"normal" populations. More recently, permanent cell lines
expressing mast cell characteristics have been reported
following transformation of midgestation embryonic placenta
(Siegel et al., 1985) and third trimester fetal liver
targets (Pierce et al., 1985).
Recent reports from several laboratories indicate that
retroviruses may alter the growth factor requirement of
cells of several lineages. Rapp and colleagues (1985)
reported the development of factor-independent cell lines
following transformation of interleukin 2-dependent T
lineage and interleukin 3-dependent myeloid cell lines by
recombinant viruses bearing the v-myc oncogene. Abelson

5
virus was similarly capable of abrogating the interleukin
3-dependence of both myeloid (Greenberger et al., 1979;
Cook et al., 1985) and mast (Pierce et ai., 1985; Chapter
III, this dissertation) lineages. Taken with the
observation of erythropoietin-independent evthroid cells
(Waneck and Rosenberg, 1981) and interleukin 3-independent
mast cells (Pierce et al., 1985; Siegel et al., 1985) from
A-MuLV-infected primary cell populations, the data suggest
that A-MuLV may alter the requirements of these cells for
growth factors while permitting the cells to differentiate.
Although the precise role of the v-abl oncogene in
maintaining factor-independent proliferation is not yet
known, it is interesting to note that the cellular
homologue, c-abl, is transcribed at its highest level in
the developing embryo at the same time that the number of
A-MuLV targets reaches its peak. At least two retroviral
oncogene products have recently been shown to have
analogous structures in normal cells. The epidermal growth
factor receptor exhibits striking similarity to the erb B
oncogene product of avian erythroblastosis virus (Downward
et al., 1984), while the v-sis oncogene of simian sarcoma
virus encodes a protein structurally and immunologically
related to platelet-derived growth factor (Doolittle et
al., 1983; Robbins et al., 1983; Waterfield et al., 1983).
Although the function of c-abl has not been established, it
is known that all of the hematopoietic lineages sensitive

6
to A-MuLV transformation are also sensitive to the
proliferative effects of interleukin 3 (reviewed in Iscove
and Roitsch, 1985; Rennick et al., 1985). Seminal studies
on the culture of mast cells revealed that cells of this
lineage would proliferate in the presence of media
conditioned by lectin- or antigen-stimulated T lymphocytes
or by the myelomonocytic leukemia cell line WEHI-3B
(Hasthorpe, 1980; Nabel et al., 1981; Nagao et al., 1981;
Schrader, 1981; Tertian et al., 1981). More recently,
interleukin 3, the proliferative factor in the conditioned
media, was purified to homogeneity (Ihle et al., 1983;
Razin et al., 1984a). Subsequent studies performed with
the glycoprotein product of cloned interleukin 3 gene have
substantiated the proliferative activity of the factor
(Yokota et al., 1984; Rennick et al., 1985).
Mast Cell Function, Origin, Ontogeny, and Heterogeneity
Contemporary knowledge and interest in the mast cell
has its roots in the midnineteenth century. The earliest
description of these cells is found in the work of von
Recklinghausen (1863), who observed and illustrated the
mast cell in the unstained mesentery of the frog. Credit
for the discovery of the mast cell, however, is generally
assigned to Paul Ehrlich, a young physician-scientist who
was then interested in the differential staining affinities
of certain tissue cells and their organelles. Ehrlich
(1877) first described mast cell-like cells in several

7
species as elements which stained atypically red-violet
with the blue basic analine dye dahlia. The term mast cell
is derived from Ehrlich's nomenclature (Mastzellen), which
he assigned to granular cells which were abundant in well-
nourished tissues of frogs (Ehrlich, 1879). In the same
work, Ehrlich first used the term "Metachromasie," or
metachromasia, to describe the anomalous staining of these
connective tissue cells. Aside from their role as
histochemical curiosities, however, few references to mast
cell derivation, functions, and heterogeneity were
published in the ensuing fifty years (Selye, 1965).
Mature mast cells have been attributed with a number
of physiologic functions, many of which have been reviewed
in the recent literature (Austen, 1984; Shanahan et al.,
1984; Katz et al., 1985a; Lagunoff, 1985). The most widely
known mast cell function is the anaphylactic response,
which was first described by Selye in 1937 (cited in Selye,
1965). The response was originally experimentally induced
in rats by intraperitoneal injection of egg white and
produced hyperemia and edema of the lips, ears, paws, and
genitalia which were aggravated by adrenalectomy and
ablated by stressors like formaldehyde, which induce
adrenocortical hyperplasia. The release of histamine
during anaphylactoid inflammation was hypothesized at this
time, although release of the mediator was not
experimentally associated with mast cells until 1954

8
(Benditt et al., 1954). Other substances shown to be
released during the anaphylactic response include heparin
and related proteoglycans, leukotrienes, prostaglandins, 5-
hydroxytryptamine and other amines, and neutral proteases
(reviewed in detail in Austen, 1984).
Mast cells have been directly or indirectly implicated
in a number of other physiologic roles. Histamine has been
associated with modulation and regulation of the immune
system (Askenase et al., 1981) including reduction of T
ceil effector function (Plaut et al., 1973; Schwartz et
al., 1980) and decreased lymphokine production (Rocklin,
1976). Mast cells have also been implicated in delayed-
type hypersensitivity reactions (Askenase, 1977), immune
complex formation (Benveniste et al., 1972), natural
cytotoxicity (Farram and Nelson, 1980; Henderson et al.,
1981), and parasite resistance (Capron et al., 1978), as
well as elaboration of a factor akin to interleukin 1 which
potentiates inflammation and collagenase activity in
synovial cells (Van Den Hoof and Tichelar-Guttar, 1983;
Yoffee et al., 1984, 1985).
Nonimmune functions have aLso been ascribed to mast
cells. "Microenvironmental hormones" (Lewis and Austen,
1981) produced by mast cells have been implicated in tissue
growth and repair (Kahlson and Rosengren, 1968) and
thyroxine secretion by the thyroid (Melander, 1977).
Histamine was shown to be required for timely blastocyst

9
implantation (Nalbandov, 1971; Dey and Johnson, 1980a,
1980b; Dey, 1981), as were prostaglandins (Kennedy, 1977),
but the number of detectable mast cells in the gravid
uterus was shown to decrease after implantation
(Shelesnyak, 1960; Brandon and Bibby, 1979). Mast cell
association with nerve tissue was first noted in 1878
(cited in Selye, 1965) and intimate contact between nerve
endings and mast cell plasma membranes was documented by
Weisner-Menzel and colleagues (1981) and Newson and
colleagues (1983). Histamine release has been attributed
to stimulation of cutaneous nerves (Kiernan, 1972), thus
establishing a neuroendocrine-mast cell axis. Mast cells
in alimentary tract mucosa have been implicated in
promotion of gut mobility (Erjavek et al., 1981; Fjellner
and Hagermark, 1981); histamine-induced gastric secretion
was shown to be blocked by histamine H2 receptor agonists
(Soli et ai., 1981) and enhanced by glucocorticoids
(Sathiamoorthy et al., 1976). The latter observation is
contradictory to published reports of glucocorticoid-
induced suppression of intestinal anaphylaxis (King et al.,
1985), and may indicate different modes of action of
steroids on sensitized and nonsensitized mast cells. Thus,
Daeron and colleagues (1982) noted that glucocorticoids
inhibited antigen-induced, but not calcium ionophore
A23187-induced, histamine release from mast cells.

10
The derivation of mast cells in mammalian tissues has
been the subject of considerable investigation, and
sometimes heated debate, since the early nineteen sixties.
Because the developmentaliy earliest described mast cells
were observed in connective tissue, and a gradation of
"immature" to "mature" forms of mast cells could be
isolated from this source, several investigators proposed
that mast cells were derived directly from connective
tissue precursors (Burton, 1963; Michels, 1963). Combs and
colleagues (.1965) observed the development of mast ceils in
embryonic rats between fifteen and twenty-three days post
coitum. The cells appeared to arise in undifferentiated
mesenchymal tissue and progressed through a gradation of
intermediates to characteristic mast cells. Asboe-Hanson
(1971) further noted that mast cells in the skin appeared
to differentiate locally from mesenchymal elements.
Based on observations of profound mastocytosis
associated with immune and neoplastic lymphocyte
proliferation, a second faction proposed the hematopoietic
origin of mast cells. Accordingly, Ginsburg (1963) removed
thymuses from mice and cultured them with embryonic skin
monolayers. The mast cells so derived led the author
(Ginsburg, 1963; Ginsburg and Lagunoff, 1967) to propose
that mast cells were derived from thymocytes, an
observation subsequently confirmed in the rat (Ishizaka et
al., 1976). Burnet (1965, 1975, 1977), observing that mast

11
cells exhibited evidence of thymic origin or dependence,
participated in immunologic reactions, were similarly
activated by lectins and immune stimuli, and were
relatively amitotic, speculated that the mast cells were
"post-mitotic" T lineage cells.
Other hematopoietic cell types have been postulated to
be mast cell by precursors in vivo and in vitro cultivation
techniques. Desaga and colleagues (1971) performed
repeated peritoneal lavages on rats to deplete them of
mature serosal mast cells. Mast cell-deficient
peritoneal exudate cells, cytochemically identified as
monocytes at the beginning of in vitro growth, developed
into metachromatic, granulated mast cells within two days
of harvest. Similarly, Czarnetzki and Behrendt (1981)
reported that peritoneal exudate cells from mast cell
depleted rats (injected with sterile water
intraperitoneally) resembled mononuclear phagocytes both
morphologically and cytochemically before and shortly after
culture in L-cell conditioned media. The in vitro-
propagated population, however, differentiated into mast
cells which were identified morphologically and which
contained granules with histamine and alpha-naphthol
acetate esterase.
The hematopoietic origin of some, and perhaps all,
mast cells was defined in great detail by Kitamura and
colleagues in a series of reports beginning less than ten

12
years ago. Initially, they demonstrated that irradiated,
mast cell-deficient mice could be reconstituted by
injection of bone marrow from untreated donors (Kitamura et
al., 1977). Since irradiation did not eradicate all
recipient mast cells prior to reconstitution, a donor
strain with phenotvpically distinct mast cell granules
(beige) was used to definitively demonstrate the origin of
the cells which were detected. Similar experiments, using
unirradiated, genetically mast cell-deficient W/Wv mice,
demonstrated that adult bone marrow (Kitamura et al., 1978;
Hatanaka et al., 1979), blood (Kitamura et al., 1979a) and
spleen (Kitamura et al., 1979d) were rich reservoirs of
mast cell precursors which were also found in smaller
numbers in thymus, lymph node, and Peyer's patches
(Kitamura et al., 1979d). Mast cells were also detected in
fetal liver populations despite the apparent lack of mature
mast cells in that tissue (Kitamura et al., 1979c).
The hematopoietic nature of the ini vivo mast ceil
precursor was further defined bv the same group. in
preliminary studies, Kitamura and colleagues (1981) showed
that genetically mast cell-deficient W/Wv mice could be
reconstituted with cells from individual spleen colonies of
normal (C57BL/6) mice which had been irradiated and
subsequently reconstituted with bone marrow cells. Having
assured themselves of the clonality of each donor spleen
colony (by injecting mixed phenotypically distinct beige

13
and wild type bone marrow cells into the primary,
irradiated recipients and screening the colonies for cells
with only one type of granule), the authors were able to
conclude that the colony forming unit of the spleen (Till
and McCulloch, 1961) was the ultimate progenitor cell for
mast cells in the spleen, stomach, caecum, and skin as well
as for peripheral blood granulocytes and erythrocytes.
These results were corroborated by Sonada and colleagues
(1983), who demonstrated that late (twelve days post
injection) spleen colonies, which included both erythroid
and myeloid elements, contained mast ceil precursors which
differentiated in secondary recipient skin.
The ontogeny of embryonic mast cells in mice is
meagerly defined in the literature. As previously
mentioned, iji vivo mast cell precursors are present,
despite the absence of more mature forms, in the mouse
fetal Liver thirteen and more days post coitum (Kitamura
et ai., 1979c). Embryonic mast cell ontogeny was better
defined in the rat. Csaba and Kapa (1960) demonstrated the
presence of mast cells, which incorporated exogenous
heparin, in the thymus, spleen, lymph nodes, myocardium,
and kidney of day sixteen to seventeen rat embryos. By
studying sections through rat embryos between fifteen and
twenty-three days of gestation, Combs and colleagues (1965)
placed mast cells into four stages of differentiation based
upon their staining characteristics with alcian blue and

14
safranin, dyes which preferentially bind to the
glycosaminoglycan components of mast ceil granules. By
this technique, alcian blue binds to poorly sulfated
glycosaminoglycans like chondroitin sulfate, while safranin
binds to highly sulfated molecules like heparin. A
gradation of cells, beginning with large, lymphocvte-like
elements with few, alcian blue-stained granules (Stage I),
and progressing through characteristic mature mast cells
with small nuclei and large numbers of safranin-staining
granules (Stage IV), was documented. Mast cell stages were
also differentiated by nuclear characteristics (mitotic
figures), granule heparin (periodic acid-Schiff staining),
granule glycosaminoglycan synthesis (sodium sulfate uptake),
granule histamine (diazotized parabromoaniline reaction),
and granule protease (phenyiproprionyl naphthol AS
reaction) content. The first recognizable mast cells
(Stage I) were found in the head mesenchyme at fifteen days
of gestation. Mast cell numbers rapidly increased during
the sixteenth day of gestation, with Stage II mast cells
found in the connective tissue of the dorsal vertebrae.
Subcutaneous (Stage III) mast cells were identified in
tissues of embryos eighteen and nineteen days post coitum.
Mature Stage IV mast cells were first present shortly
before birth and were indistinguishable from adult
connective tissue mast cells.

15
The study of mast cells, which has accelerated
dramatically in the past quarter century, has revealed that
Ehrlich's "Mastzellen" are heterogeneous both
evolutionarily (interspecies) and functionally
(intraspecies). Mast cell heterogeneity has been the topic
of numerous general reviews in recent years (Enerback,1981;
Bienenstock et al., 1982, 1983; Pearce, 1982, 1983;
Shanahan et al., 1984; Jarrett and Haig, 1984; Austen,
1984: Katz et al., 1985a; Lagunoff, 1985; Pearce et al.,
1985). From this sea of literature, the differences
between mast cells of various species are apparent. The
multipotent biogenic compound of mammalian mast cells,
histamine, has not been detectable in fishes and
amphibians, while serotonin is apparently unique to rodent
and dopamine to bovine mast cells. Heparin proteoglycan
from porcine, bovine, rat, and human sources is
heterogeneous in molecular weight and charge (Stevens and
Austen, 1981). The proteolytic enzymes of mast cells are
also phylogeneticallv disparate; rodent mast cells contain
an alpha-chymotrypsin-like activity, while dog, human, and
turtle mast cells have trypsin-like activity and bird and
fish mast cells have no esteroprotease activity (Woodbury
and Neurath, 1980; Lagunoff, 1985). Ultrastructurally, the
granules of human mast cells appear to be organized in
crystalline scrolls, while rat mast cell granules are

16
dense, homogeneous spheres which dissociate into fibrillar
structures in hypertonic salt solution (Lagunoff, 1972).
The initial observation of intraspecies mast ceil
heterogeneity is generally attributed to Maximow (1906),
who reported that the rat intestine was replete with mast
cells which differed from other rat mast cells in
morphology and stain affinity. These differences were
reinvestigated by Enerback, who, sixty years after
Maximow's observations, published a series of reports which
described in detail the differences in morphology, stain
affinities (Enerback, 1966a, 1966b), and sensitivity to
degranulating agents (Enerback, 1966c, 1966d) between
dermal mast cells (representative of the connective tissue
or serosal subset) and intestinal mast cells
(representative of mucosal or atypical mast cells). Thus,
mucosal mast cells were shown to be smaller, possess uni-
or bilobed nuclei, and be less granulated than serosal mast
cells, and the granules of the former population were far
more heterogeneous in size than those of the latter.
Enerback also observed that the mucosal mast cells stained
red with acidic toluidine blue, while serosal mast cells
stained purple. He noted that standard formaldehyde
fixatives used to preserve serosal mast cell granules were
ineffective on mucosal mast cells, and selected and
adapted several fixatives (such as Carnoy's and Mota's
preparations) to more adequately preserve the more labile

17
mucosal mast cell granules. Mucosal mast cells also
required higher concentrations of thiazine dyes, like
toluidine blue, and azure A dyes, as well as prolonged
staining times, when compared to serosal mast cells, while
the granules of the former cells had higher affinity to
copper phthalocyanine dyes such as Astra blue at ph 0.3.
Enerback therefore concluded that the mucosal mast and
connective tissue mast cells differed not only
morphologically, but biochemically as well, and offered
that the mucosal mast cells contained less highly sulfated
mucopolysaccharides than the dermal cells. Finally,
Enerback noted that in rats systemicallv exposed to the
histamine releasing agent 48/80, serosal mast cells in the
mesentery, tongue, and skin were degranuiated and therefore
undetectable, while mast cells in the duodenal mucosa were
unaffected and perhaps greater in number. While being
unable to explain the latter observation, Enerback was able
to conclude that mucosal mast cells differed from their
serosal counterparts functionally as well.
The differences between mucosal and serosal mast
cells, first noted by Maximow and then Enerback, have
since been appended by the observations of numerous
investigators and extend beyond those mentioned to surface
markers, histamine content, IgE receptors and
internalization of bound IgE, proteoglycan composition,
proteases, sensitivity to histamine secretagogues, effects

18
of neuropeptides and endorphins, thymus dependence, and
life span. These characteristics have been surveyed in
detail in reviews previously cited. We will therefore only
briefly survey the literature which is cogent to the
ultimate topic of this discussion, the in vitro, culture-
derived mast cell.
Connective Tissue Mast Cells
Although typical mast cells have been isolated from a
variety of connective tissue sources throughout the rodent
body, the most frequently studied member of this subset is
that which is isolated, free of extraneous tissues, from
the serosal surfaces of the peritoneal cavity. As
previously discussed, by injecting bone marrow into mast
cell-deficient hosts, Kitamura and colleagues (1977) were
able to demonstrate the relationship of hematopoietic
precursors to the serosal mast cells. The ultimate
precursor cell in the bone marrow was shown to be the
colony-forming unit of the spleen (Kitamura et al., .1981;
Sonoda et al., 1983). From the bone marrow, mast cell
precursors migrate through the blood (Kitamura et al.,
1979a; Zucker-Franklin et al., 1981; Sonoda et al., 1983)
and subsequently proliferate and differentiate in
connective tissue (Hatanaka et al., 1979; Kitamura et al.,
1979b,1979d). Connective tissue mast cells in rodents have
been found to proliferate and differentiate independently of
thymic influences. Thus, the athymic nude mouse has mast

19
cells in its connective tissues (Wlodarski, 1976; Reed et
al., 1982). Aldenborg and Enerback (1985) recently reported
that congenitally athymic rnu/rnu rats have at least as many
(or more) peritoneal mast cells as normal controls for the
first fourteen weeks of life; adult rnu/rnu rats, however,
have fewer peritoneal mast cells than their wild type
counterparts. These results mav indicate that peritoneal
mast cell populations may be subject to thymic influences
later in life or may simply reflect a separate, thymus-
independent defect inherent in the athymic rat. Further
studies will be necessary to elucidate the apparent
contradiction.
The proteoglycan composition of serosal mast cell
granules has been the subject of study since the initial
discovery of heparin in the canine liver by Jorpes in 1937
(cited in Selye, 1965). Subsequently, heparin has been
identified in rat peritoneal mast cells (Tas and
Berndsen, 1977; Yurt et al., 1977; Stevens and Austen,
1982), human lung mast cells (Metcalfe et al., 1979), and
mouse peritoneal mast cells (Razin et al., 1982c).
Mucosal Mast Cells
Following the development of improved methods for
their fixation and staining by Enerback (1966a, 1966b), the
study of the atypical, or mucosal, mast cell accelerated
significantly. Early reports of similarities between
cultured, thymus-derived mast cells of the mouse (Ginsburg,

20
1963; Ginsburg and Lagunoff, 1967) and the rat (Ishizaka et
al. 1976) and Enerback's atypical mast cells of the lamina
propria spurred erroneous theories that the mucosal mast
cell was derived from the thymus (Burnet, 1965, 1975,
1977). Based upon observations of mucosal mastocytosis
following experimental infection of rats and mice with the
nematodes Nippostrongylus brasiliensis and Tnchinella
spiralis, a lymphoid origin of these cells was also
proposed by other investigators (Rose et al., 1976; Befus
and Bienenstock, 1979; Mayrhofer, 1979a,1979b; Nawa and
Miller, 1979).
The derivation of mucosal mast cells was ultimately
resolved by Crowle (1982), who reconstituted mucosal mast
cell-deficient mice with cells derived from a variety of
hematopoietic tissues. Crowle observed that W/Wv mice
could be reconstituted by bone marrow and spleen cells but
not by thymocytes or thymus grafts, while athvmic mice
could be reconstituted by thymus grafts, thymocytes or
splenocytes. Crowle proposed that the W/Wv mice were
defective in mucosal mast cell precursors which were
present in bone marrow (and spleen) of normal mice, while
athymic mice possessed the precursor population and needed
a thymus-related component to effect differentiation.
Crowle concluded that mucosal mast cells were derived from
bone marrow and required a thymic influence for
accumulation in mucosal surfaces. These relationships

21
were further reinforced by the same investigator (Crowle
and Reed, 1984). Reconstitution of athymic mice was
ablated by pretreatment of wild type mouse bone marrow
cells or splenocytes with anti-Thy 1 and complement, while
similar treatment of beige bone marrow or spleen cells
still resulted in the detection of some mast cells, albeit
fewer, in the mucosa of thymus-intact W/Wv mice.
The thymic dependence of mucosal mast cells, in
contrast to the thymus-independent growth and development
of serosal mast cells, has been documented bv a number of
other investigators. Prior to the reports of Crowle
(1982,1984), Ruitenberg and Elgersma (1976) observed that
nude mice infected with Trichinella spiralis experienced no
intestinal mucosal mast cell response unless reconstituted
by thymus or parasite-immune thoracic duct cell grafts,
concluding that thymus-derived T-lineage cells were required
for mucosal mastocytosis. These results were substantiated
by a number of other researchers (Olson and Levy, 1976;
Mayrhofer and Bazin, 1981; Reed et al., 1982). Similar
studies were performed in the rat. Mayrhofer (1979a) noted
that the number of mucosal mast cells in Nippostrongylus
brasiliens is-infected rats increased in a pattern similar to
primary and secondary immune responses. Adult thymectomy or
chronic thoracic duct drainage several months prior to
nematode challenge (to deplete mature T cells) resulted in
significantly depressed intestinal mastocytosis, while

22
thymectomy shortly before challenge was ineffective in
ablating the mast cell response (Mayrhofer, 1979b). These
last observations indicated that the thymus, per se, is not
the immediate source of mast cells or mast cell growth
factors. Similar depression of mucosal mast cell response
and poor clearance of intestinal parasites were observed in
B rats, thymectomized, irradiated animals which were
reconstituted with bone marrow of T cell-depleted (chronic
thoracic duct drainage) donors (Mayrhofer and Fisher, 1979).
Interestingly, contrary to the reports of Crowle (Crowle,
1982; Crowle and Reed, 1984), unchallenged B rats, as well
as athymic nu/nu mice, were reported to have normal numbers
of mucosal mast cells when compared to appropriate controls
(Mayrhofer and Bazin, 1981).
Three other independent lines of evidence fortified
the hypothesis that mucosal mast cells were dependent on a
T cell-derived proliferation-differentiation factor.
First, the primary mucosal mast cell response to
Nippostrongylus brasiliensis in the rat was enhanced by
adoptive transfer of immune T cells (Nawa and Miller,
1979). Second, mucosal mastocytosis was demonstrated in a
variety of other immune scenarios, including the
inflammatory reactions of ulcerative colitis, Crohn's
disease, and pulmonary fibrosis (Askenase, 1980). Third,
Guy-Grand and colleagues (1984) recently reported the
direct stimulation of intestinal mucosal mast cell

23
precursors in BALB/c mice bearing the myelomonocytic
leukemia WEHI-3, a constitutive producer of the mast ceil
growth factor interleukin 3, which is identical to the mast
cell growth factor produced by activated T lymphocytes
(Yung et al., 1981).
A variety of other distinguishing characteristics have
been ascribed to the cells alternately called mucosal mast
cells, atypical mast cells, and histaminocytes (Code,
1977). Early studies of mucosal mast cells were performed
on tissue sections or on heterogeneous populations of cells
isolated from the gut mucosa. The previously cited
methodologies of Enerback (1966a, 1966b) were later
optimized by the inclusion of techniques which further
stabilized the granules (neutral formalin fixation) and
enzymatically (.with trypsin) stripped stain-retarding
proteins from glutaraldehyde-treated preparations (Wingren
and Enerback, 1983). The recent development of methods for
isolating such cells from the small intestine, which
exploited the mucosal mastocytosis induced by parasitic
infection, was reported by Befus and colleagues (1982a),
and made it possible to analyze mucosal mast cells in the
absence of extraneous elements and to confirm some previous
observations. In contrast to peritoneal mast cells,
isolated mucosal mast cells are smaller and have a shorter
lifespan. Mucosal mast cells have fewer granules which
contain nonheparin, lower sulfated proteoglycans (as yet

24
biochemically undefined), less histamine, and serotonin
(Bienenstock et al., 1983).
The response of mucosal mast cells to a variety of
secretagogues has been the subject of a number of studies.
Similar to serosal mast cells, mucosal mast cells are
responsive to the degranulation effects of IgE and antigen,
IgE and anti-IgE, concanavalin A, ionomycin, and compounds
23187 and Br-X537A, albeit with the release of less of
their total histamine content (Befus et al., 1982a, 1982b;
Pearce et al., 1982). Enerback's early observations on the
insensitivity of rat mucosal mast cells to degranulation by
compound 48/80 jui vivo were confirmed by other
investigators in canine (Lorenz et al., 1969; Rees et al.,
1981) and murine (Enerback, 1981) models. This
unresponsiveness was confirmed in isolated mucosal mast
cells and extended to the secretagogue Bee Venom Peptide
401 (Befus et al., 1982a, 1982b), for which membrane
receptors were found to be absent on mucosal, but not
serosal, mast cells (Pearce et al., 1982). The same group
also reported that, unlike serosal mast cells, mucosal mast
cells were unresponsive to enhanced, antigen-induced
secretion of histamine mediated by phosphatidyl serine.
Mucosal mast cells were also shown to be distinct from
serosal mast cells in their responsiveness to secretory
antagonists disodium chromoglycate, theophylline, and
AH9679, while both subsets were equally sensitive to the

25
degranulation inhibitory effects of Doxantrazole (Befus et
al., 1982b; Pearce et al., 1982, 1985).
Basophils
Cells with membrane receptors for IgE are not limited
to the mast cell lineage. Both lymphocytes (Gonzales-
Molina and Spiegelberg, 1978) and macrophages (Melewicz et
al. 1982) have been reported to bind IgE; the affinity of
the membrane receptors of the non-mast cells, however, was
ten to one-hundred times Less than that of mast cells
(Ogawa et al., 1983). The best known of the mast-like
cells, however, are the basophils (basophilic
granulocytes), polymorphonuclear leukocytes which are
present in the blood of several mammalian species (Lagunoff
and Chi, 1980). Like mast cells, basophils contain
metachromatic granules and express surface membrane
receptors for IgE. Histamine is released by antigenic
challenge of IgE-bearing basophils (Lagunoff and Chi,
1980). The granules of basophils of several species have
been reported to contain chondroitin sulfate proteoglycan
(Olsson et al., 1970; Orenstein et al., 1978; Metcalfe et
al., 1980b) similar in its degree of sulfation to the
proteoglycan of mucosal mast cells (Tas and Berndsen,
1977). Basophilic granulocytes, however, are apparently
absent from mouse peripheral blood (Lagunoff and Chi,
1980). The relationship of these cells to culture-derived

26
mast cells in several species, including the mouse, will be
further discussed in a later section of this review.
Mastocytomas
The study of mastocytomas has contributed to the
understanding of mast cell growth, differentiation, and
function, and has provided the bridge between complex in
vivo investigations and better-defined, clonal population
analyses of in vitro cultures. Spontaneous mastocytoma,
while common in such species as dogs (Cobb et al., 1975;
Yoffee et al., 1984, 1985), is a more infrequent condition
in other species, notably rats and mice (Lagunoff, 1985).
Efrati and colleagues (1957) described human mastocytoma
cells as large, lymphocyte-like elements similar to the
early mast cell progenitors described in lower mammals
(Maximow, 1906) .
The link between m vivo and in vitro mast cell
studies was established in 1959 when Schindler and colleagues
(1959) reported the successful adaptation of the
methylcholanthrene-induced, murine mastocytoma P815 (Dunn
and Potter, 1957) to growth in culture. The latter
investigators had isolated the neoplasm from a disseminated
disease with foci in the spleen and subcutaneous tissue and
had subsequently adapted it to a highly transplantable
ascites form from which a number of observations were made.
Variations in cell morphology, granule size and density,
and nuclear morphology were noted in the descriptive study.

27
In vitro analysis (Schindler et al., 1959) demonstrated
that the cells synthesized histamine and serotonin.
Through extensive culture, some sublines of P815 lost both
granules and intracellular histamine content; in some
sublines, however, the condition was reversed by addition
of sodium butyrate to the culture medium (Mori et al.,
1979) .
Focal mastocytomas have frequently been associated
with the presence of lymphocytes (Galli and Dvorak, 1979;
Askenase, 1980), supporting the once popular hypothesis
that the latter cells were precursors to the former
(Burnett, 1965, 1975, 1977). It was also noted that some
mastocytomas, like basophils and culture-derived mast
cells, synthesize chondroitin sulfate proteoglycan rather
than the heparin (Lewis et al., 1973) found in serosal mast
cells.
Some mastocytomas have been reported during the course
of experiments analyzing Abelson murine leukemia virus-
induced lymphomagenesis (Mendoza and Metzger, 1976; Risser
et al., 1978). The mastocytomas generally arose in
peritoneal oil granulomas evoked by tetramethylpentadecane
(Pristane) in mice which were inoculated with Abelson virus
thirty to forty days after administration of the oil
(Pierce et al., 1985). The tumors were generally
transplantable into syngeneic hosts and frequently could be
adapted to growth jji vitro. In a yet unexplained, but

28
perhaps related note, Hasthorpe (1980) reported the
isolation and adaptation to in vitro growth of a factor-
dependent mast-like cell line from splenocvtes of a DBA-2
mouse infected with Friend erythroleukemia virus. Although
electron microscopy established the presence of budding C-
type virus particles on the highly granular cells,
Hasthorpe's FMP1.1 cell line was nontumorigenic upon
intraperitoneal or subcutaneous administration to syngeneic
hosts and could not proliferate in the absence of exogenous
growth factor in vitro.
Culture-Derived Mast Cells
The growing body of knowledge concerning mast cells is
due, in part, to the development of methodologies for the
selection, enrichment, and maintenance of culture-derived
mast cells. These cells possess many of the
characteristics of their in vivo correlates, the mucosal
mast cells, as demonstrated in the following text. A note
of caution, however, must be interjected into the seemingly
logical flow between naturally occurring fn vivo mast cells
and culture-derived mast cells. Although similar by a
variety of criteria including morphology, biochemistry,
function, and growth factor dependence, culture-derived
mast cells are not "normal" in the sense that they have
been produced in anatomically foreign, although perhaps
physiologically sufficient, conditions. They may therefore

29
be considered models of their in vivo homologues until
definitive evidence permits us to conclude that the two
populations are completely identical. We will therefore
continue to use the term "culture-derived" mast cells, and
similarly distinctive terms, to maintain the tenor of this
caveat throughout the following discussion.
Adherent Feeder Layer Studies
The earliest reports of culture-derived mast cells by
Ginsburg (Ginsburg,1963; Ginsburg and Sachs, 1963) involved
a complex system of mouse thymocytes cultured on feeder
layers of mouse embryonic fibroblasts. It was apparent to
the authors that the monolayer was essential for
proliferation of mast cells. Culture of thymocytes in the
absence of the feeder layer failed to produce mast cells.
On the other hand, culture of embryonic skin fibroblasts
from eighteen day fetuses, without additional thymocytes,
infrequently resulted in mast cell outgrowth. The tissue
source of the mast cells, therefore, was disputable, and
quite possibly both thymocytes and embryonic feeder layers
contributed progenitors to the mast cell culture. The
issue was better defined several years later when the same
group reported that irradiated embryonic fibroblast
monolayers, which could no longer produce mast cells when

30
cultured alone, could support the growth of culture-derived
mast cells from cocultured thymocytes (Ginsburg and
Lagunoff, 1967).
Thus, the culture-derived mast cell era was ushered in
with the observation that at least some mast cell
progenitors were present in lymphoid tissue and that
adherent cells were required for jin vitro mast cell
differentiation and growth. In seminal attempts to clone
mast cell precursors in soft agar, Pluznik and Sachs (1965)
reported that embryonic feeder layers were again required
for outgrowth of mast cells from disaggregated splenocytes.
Ishizaka and colleagues (1977), investigating rat thymus-
derived mast cells in a system modeled after that of
Ginsburg, observed that clonal expansion was more prolific
in the presence of a feeder layer, although mast cells were,
indeed, isolated from cultures containing only thymocytes.
In the latter case, however, the mast cells were observed
to be associated with islands of fibroblast-like adherent
cells which arose in the thymocyte cultures.
The origin of mast cells in feeder layer cultures was
better resolved, almost twenty years after its initial
identification, by the definition of two morphologically
distinct populations of mast cells in mixed cultures of
adult lymphoid and embryonic feeder layer cells (Ginsburg
et al., 1982). Mast cells derived from the feeder layer
were morphologically similar to those found in connective

31
and serosal tissues, while those of lymphoid origin (lymph
node and thoracic duct) resembled mucosal mast cells, being
smaller in size with sparser, but larger granules than the
former cells. Pure "mucosal mast cells" (in fact, culture-
derived mast cells of lymphoid origin) could be grown on
selected feeder layers which were free of serosal type
precursors. The "mucosal mast cells" persisted, however,
only in the presence of T cell-derived factors. In
contrast, the mast cells derived from embryonic feeder
layers continued to persist, albeit without further
expansion, for six months or longer in the absence of
exogenous factors.
The same group (Davidson et al., 1983) later reported
that lymph node ceils derived from unimmunized, horse
serum-immunized, and helminth-infected mice, grown in the
presence of conditioned media (from antigen-stimulated
mesenteric lymph node cells) but in the absence of
irradiated embryonic mouse fibroblast feeder layers,
proliferated (as large, vacuolated cells) but failed to
develop granules. When the undifferentiated, culture-
derived cells were transferred to fibroblast monolayers,
however, the cells developed metachromatic granules
containing histamine within seven days. Intimate contact
between the two populations of viable cells was apparently
essential to granule maturation, as neither fibroblast
conditioned media, fibroblast homogenates, glutaraldehyde-

32
fixed fibroblasts, nor separation of fibroblasts from
"large lymphocyte" mast cell precursors by a membrane could
effect the change.
Despite the initial success in culturing mast cells
from lymphoid tissue cocultivated with adherent cell feeder
layers, reports in the literature of the technique's use
were limited to those of the previously cited groups. Two
factors probably contributed to the limited use of adherent
cell monolayers in mast cell culture. First, the system
was quite complex, requiring considerable time and
extensive subculture (or irradiation) of feeder layers to
eliminate the contribution of connective tissue type mast
cell precursors. Secondly, and perhaps more significantly,
the development of culture-derived mast cells in media
conditioned by activated lymphocytes by at least three
independent groups provided the opportunity to maintain
mast cells in the absence of a continuous monolayer of
feeder cells. As previously noted, however, even in the
absence of fibroblast feeder layers, islands of adherent
cells are observed in early cultures of lymphoid and
hematopoietic tissue-derived mast cells (confirmed in our
studies; see Chapters ill and IV). Since the adherent
cells are present in such cultures before the selection and
enrichment of mast cells, it is, at this juncture,
plausible to speculate that the adherent cells may assume a
transient, maturational role in mast cell differentiation.

33
Conditioned Media-Dependent Mast Cells
The development of techniques for culturing mast cells
from hematopoietic and lymphoid tissues has led to an
exponential increase in mast cell research and literature
citations. With the burst of scientific activity, however,
has come a concurrent increase in the number of terms used
to describe culture-derived mast cells, including P
(persisting) cells (Schrader and Nossal, 1980), histamine-
containing granular cells (Sredni et al., 1983), mucosal
mast cells, basophil/mast cells, and atypical mast cells.
Despite the discrepancy of terms, however, the long term
suspension cultures of mast cells appear to be strikingly
similar. Mast cells have thus been derived from mouse bone
marrow (Tertian et al., 1980, 1981; Nagao et al., 1981;
Razin et al., 1981a, 1982a,b,c; Schrader, 1981; Schrader et
al., 1981; Galli et al., 1982b; Crapper and Schrader, 1983;
Sredni et al., 1983; Wedling et al., 1983, 1985; Yung et
al., 1983; Suda et al., 1985), spleen (Hasthorpe, 1980;
Schrader and Nossal, 1980; Schrader et al., 1980, 1981;
Schrader, 1981; Tertian et al., 1981; Crapper and Schrader,
1983; Sredni et al., 1983; Pharr et al., 1984), fetal liver
(Nabel et al., 1981; Razin et al., 1984b), peripheral blood
(Crapper and Schrader, 1983; Suda et al., 1985), thymus
(Tertian et al., 1981; Schrader, 1981; Davidson et al.,
1983), lymph nodes (Ginsburg et al., 1978; Crapper and
Schrader, 1983), and intestinal mucosa (Schrader et al.,

1983b). Similar cells have been cultured from rat bone
marrow (Haig et al., 1982, 1983), peripheral blood (Zucker-
Franklin et al., 1981; Czarnetzki et al., 1983), and thymus
(Ishizaka et al., 1976, 1977) as well as human fetal liver
(Razin et al., 1981b), umbilical cord blood (Ogawa et al.,
1983), and adult peripheral blood (Denburg et al., 1983;
Czarnetzki et al., 1984).
The development of m vitro methods for mast cell
culture provided additional means of detecting embryonic
mast cell precursors. As previously noted, Ginsburg (1963)
observed occasional mast cell outgrowth in cultures of day
eighteen embryonic mouse skin. Similar experiments with
the rat model demonstrated that embryonic rat thymus,
isolated between eighteen and twenty days post coitum and
cocultured with adult rat thymocytes or thymocyte
conditioned media, contained cells capable of
differentiating into mast cells (Ishizaka et al., 1976). A
third group (Nabel et al., 1981; Galli et al., 1982a) was
able to culture murine mast cells derived from day thirteen
fetal liver suspensions cultured in lymphocyte conditioned
media. Using the adherent cell system, Ginsburg and
colleagues (1982) were also able to demonstrate that mast
cells could be derived in culture from disaggregated mouse
embryos between ten and thirteen days of gestation. It was
thus apparent that the precursors of culture-derived mast
cells were present in the mouse embryo at ten days post

35
coitem, several days before mast cells per se were
observable in the embryo (Kitamura et al., 1979c).
Interleukin 3 From Lymphocyte-Conditioned Media And Other
Sources
The analysis of culture-derived mast ceils and their
precursors evolved from studies of hematopoietic cell
growth factors produced by lymphocytes. Reports of mast
cell-supporting factors (which, for the sake of convention,
we will commonly call interleukin 3) in the supernatants of
mitogen-stimulated spienocytes began to surface at the
beginning of the present decade (Burgess et al., 1980;
Hasthorpe, 1980). Since that time, a number of other
investigators have utilized media conditioned by a variety
of means to support the differentiation and growth of
culture-derived mast cells. Such media have thus been
derived from spienocytes activated by concanavalin A
(Clark-Lewis and Schrader, 1981; Tertian et al., 1981; Yung
et al., 1981; Schrader et al., 1981; Nakahata et al.,
1982b; Yung and Moore, 1982; Sredni et al., 1983), by
pokeweed mitogen (Hasthorpe, 1980; Nakahata et al., 1982b,
Wedling et al., 1983, 1984; Pharr et al., 1984), by
phytohemagglutinin A (Ogawa et al., 1983), by bacterial
lipopolysaccharide (Nakahata et al., 1982b), and by mixed
lymphocyte reactions augmented by lectin (Razin et al.,
1981a, 1982a, 1982c). Conditioned media with analogous
activity have been elicited from concanavalin A-stimulated

36
mesenteric lymph node cells of parasitized animals
(McMenamin et al., 1985). Phytohemagglutinin A- or
concanavalin A-stimulated human blood lymphocytes also
produced activities which supported the growth of human
basophilic cells and a growth factor-dependent mouse cell
line (Tadokoro et al., 1983; Stadler et al., 1985).
The phenotype of the murine lymphoid cell which
produces interleukin 3 was deduced from the activities of
conditioned media of a number of related T cell clones. In
contrast to Lyt 1+2+, Lyt 1-2+, and Lyt 1-2- cells, which
did not support culture-derived mast cell growth, the
supernatants of Lyt 1+2- T cell clones, corresponding to
the inducer T lymphocyte subset, supported the
proliferation of such cells (Nabel et al., 1981). The
observations of Nabel and colleagues were subsequently
confirmed by other investigators (Fung et al., 1984; Yokota
et al., 1984), and by the observation that rat mesenteric
lymph node cells expressing differentiation markers of
helper T lymphocytes (0X19+, W3/25+, 0X8-) were responsible
for the production of a factor with analogous activity to
mouse interleukin 3 (McMenamin et al., 1985).
A number of permanent cell lines also produce factors
which support the growth of culture-derived mast cells.
The best known of these cell lines is the myelomonocytic
WEHI-3 line, which constitutively produces high levels of
interleukin 3 (Nagao et al., 1981; Schrader et al., 1981;

37
Yung et al., 1981; Yung and Moore, 1982). Mast cell-
promoting activities have also been demonstrated in
conditioned media from concanavalin A-stimulated T cell
hybridoma cells (Clark-Lewis and Schrader, 1981), cloned T
cell lines (Nabel et al., 1981), lectin-stimulated T
leukemias (Yung et al., 1981; Yung and Moore, 1982; Metcalf
and Kelso, 1985), and B lymphoma cells (Clark-Lewis et al.,
1982) .
The biologically active factor in lymphocyte and WEHI-
3 conditioned media has been given a variety of names. It
was first termed "multi-CSF" by Burgess and colleagues
(1980), due to its ability to support the differentiation
of multiple hematopoietic lineages fn vitro. Schrader and
colleagues (Schrader and Nossal, 1980; Schrader, 1981;
Schrader et al., 1981; Clark-Lewis and Schrader, 1981)
reported that "P cell stimulating factor" (PSF), which
supported the growth of persisting, mast-like cells in
vitro, was present in concanavalin A spleen conditioned
media. Ihle and colleagues (1981, 1982) proposed the name
"interleukin 3", or IL 3, for the factor which induced the
enzyme 20-alpha-hvdroxysteroid dehydrogenase in nude mouse
spleen cells as well as effecting a number of
differentiative and supportive activities in multiple cell
lineages. Although Ihle's terminology has achieved the
greatest usage in recent literature, alternative
nomenclature for the same activity has been proposed by

38
Bazill and colleagues ("multi-hematopoietic cell growth
factor", MCGF; Bazill et al., 1983) and Iscove
("multilineage hematopoietic growth factor", multi-HGF;
Iscove, 1985).
Interleukin 3, derived from a variety of sources, has
been purified to homogeneity (Yung and Moore, 1982; Ihle et
al., 1982b; Bazill et al., 1983; Clark-Lewis et al., 1984)
and, more recently, the genes for IL 3 have been molecularly
cloned and expressed (Fung et al., 1984; Yokota et al.,
1984; Rennick et al., 1985). Extensive reviews of the
literature describing interleukin 3 (per se, and the
related activities called by various other names) have been
published recently and should be consulted for additional
information (Clark-Lewis et al., 1985; Ihle, 1985; Iscove,
1985; Schrader et al., 1985; Whetton et al., 1985; Yung and
Moore, 1985).
Interleukin 3-Independent Mast Cells
Several reports of interleukin 3-independent mast cells
have appeared in the literature in recent years.
Schrader's group (Schrader and Crapper, 1983; Schrader et
al., 1983a) observed the emergence of factor-independent
variants from factor-dependent cells. In one experiment,
factor-dependent cells were plated in agar in the absence
of exogenous interleukin 3. From these cultures, several
colonies of autonomous culture-derived mast cells (P cells)
arose which, after several weeks, were subsequently adapted

39
to growth in interleukin 3-free liquid media. The
autonomous colonies secreted interleukin 3 into the culture
media, but also retained their receptors for the factor.
Furthermore, the autonomous cells generated more colonies
when plated at low density in the presence of exogenous
interleukin 3 in agar than in the absence of the growth
factor. It seems likely that the "autogenous cells were
not truly factor-independent, but rather were variants
which were able to proliferate, albeit at lower efficiency,
at the low levels of interleukin 3 provided by an autocrine
mechanism.
Similar factor-independent culture-derived mast cells
have been reported by a second group (Ball et al., 1983;
Conscience and Fisher, 1985). Several long-term bone
marrow-derived cultured mast cell lines were found, after
eleven months in culture, to contain variants which
proliferated at higher rates than similar cultures in the
presence of interleukin 3. The more proliferative cells
were able to continue cell growth in the absence of
exogenous interleukin 3, but the doubling time was
increased 160 percent when compared to cells of the same
line maintained in conditioned media. In contrast to the
autogenous cells of Schrader, the media conditioned by the
factor-independent mast cells described in the more recent
studies failed to support the growth of other factor-
dependent culture-derived mast cells. It is possible,

40
however, that the cells were able to maintain their growth
in the presence of interleukin 3 at levels below those
detected in the assay. interestingly, both sets of factor-
independent mast cells were tumorigenic in syngeneic mice.
Although no retroviral particles were observed in one of
these cell lines (Ball et al., 1983), the observations are,
by the criteria of tumorigenicity and factor independence,
similar to those of a recent report of Abelson murine
leukemia virus-transformation of culture-derived mast cells
(Pierce et al., 1985). Two possible mechanisms could
reconcile the yet unexplained results. First, the
activation of a latent replication-defective viral genome
in the Long-term culture-derived mast cells of Ball and
colleagues could be the missing link. Under such
circumstances, no viral particles would be detected, but
the cells could become both factor independent and
tumorigenic by virtue of the viral transforming gene
product. Similarly, the activation of a cellular homologue
of a viral transforming gene, like c-abl (the normal
function of which is unknown), could be invoked to activate
the mechanisms necessary to generate the phenotype of the
factor-independent, tumorigenic mast cells.
Mast Cell Precursors
The development of in vitro techniques for the
differentiation and maturation of mast cells from
phenotypically immature progenitors permitted the analysis

41
of mast cells based upon a number of criteria. First, the
number of culture-derived mast cell precursors in
particular tissues was determined quantitatively. Prior to
the development of mast cell culture techniques which
utilized sources of interleukin 3 for mast cell
proliferation, Pluznik and Sachs (1965) enumerated mast
cell clones in soft agar with feeder layers. The authors
reported approximately thirty mast cell colony forming
units per million spleen cells seeded, while the frequency
of culture-derived mast cell precursors in embryos, thymus,
and lymph nodes were five per million, three per hundred
million, and less than one per fifty million cells,
respectively. Schrader and colleagues (1981) cloned mast
cell precursors from mouse bone marrow in soft agar with
WEHI-3 conditioned media and found thirty to two-hundred
progenitors per million Thy 1-negative ceils. These
results were contradicted by a more recent report (Sredni
et al., 1983) which found 600 to 700 bone marrow
precursors, 400 to 500 spleen precursors, 25 to 30 thymus
precursors, and 100 to 200 Lymph node precursors per
million seeded cells. The latter results, however, were
generated in a system using concanavalin A-activated
splenocvte conditioned media as a source of interleukin 3
and different strains of mice, thereby making comparison
difficult. Interestingly, the latter authors also observed
that athymic nude mice had as many mast ceil precursors as

syngeneic wild type controls. Since athymic mice do not
exhibit the profound mucosal mastocytosis found in
appropriate controls (Olson and Levy, 1976), the last
observations could be interpreted to indicate that athymic
mice lack the inductive conditions required for the
proliferation of seemingly normal numbers of mast cell
precursors.
Mast cell precursors were enumerated by Nakahata and
colleagues (1982), using a modification of the semisolid
methylcellulose media culture system previously used to
identify erythroid and myeloid precursors. In the latter
and subsequent report using this system (Pharr et al.,
1984), which used pokeweed mitogen activated spleen
conditioned media for a source of interleukin 3, the
investigators found between twenty and 140 mast cell
precursors per million BDF1 mouse spleen cells and 200 mast
cell precursors per million bone marrow cells. Suda and
colleagues (1985), using the Nakahata culture system,
demonstrated that W/Wv mice, which are severly deficient in
mast cells of both serosal and mucosal subsets, had the
same number of peripheral blood mast cell precursors as
wild type mice (approximately thirty per million nucleated
cells), thus indicating that the mast cell defect was in a
homing or developmental step, rather than at the stem cell
or migratory level.
A third method for the enumeration of culture-derived

43
mast cell precursors from various tissues was reported by
Crapper and Schrader (1983). Using limiting dilution
analysis of cells in liquid culture containing WEHI-3
conditioned media, The authors were able to enumerate mast
cell precursors in bone marrow, spleen, mononuclear
peripheral blood cells, and lymph nodes. All of the data
recorded in the latter experiments concurred with the
previously cited results of Schrader and colleagues (1981)
as well as those reported by the Nakahata group (Nakahata
et al, 1982b; Pharr et al., 1984). Furthermore, Crapper
and Schrader were able to substantiate the findings of Suda
and colleagues that mast cell deficient mice had similar
numbers of culture-derived mast cell precursors (in bone
marrow and spleen) when compared to appropriate wild-type
controls, although the former authors used W^/W^ and the
latter authors used W/Wv.
The development of techniques for the propagation of
culture-derived mast ceils has also permitted the
characterization of such cells at various stages of
differentiation. Thus, Ginsburg and colleagues (Ginsburg,
1963; Ginsburg and Sachs, 1963; Ginsburg and Lagunoff,
1967; Ginsburg et al., 1982; Davidson et al., 1983)
reported a progression of characteristics of cultured mast
cells, starting with large, mononuclear "stem" cells.
After six to ten days in culture, large, lymphocvte-like
"mastoblasts" with round and bilobed nuclei and a narrow

44
rim of metachromatic cytoplasm, similar to those described
by Maximow (1906) in stained tissue sections, arose and
became dominant in cell culture. The cytoplasm of the
monoblasts continued to increase in size as the nucleus
became more distinctly indented, with a chromophobic region
in the concave aspect of the nucleus. At twelve to
thirteen days in culture, a foamy region in the cytoplasm
was seen to spread, sometimes encompassing the entire
cytoplasm. Metachromatic material first appeared in the
foamy region as faint, amorphous substance in vacuoles,
later increasing in size and staining intensity to well-
defined granules. These latter cells were described as
"young mast cells", possessing granular, metachromatic
cytoplasm, with actively mitotic, kidney-shaped, round or
oval nuclei; such cells dominated the cultures between days
twelve and twenty-two. After three weeks in culture, the
majority of the cells were mature, round mast cells with
round to oval, eccentric or centered amitotic nuclei and
abundant, metachromatic cytoplasmic granules, similar in
morphology and histochemistry to mucosal mast cells.
Similar staging of rat culture-derived mast cell precursors
and intermediates has been reported (Ishizaka et al., 1976;
Zucker-Franklin et al., 1981; Sterry and Czarnetzki, 1982;
Czarnetzki et al, 1983), corroborating iji situ observations
(Combs et al., 1965).

45
Characteristics of Culture-Derived Mast Cells
The development of refined analytical methods, first
applied to other hematopoietic cells, aided in the
characterization of culture-derived mast cells. Yung and
colleagues (1983) analyzed bone marrow cells by
centrifugation techniques and reported that interleukin 3-
responsive cells could be isolated by nature of their
median buoyant density (1.033) from interleukin 2-
responsive ceils (1.075). The authors also noted that the
buoyant densities of long-term bone marrow-derived cultured
mast cells (1.062 to 1.095 g/ml) were similar to those
determined by Pretlow and Cassidy (1970), who analyzed
heterogeneous populations of freshly isolated peritoneal
mast cells and reported that immature mast cells have a
median buoyant density of 1.087. Interleukin 3-
responsive cells were separated by sedimentation velocity
analysis from the in vitro precursors of macrophages (CFU-
M) and the pluripotent colony-forming cell of the spleen
(CFU-S), but not the bipotent precursor of granulocytes and
macrophages (CFU-GM).
Culture-derived mast cells and their precursors have
been characterized for the expression of a broad variety of
hematopoietic differentiation markers in attempts to assign
them to a particular lineage. Bone marrow and intestinal
precursors to culture-derived mast cells were observed to
lack the T lineage antigens Thy 1, Lyt 1 and Lyt 2

46
(Schrader et al., 1983b; Guy-Grand et al., 1984). Like
their precursors, more differentiated culture-derived mast
cells are deficient in Thy 1, Lyt 1, and Lyt 2 ( Ginsburg
et al., 1981; Nabel et al., 1981; Schrader, 1981; Schrader
et al., 1981; Tertian et al., 1981; Davidson et al., 1983;
Sredni et al., 1983; Ghiara et al., 1985), although
Schrader and colleagues (1982) reported that Thy 1 may be
transiently expressed on these cells. Similarly, culture-
derived mast cells lack surface immunoglobulin, a B lineage
marker (Ginsburg et al., 1981; Schrader et al., 1981;
Tertian et al., 1981; Sredni et al., 1983), NK-1, a marker
of natural killer cells (Nabel et al., 1981), complement
receptors and MAC-1, a differentiation antigen of
mononuclear phagocytes which is also expressed by some
natural killer and T lymphoma cells (Tertian et al., 1981).
Like their ini vivo correlates, murine culture-derived
mast cells express surface receptors for IgE (Ginsburg et
al., 1978; Nagao et al., 1981; Schrader, 1981; Schrader et
al., 1981; Tertian et al., 1981; Ginsburg et al., 1982;
Nakahata et al., 1982b; Sredni et al., 1983; Wedling et
al., 1983, 1985), which induce the anaphylactic release of
histamine when cross-linked by IgE and homologous antigen
(Ginsburg et al., 1978; Sredni et al., 1983) or anti-IgE
(Ginsburg et al., 1982). The number of IgE receptors per
cell has been estimated to be 2 to 3 x 10^, similar to the
number on serosal and mucosal mast cells (Razin et al..

47
1981a; Ginsburg et al., 1982). Some investigators have
also noted the presence of receptors for IgG (Schrader,
1981; Tertian et al., 1981); the cells, however, did not
phagocytose opsonized or unopsonized targets (Schrader,
1981; Sredni et al., 1983). Liquid cultured mast cells in
conconavalin A-stimulated spleen conditioned media also
express the lymphocyte marker Ly 5 (Nabel et al., 1981;
Tertian et al., 1981). Contradictory observations of
histocompatability Class II (la) antigens, or the lack
thereof, on culture-derived mast cells were resolved by
Wong and colleagues (1982), who showed that such cells,
grown in the presence of immune interferon (interferon
gammma, found in the supernatants of concanavalin A-
stimulated splenocytes) expressed the marker while cells
grown in the absence of interferon (as in WEHI-3
conditioned media) were devoid of la. Culture-derived mast
cells were also shown to express Class 1 histocompatability
antigens and receptors for peanut agglutinin (Schrader,
1981; Schrader et al., 1981; Tertian et al., 1981).
Russell and colleagues developed a panel of rat
monoclonal antibodies against murine mononuclear phagocytes
which could discriminate between culture-derived mast cells
and connective tissue mast cells (Leblanc et al., 1982).
The same group observed that culture-derived mast cells
expressed the phenotype B1.1-/B23.1+/ B54.2T, while in
contrast peritoneal mast cells were B1.1T/ B23.1-/B54.2T

48
(Katz et al., 1983). Although the Forsmann glycolipid
recognized by monoclonal antibody B1.1 is undetectable on
culture-derived mast cells, the latter cells do express the
antigen precursor, globotetrasylceramide (Katz et al.,
1985b), and may therefore be deficient or defective in the
giycosyltransferase required for the synthesis of the
mature antigen.
Culture-derived mast cells have been extensively
characterized biochemically as well. The histamine content
of cultured mast cells, like that of mucosal mast cells,
has been estimated between 450 and 500 nanograms per
million cells, at least ten-fold less than the histamine
content of comparable numbers of peritoneal mast cells
(Nabel et al., 1981; Nagao et al., 1981; Razin et al.,
1981a; Galli et al., 1982b; Sredni et al., 1983; Wedling et
al., 1985). Mouse culture-derived mast cell (and mucosal
mast cell) granules stain blue when treated with alcian
blue and safranin (Ginsburg and Lagunoff, 1967), indicating
the presence of weakly sulfated mucopolysaccharides,
whereas serosal mast cell granules, containing strongly
sulfated heparin proteoglycan, stain red. Razin and
colleagues (1982c) analyzed the proteoglycan of murine
culture-derived mast cells and found they contained
glucuronic acid-N-acetyigalactosamine-4,6-disulfate, or
chondroitin sulfate proteoglycan E, a unique
glycosaminoglycan which could not be detected in basophilic

leukemia cells, peritoneal mast cells, or chondrocytes.
Chondroitin sulfate proteoglycan E was shown in this study
to be chemically distinct from heparin by a number of
criteria including sensitivity to enzymatic degradation and
molecular weight (chondroitin sulfate proteoglycan E has an
estimated molecular weight of 200 kilodaltons, in contrast
to heparin, which has a molecular weight of 750
kilodaltons). These results have been confirmed in the
literature (Razin et al., 1983; Sredni et al., 1983).
Mouse culture-derived mast cell granules also contain a
number of other in vivo mast cell-associated biological
mediators, including serotonin (5-hydroxytryptamine) and
dopamine (Tertian et al., 1981).
Arachidonic acid metabolites, the prostaglandins and
leukotrienes, are important biological mediators
asssociated with metachromatic cells. IgE-dependent
activation of mouse culture-derived mast cells results in
the synthesis and release of leukotriene C4 (Razin et al.,
1982b, 1983), a component of the slow releasing substance
of anaphylaxis (Austin, 1984). These studies also showed
that bone marrow-derived cultured mast cells generated
approximately twenty-five times more leukotriene C4 than
prostaglandin D2 upon activation by calcium ionophore
A23187 or IgE receptor-mediated pathways. In contrast, rat

50
peritoneal mast cells preferentially synthesized and
released prostaglandin D2 in forty-fold excess over
leukotriene C4.
Culture-derived mast cells are ultrastructurally
distinct from serosal mast cells. Cells of the former
category thus possess granules which are more heterogeneous
in size and electron density than the latter (Ginsburg and
Luganoff, 1967; Ginsburg et al., 1978; Nabel et al., 1981;
Galli et al., 1982b; Wedling et al., 1985). Granules in
culture-derived mast cells are ovoid and are frequently
associated with small vessicles (Sredni et al., 1983) and a
well-developed Golgi apparatus (Ginsburg and Lagunoff,
1967). The substance of mouse culture-derived mast cell
granules is crystalline (Razin et al., 1982a), while that
of human origin (resembling basophils, rather than mast
cells) is more particulate (Razin et al., 1981b). Mouse
bone marrow-derived mast cells form membrane channels after
activation with IgE and anti-IgE through which granules may
reach the cell surface (Razin et al., 1982a), similar to
human lung mast cells (Caulfield et al., 1980). The
cytoplasmic membrane of culture-derived mast cells is
characterized by numerous, fine protrusions (Galli et al.,
1982b; Wedling et al, 1985) which are absent from
peritoneal mast cell membranes.
The response of culture-derived mast cells to
secretagogues is similar to that of mucosal mast cells, but

51
not serosal mast cells. As previously discussed,
leukotriene C4 is synthesized and released as a result of
activation of the IgE receptor-mediated pathway. In
addition, histamine, chondroitin sulfate proteoglycan E,
and beta-hexosaminadase are released by immune activation
(Razin et al., 1983). Like both serosal and mucosal mast
cells, culture-derived mast cells are induced to
degranulate by the calcium ionophore A23187 (Razin et al.,
1982a, 1982b; Sredni et al., 1983; Robin et al., 1985).
Culture derived mast cells, however, mimic mucosal mast
ceils in their lack of response to compound 48/80 (Sredni
et al, 1983). In contrast, mouse peritoneal mast cells are
degranulated by compound 48/80.
Many of the characteristics previously described for
mouse culture-derived mast cells have been reported in
analogous rat and human systems. Ishizaka and colleagues
(1976) cultured rat thymocytes in the presence of rat
embryonic fibroblast monolayers and observed the outgrowth
of cells with receptors for IgE and metachromatic granules.
Haig and colleagues (1982, 1983) grew rat bone marrow-
derived cultured mast cells in the presence of media
conditioned by mesenteric lymph node cells. The
investigators observed that, similar to culture-derived
murine mast cells, rat culture-derived mast cells were
smaller than peritoneal mast cells, possessed sparse
granules of heterogeneous size, and expressed surface

52
receptors for IgE. Rat culture-derived mast cell granules
stained blue by the alcian blue-safranin technique and were
metachromatic when stained with toluidine blue. The
granule proteoglycan was identified as non-heparin,
although no report of its precise chemical composition has
been published to date. Such cells also contained
immunochemically detectable levels of rat mast cell
protease II, which was previously described as a marker of
mucosal, but not serosal mast cells. Rat mesenteric lymph
nodes (Denburg et al., 1980) and peripheral blood (Zucker-
Franklin et al., 1981) have also been shown to contain
precursors of culture-derived mast cells.
Reports of mast cells derived from human tissues are
clouded by difficulties in distinguishing between the
various types of basophilic cells when compared to in vivo
correlates, namely mucosal mast cells, connective tissue
mast cells, and basophils. Granulated cells with receptors
for IgE and low levels of histamine (50 to 450 nanograms
per million cells), thus resembling mouse culture-derived
mast cells, have been observed in cultures of human fetal
liver grown in unconditioned media (Razin et al., 1981b).
Adherent human blood mononuclear cells and pleural exudate
cells, which were propagated with L-cell conditioned media,
exhibited similar characteristics (Czarnetzki et al., 1983,
1984; Kruger et al., 1983). Cells with high affinity
receptors for IgE and slightly higher levels of histamine

53
(480 to 1600 nanograms per million cells) were cultured
from human umbilical cord blood grown in phytohemagglutinin
A-stimulated human T cell conditioned media (Ogawa et al.,
1983). Horton and O'Brien (1983) reported the culture of
granulated cells with centrally placed, round or indented
nuclei from human bone marrow harvested from a patient with
systemic mastocytosis. The culture-derived mast cells
showed no growth advantage with a number of conditioned
media, but required an adherent, bone marrow-derived feeder
layer to persist.
Other In Vitro-Derived Metachromatic Cells
As noted previously, some of the confusion in the
terminology applied to culture-derived mast cells has been
generated by the appearance of characteristics of one or
more of the in vivo basophilic cell correlates. Thus,
although basophils in mice are lacking (Lagunoff and Chi,
1980) or extremely rare (Dvorak et al., 1982), a report of
cloned, basophil-like cell lines with IgE receptors has
appeared in the literature (Galli et al., 1982a). The cell
line in question, derived from mouse splenocytes cultured
in concanavalin A-stimulated splenocyte conditioned media,
lacked histamine and had both natural killer cell
differentiation markers and natural killer activity.
In a series of studies on the rat, Czarnetzki and
colleagues described the growth of connective tissue-like
mast cells in vitro. In the earliest study (Czarnetzki et

54
al., 1979), mast cell-free peritoneal exudate cells were
harvested from rats which were previously injected with
sterile water (intraperitoneally). The peritoneal cells
were cultured in L-ceil conditioned media with sodium
butyrate. The mast cells which grew out of this
population, although initially possessing blue-staining
granules by the alcian blue-safranin technique, later had
red staining granules and released histamine in response to
compound 48/80. Despite their serosal mast cell
characteristics, these culture-derived mast cells were
similar to the classical description of mucosal mast cells
in that they contained low levels of histamine (500
nanograms per million cells) and survived for only short
periods. Similarly described cells were subsequently
isolated by the same group from rat peritoneal cells
(Czarnetzki and Behrendt, 1981), and rat mononuclear
phagocytes (Czarnetzki et al., 1981, 1982; Sterry and
Czarnetzki, 1982).
In related studies, in vitro cultivated human
peripheral blood mononuclear cells (Denburg et al., 1983)
and guinea pig bone marrow cells (Denburg et al., 1980)
developed into metachromatic cells with segmented nuclei
which were more characteristic of basophils than mast
cells. The human cells, in particular, possessed the
polymorphonuclear structure with mature chromatin, Golgi
and microtubules which are more characteristic of basophils

55
than mast cells. This conclusion, however, directly
disagreed with Zucker-Franklin (1980), who contended that
human mast cells and basophils share common ultrastrucural
organization.
Tadokoro and colleagues (1983) cultured cells with
metachromatic granules and lobulated nuclei from normal
human bone marrow in conditioned media from lectin-
stimulated blood lymphocytes. The culture-derived cells
contained 500 to 2000 nanograms of histamine per million
cells and were responsive to IgE-anti-IgE- and calcium
ionophore-mediated histamine release but were refractory to
the effects of compound 48/80. The authors concluded that
their conditioned media contained a basophil-promoting
activity which furthermore had a molecular weight of 25 to
40 kilodaltons and was distinct from interleukin 2. The
same group recently reported that media conditioned by
phytohemagglutinin A- and concanavalin A-stimulated human
blood lymphocytes could support the interleukin 3-dependent
mouse cell line 32Dcl as well as promote the growth of
human culture-derived basophils (Stadler et al., 1985).
Furthermore, the interleukin 3 and basophil-promoting
activities, which were also found in media conditioned by
the growth of E-rosetting T lymphocytes and the MoT cell
line, were biochemically distinct by at least five
different criteria. The isolated human interleukin 3 was
shown to promote the growth of mast cells which were unable

56
to proliferate in the presence of mouse interleukin 3 (in
WEHI-3 conditioned media). Thus, in the human system, the
culture-derived mast cell-basophil dilemma is no longer
solely a matter of terminology and mistaken identity, but,
in fact, apppears to involve multiple growth promoters and,
most likely, multiple progenitor cells.
Relationship of In Vivo- and In Vitro-Derived Mast Cells
A small body of evidence supports the theory that
culture-derived mast cells are more than circumstantially
related to mucosal, and perhaps serosal, mast cells. A
number of characteristics, including morphology,
histochemical fixation and staining, dependence of
proliferation on T cell-derived factors, biogenic amine
content, protease content, presence of receptors for IgE,
and sensitivity to secretogogues, have been noted in this
review and cited by many of the authors as proof of the
relationship between culture-derived mast cells and the
mast cells of the mucosal surfaces. There is preliminary
evidence that culture-derived mast cells have natural
cytotoxic activity against tumors such as WEHI-3 and Meth A
which is enhanced by interleukin 3 (Ghiara et al., 1985).
Investigators have noted that in vivo-derived mast ceils
exhibited similar tumoricidal activity (Farram and Nelson,
1980) and that in vivo-derived cells with the

57
Thy 1-/Lyt 1-/ Lyt 2- phenotype, which are demonstrably
cytotoxic, are sensitive to the proliferative activities of
interleukin 3 (Djeu et al., 1983; Lattime et al., 1983).
Evidence of more direct relationships between in
vijtro-derived mast cells and their in vivo correlates has
been elusive. Several investigators have associated the
high incidence of culture-derived mast cell precursors and
mucosal mast cells in the intestine of normal mice (Crapper
and Schrader, 1983; Guy-Grand et al., 1984). The role of
antigenic stimulation and T cell function in the
proliferation of mucosal mast cells per se has been
thoroughly described in the literature (for reviews see
Jarrett and Haig, 1984; Shanahan et al., 1984; Bienenstock
et al., 1983). Guy-Grand and colleagues (1984) also showed
that the number of mast cells which could be cultured from
intestinal mucosa increased with antigenic stimulation and
WEHI-3 tumor burden, implicating the role of interleukin 3
in the m vivo proliferation of mast cell precursors. The
results, however, associated the iji vivo and in vitro mast
cell precursors by existence in the same tissue, and did
not directly show that the populations involved were
identical.
The most suggestive evidence to date of the
relationship between in vivo- and jji vitro-derived mast
cells involves the demonstration that culture-derived mast
cells, when injected into mast cell-deficient mice,

58
populated both mucosal and connective tissue-serosal
compartments. Nakano and colleagues (1985) injected
culture-derived mast cells and partially purified (30 to 40
percent) peritoneal mast cells into W/Wv mice by various
routes. At high levels of inoculum (10^ to 10^ cells),
intravenously- or intraperitoneally-injected cultured mast
cells populated the spleen and stomach (mucosa and muscle),
while at lower levels of inoculum (10- to lO^cells), only
intraperitoneallv injected cells were able to populate the
same anatomical sites. Both cultured and peritoneal mast
cells were relatively inefficient at populating the skin,
however, possibly due to the presence of mast cells, or
their precursors, which were already present in the skin
(Kitamura et al., 1977). An interesting note to the Nakano
studies was the evolution of mucosal or serosal mast cell
characteristics from injected cells (regardless of origin)
depending on the anatomical site of subsequent lodging.
Thus, the granules of mast cells isolated from the
peritoneal cavity, spleen, skin, and gastric muscularis
propria of reconstituted animals stained preferentially
with safranin (which has an affinity for highly sulfated
mucopolysaccharides like heparin) and the fluorescent dve
berberine sulfate (which also binds to heparin), and were
ultrastructurally homogeneous in size and electron density,
while mast cells identified in the glandular stomach mucosa
stained preferentially with alcian blue and were unstained

59
by berberine sulfate. Mast cell phenotype, therefore, may
be functionally regulated at the level of the tissue
microenvironment in which a multipotent mast cell precursor
or intermediate develops.
Epilogue
Despite an apparent wealth of literature available on
the subject, the potential still exists for scholarly,
significant contributions to the body of knowledge which
describes mast cells. The lineage relationship between the
mast cells found ini vivo (mucosal and serosal) is still
poorly defined, ond only recently have preliminary studies
approached the relationship between the aforementioned
cells and their putative correlate, the culture-derived
mast cell. Little is known of the phenotype of the cells
which give rise to mast cells in culture, and the ontogeny
of the mast cell in early embryonic tissues is documented
in scant and unsystematic reports.
In the course of the remaining chapters of this
dissertation, we describe our recent contributions to the
study of the mast cell. Beginning with the observation of
cell lines with basophilic granules, we have characterized
Abelson murine leukemia virus-transformed mast cell-like
lines of midgestational, embryonic origin using panels of
monoclonal antibodies as well as biochemical and molecular
biological techniques (Chapter II). Although mast cells
were not detected in homologous, uninfected tissues,

60
culture-derived mast cells could be propagated from
embryonic sources in the presence of exogenously supplied
interleukin 3. These studies (and parallel experiments on
adult bone marrow-derived mast cells) also provide the
first detailed analysis of hematopoietic marker expression
of cultures progressing from heterogeneous to homogeneous
populations of mast cells (Chapter III). We have
subsequently analyzed the frequency of mast cell precursors
in embryonic placenta, nonplacental embryonic tissues, and
adult tissues, demonstrating the earliest reported mast
cell precursors as well as a heretofore unreported rich
source of such cells, the placenta (Chapter IV). In the
same chapter, we have characterized the cell surface of
mast cells grown in semisolid agar media and have presented
encouraging preliminary results of experiments designed to
sort mast cell precursors on the oasis of differentiation
antigen expression.

CHAPTER II
ABELSON MURINE LEUKEMIA VIRUS-INFECTED CELLS FROM
MIDGESTATION PLACENTA EXHIBIT MAST CELL AND LYMPHOID
CHARACTERISTICS
Introduction
Abelson murine leukemia virus (A-MuLV) is a
replication-defective transforming retrovirus which was
isolated from a tumor in a steroid-treated BALB/c mouse
inoculated with the Moloney murine leukemia virus (Abelson
and Rabstein, 1970). Molecular analysis of the A-MuLV
genome has revealed that the virus arose by recombination
between the thymotropic Moloney virus genome and a cellular
gene termed c-abl (Goff et al., 1980; Shields et al.,
1979). The recombinant virus can infect and immortalize
hematopoietic cells jni vivo and ini vitro, and can transform
certain fibroblast cell lines in vitro (Scher and Siegler,
1975). The virus has demonstrated the ability to transform
in vivo mature cells of the B lineage (Potter et al., 1973;
Premkumar et al., 1975) as well as those of the T (Cook,
1982), myelomonocytic (Raschke et al., 1978; Ralph et al.,
1976), and mast cell (Risser et al., 1978; Mendoza and
Metzger, 1976) lineages. In contrast, in vitro infection
of adult or embryonic primary hematopoietic tissues
followed by clonal selection in semisolid media results in
the immortalization of cells exhibiting characteristics of
the earliest stages of B lymphoid differentiation
(Rosenberg et al., 1975; Rosenberg and Baltimore, 1976b;
61

62
Boss et al., 1979; Siden et al., 1979; Alt et al., 1981).
Abelson virus can also induce agar colony-forming cells
which express erythroid characteristics (Waneck and
Rosenberg, 1981); the latter cells, however, fail to
proliferate as permanent cell lines in liquid culture.
Based upon Abelson virus's propensity to immortalize B
lineage precursors jji vitro, experiments were designed to
study early embryonic lymphoid precursors. The
midgestation embryonic placenta has been reported to be the
earliest source of B cell precursors (Melchers and
Abramczuk, 1980; Melchers, 1979) in the mouse. The
successsful development of permanent cell lines from
midgestation embryonic placenta transformed iua vitro by A-
MuLV has recently been reported (Siegel et al., 1985).
Primary agar colony counts indicated that the frequency of
A-MuLV targets is highest at ten days of gestation. Unlike
previously reported A-MuLV-transformed embryonic cell
lines, the genomes of the placental cells contain a
germline immunoglobulin heavy chain locus characteristic of
nonlvmphoid cells and perhaps very immature lymphoid cell
precursors.
We proceeded to analyze this novel group of A-MuLV
embryonic transformants to better ascribe them to cells of
a particular lineage. This chapter summarizes our efforts
to characterize the A-MuLV transformants derived from
midgestation embryonic tissues and presents several

63
significant observations. First, unlike previously
reported Abelson virus-transformed embryonic cells, the
placental cell lines isolated in our laboratory exhibit
many characteristics of culture-derived and mucosal mast
cells including differentiation antigens, high affinity
receptors for IgE, and metachromatic granules containing
histamine and sulfated proteoglycans. Second, we describe
the development of a simple, sensitive, nonisotopic,
nonfluorometric method to detect membrane receptors for
immunoglobulins. Third, all of the cell lines analyzed had
at least one, and sometimes more than one integrated A-MuLV
provirus. Finally, the cell lines proliferated independent
of exogenous mast cell growth factor, and no growth factor
could be detected in either cell lysates or the
supernatants of exponentially growing cultures maintained
at high cell density.
Materials and Methods
Cell Lines
Cloned A-MuLV-transormed embryonic cell lines were
established in our laboratory as previously reported
(Siegel et al., 1985). All had been adapted to growth in
liquid culture and had been successfully maintained in 10P
(RPMI 1640 (GIBCO, Grand Island, NY) with 5x10^ M 2-

64
mercaptoethanol (Sigma Chemical Co., St. Louis, MO)) and 10
percent heat-inactivated fetal bovine serum (Sterile
Systems, Logan, UT)) for at least two years with twice
weekly passages prior to analysis. The nomenclature used
to designate each cell line included the number of days of
gestation (detection of vaginal plug on day 0) followed by
a suffix ("P" for placenta of (BALB/cAN x BIO.BR/SgSn)Fl
origin, "PC" for placenta of (BALB/cAN x CBA/J) origin, and
a clone number. Cell line 10P12, for example, was the
twelfth clone isolated from 10 day placental cells derived
from matings of BALB/cAN females and B10.BR males.
Mouse tumor cell lines used as experimental controls in
the studies were chosen to represent the major
hematopoietic lineages:
1. B 1ymphoid
a. FLEI-4, an pre-B cell line derived by E.J.
Siden by infection of day 15 BALB/c fetal
liver with A-MuLV strain P120;
b. 18-81 (Siden et al., 1979), a pre-B cell line
induced by infection of bone marrow cells with
A-MuLV strain P120;
2. T lymphoid
a. RLd 11, a radiation-induced leukemia
obtained from N. Rosenberg;
b. B2-4-4, a Moloney virus-induced leukemia
obtained from N. Rosenberg;

65
c. YAC-1, a Moloney leukemia virus-induced
lymphoma obtained from R. Weiner (University
of Florida);
3. Monocyte-macrophage
a. WEHI-3, a myelomonocytic leukemia obtained
from M. Norcross (University of Florida);
b. P388D1, a methylcholanthrene-induced monocytic
tumor obtained from American Type Culture
Collection (Rockville, MD);
4. Basophil-mast cell
a. P815, a methylcholanthrene-induced mastocytoma
obtained from S. Noga (University of Florida);
b. CB6ABMC4, an A-MuLV-induced mastocytoma
obtained from M. Potter (National Cancer
Institute, Bethesda, MD);
c. BALABMC20, an A-MuLV-induced mastocytoma
obtained from M. Potter.
All of the above cell lines were maintained in 10P with
twice-weekly passage.
Cell line DA-1, which was used in the analysis of
interleukin 3 production, was generously supplied by J.N.
Ihle (National Cancer Institute, Frederick, MD) and was
maintained in a one-to-one mixture of WEHI-3 conditioned
media and enriched media (EM; Razin et al., 1984a).

66
Analysis of Histamine Content
Histamine content was determined by a modification of
the enzymatic-isotopic microassay of Taylor et al. (1980).
Histamine methyltransferase (HMT) reagent was prepared from
BALB/cAN mouse brain which was homogenized in iced 5mM
sodium phosphate buffer, pH7.9 (10 ml per gram of brain)
with an iced, sintered glass homogenizer. The crude
homogenate was transferred to an Oak Ridge type tube and
cleared centrifugally at 50,000xg for ten minutes at 4C.
Histamine standard solutions and tumor cell lysates were
prepared in PIPES-BSA which contained 25mM PIPES, pH 7.4
(Sigma Chemical Co., St. Louis, MO), 0.4 mM magnesium
chloride, 5 mM potassium chloride, 120 mM sodium chloride,
1 mM calcium chloride, 5.6 nm glucose (Fisher Scientific
Co., Fairlawn, NJ), 0.1 percent BSA (Sigma); standard
solutions and cells were boiled for ten minutes and cleared
for five minutes in a microfuge (Brinkmann Instruments,
Westbury, NY). Immediately before starting the reaction,
HMT-SAMe was formulated: 500 microliters of HMT reagent
was mixed with 10 microliters (5 microcuries) S-[methyl-
^H]-adenosylmethionine (76.1 Ci per mMol; New England
Nuclear, Boston, MA) and 5 microliters unlabeled S-
adenosylmethionine (SAMe, Sigma Chemical Co.; 16 micrograms
per ml). Each reaction mixture consisted of 10 microliters
of HMT-SAMe and 20 microliters of histamine standard
solution or tumor cell lysate. Following incubation for

67
two hours at 37C, each reaction was stopped with 0.1 ml of
1 M sodium hydroxide and saturated with solid sodium
chloride. The contents of each tube were extracted with
0.2.5 ml chloroform in the presence of 10 microliters of
unlabeled SAMe (16 micrograms per ml)and the phases were
separated by brief centrifugation. The aqueous phase was
carefully removed with a pasteur pipet and the organic
phase was re-extracted with an equal volume of 1 M sodium
hydroxide and unlabeled SAMe. Aliquots (150 microliters)
of the organic phase were added to toluene-based
scintillation fluid (10 ml) and counted on a Beckman liquid
scintillation counter (Beckman Instruments, Palo Alto, CA).
All samples and standards were prepared in duplicate or
triplicate. Histamine content was determined by comparison
of tritiated counts in dilutions of tumor lysates to those
in histamine standard solutions and were expressed as
nanograms (ng) per 10^ cells.
Cytological Staining
Staining procedures were performed on air dried
cytocentrifuge (Shandon Scientific Company, Ltd., London,
U.K.) preparations on 25x75 mm precleaned glass slides.
Slides were fixed in methanol for one to two minutes prior
to staining with either Wright's Giemsa (Schalm et al.,
1975) or May-Gruenwald Giemsa (Thompson and Hunt, 1966).
Metachromatic cell granules were identified by staining for
five minutes with 0.1% w/v toluidine blue in 30% v/v

68
ethanol, pH 0.5 after fixation for two minutes in Mota's
fixative (lead subacetate in acidic ethanol)(Yam et al.,
1971). All stained slides were coverslipped with Permount
(Fisher Scientific Co.) prior to observation by light
microscopy.
Antibodies
Lineage-specific determinants on the surfaces of
placental and control cell lines were probed with a panel
of monoclonal (mAb) and polyvalent antibodies whose
significant features are summarized in Table II-l. All
monoclonal antibody preparations, unless otherwise noted,
were used as filtered hybridoma supernatants from
stationary phase cultures grown (in our facility) in
Dulbecco's Modified Eagle's Minimal Essential Media (GIBCO)
with 10 percent heat-inactivated fetal bovine serum and
5x10~5 m 2-mercaptoethanol. Preliminary work with mAb
B23.1 was performed with partially purified antibodies
generously supplied by P. LeBlanc (University of Florida);
later studies were performed with filtered hybridoma
supernatants which had been grown in RPMI 1640 with 10
percent fetal bovine serum, generously supplied by G. Place
in the laboratory of S. Russell (University of Florida).
Monoclonal antibody FT-1 was generously supplied by M.
Kasai (National Institute of Health, Tokyo, Japan); the
nature of its production and processing have been
previously reported (Kasai et ai., 1983).

69
Table II-l.
Designation
(isotype)
14.8
(IgG2b)
RA3-2C2
(IgM)
RA3-3A1
(IgM)
M6
(IgM)
anti-Asialo
(polyclonal)
T24/31.7
(igG)
5H1
(IgM)
B23.1
(IgM)
Lineage-Specific Antibodies Used in Surface
Marker Analysis
Specificity (Literature Citation)
200 Kd Ly5 antigen on lymphoid cells from spleen,
lymph node, and bone marrow (Kincade et al., 1981b).
Ly5 antigen on lymphoid cells from spleen, bone
marrow, lymph node, and plasma cells but not on
thymocytes or CFU-S (Coffman and Weissman, 1981a).
220 Kd Ly5 variant (B220) on B lineage cells on
spleen, lymph node, and bone marrow, but not on
thymocytes (Coffman and Weissman, 1981b).
Probably recognizes Dolichus biflorus agglutinin
receptor on early fetal thymocytes and on some
thymic leukemia cells (Kasai et al., 1983).
GM1 Neutral glycolipid asialo GM1 on early fetal
thymocytes, some fetal liver cells, and few adult
bone marrow, spleen, and lymph node cells as well
as some thymic leukemia cells and natural killer
cells. Not on adult and embryonic, Thy-1 positive
thymocytes (Habu et al., 1980; Kasai et al., 1980;
Young et al., 1980).
Thy 1 glycoprotein on thymocytes and T cells from
spleen but not on bone marrow prothymocytes
(Dennert et al., 1980).
Abelson transforming antigen on bone marrow targets
of A-MuLV, on most thymocytes, some bone marrow and
spleen cells, fetal liver erythrocytes and pre-B
cells, and bone marrow pre-B cells, but not on lymph
node cells, CFU-S or stem cells committed to myeloid
lineages (Shinefeld et al., 1980).
Antigen on resident and elicited macrophages, adherent
cultured bone marrow cells and macrophage-like cell
lines as well as on culture-derived mast cells but not
on resident peritoneal mast cells (Katz et al., 1983;
Leblanc et al., 1982).

70
Rabbit anti-asiaio GM1 was received from W.W. Young
(University of Virginia Medical Center) as a delipidated
serum. Rabbit anti-rat immunoglobulins was purchased as a
lyophilized powder (IgG fraction of rabbit anti-rat IgG
(heavy and light chains), Miles Laboratories, Elkhardt, IN)
and reconstituted per manufacturer's specifications.
Rabbit anti-mouse immunoglobulins was purchased from
Gateway Biologicals (St. Louis, MO).
Normal rat serum was prepared from cardiac blood of an
unimmunized animal. Normal mouse serum was prepared from
pooled specimens of multiple unimmunized BALB/cAN mice.
Cell Surface Markers
Cell surface differentiation antigens were detected by
a modification of the method of Uchanska-Ziegler and
colleagues (1982). Formalin-treated, heat killed
Staphylococcus aureus (S.aureus) Cowan I (The Enzyme
Center, Inc.,Boston, MA, and later a generous gift of Dr.
Michael D.P. Boyle) were first coated with rabbit anti-rat
IgG (RAMIgG, Miles Laboratories, Elkhart, IN) and then with
rat monoclonal antibodies specific for mouse
differentiation markers. Either hybridoma cell culture
media or partially purified immunoglobulins were used as a
source of the latter antibodies.
Mouse cells (1x10^ or fewer) selected for analysis
were pelleted in round-bottom, PVC microtiter wells
(Dynatech, Alexandria, VA) and resuspended in a small

71
volume (5 microliters) of S.aureus-RAMIgG-monoclonal
antibody sandwiches. Following a thirty-minute incubation
on ice, the contents of each well were washed six times and
a sample was prepared as a cytocentrifuge mount. Slides
were stained with May-Gruenwald Giemsa or Wright's Giemsa.
Fc Receptor Assays
Receptors for the Fc domain of mouse igE and IgG were
detected by a novel modification of the S. aureus method
used to detect other surface antigens (Siden and Siegel,
1986). Indicator bacteria were prepared by incubating 25
microliters of packed S. aureus or Escherichia col i (E.
coli) with 0.175 ml of 2,4,6-trinitrobenzene sulfonic acid
(3.7 mg per ml in 0.28M cacodylate buffer, pH6.9) for 10
minutes at room temperature. The 1.5 ml microfuge tube
containing the reactants was wrapped in foil to retard
photodecomposition and taped to a rotator during
incubation. After four washes with 0.01 M phosphate-
buffered 0.15 M sodium chloride, pH 7.3 (PBS; Mishell and
Shiigi, 1980), the TNP-S. aureus or TNP-E. coli were
resuspended to their orginal volume in balanced salt
solution (BSS; Mishell and Shiigi, 1980) with 10 mM HEPES
(pH 7.35), 0.1% w/v sodium azide, 5x10^ M 2-
mercaptoethanol (Sigma Chemical Co.) and 1% v/v fetal
bovine serum (H10BNF1).
Hybridoma supernatants containing mouse IgE anti-
(DNP)2 (IgELa2, American Type Culture Collection) and

72
partially purified mouse IgG anti-TNP (generous gift of M.
Rittenberg, Oregon Health Center University) were cleared
by centrifugation at 12,000xg for 15 minutes at 4C. The
latter was diluted in Dulbecco's modified Eagle's minimal
essential medium (GIBCO) with 10% v/v heat-inactivated
fetal bovine serum, and both contained 0.1% w/v sodium
azide. Cells to be analyzed were suspended in H10BNF1 at
1x10^ per ml, aliquoted at 0.1 ml per well of a 96-well PVC
cluster (Dynatech, Inc., Alexandria, VA) and pelleted by
centrifugation (2 minutes, lOOxg, 4C). The cluster was
"flicked" and briefly vortexed (five to ten momentary
touches) and the cells were resuspended in 0.1 ml of
cleared IgE or IgG anti-TNP. Clusters were covered with
plastic wrap and placed in a 37C, 5% carbon dioxide
incubator for one hour. Following incubation, the treated
cells were washed twice with H10BNF1 by centrifugation.
The cell pellets were dispersed by vortexing and 5
microliters of 10% w/v TNP-S_^ aureus or TNP-E. col i were
added to each well. The covered microtiter clusters were
placed on ice for thirty minutes and the contents of the
wells were washed six times as previously described. The
pellets were resuspended in 0.1 ml of H10BNF1. Ten to
thirty microliter samples of each well were
cytocentrifuged, stained with May-Gruenwald Giemsa and
observed by bright field microscopy.

73
Mice
BALB/cAn mice used in these studies were bred in our
colony. B10.BR/SgSn and CBA/J mice were purchased from The
Jackson Laboratory (Bar Harbor, ME). For studies involving
timed pregnancies, females were placed in the cages housing
one or two males at ratios of one to three females per
maLe. Females were observed for vaginal plugs following
overnight cohabitation and the date of detection was noted
as day zero of gestation.
Analysis of A-MuLv Proviral Integration
High molecular weight DNA was extracted from tumor
cells by the method of Steffan et al. (1979). Tumor cells
were harvested from liquid culture by centrifugation and
washed in cold PBS. The cells were resuspended at ten
million per milliliter in TES (10 mM Tris, 5 mM EDTA, 100 mM
sodium chloride, pH 7.5) and added dropwise to an equal
volume of lysis buffer consisting of TES with one percent
w/v SDS (Sigma Chemical Co.) and 0.04 percent Proteinase K
(Fisher Scientific Co.). The lysates were digested
overnight at 37C with gentle mixing. DNA was extracted
from the lysate, twice for thirty minutes with an equal
volume of glass-distilled phenol and twice again with
chloroform containing 4 percent isoamyl alcohol (Fisher
Scientific Co.). The extracted DNA was precipitated in 2.5
volumes of 95 percent ethanol or isopropanol, dissolved in
T10E1 buffer (10 mM Tris, 1 mM EDTA, pH 7.5), and dialyzed

extensively against the same buffer. Normal embryonic
tissue, prepared in an identical manner, provided control
DNA. All DNA was quantitated spectrophotometrically by the
OD260/OD28O methd (Maniatis et al., 1982).
Ten micrograms of DNA were incubated with 10 units of
restriction endonuclease BAM HI (New England Biolabs,
Beverly, MA) in TA buffer (O'Farrell et al., 1980);
completeness of digestion was monitored by the addition of
one microgram of bacteriophage lambda DNA to a duplicate
sample. The digested DNA was mixed with sample buffer (50 mM
Tris, pH 7.5, 5 mM EDTA, 25% w/v Ficoll, 0.05% w/v
bromophenol blue, at 5X concentration) and electrophoresed
for eighteen hours at 40 volts D.C. on an 0.8% agarose gel
in TEA buffer (40 mM Tris-acetate, 2 mM EDTA, pH 7.8).
Both the gel and the electrophoresis buffer contained 0.5
micrograms of ethidium bromide per milliliter. Samples
were organized such that a set of digested cellular DNAs
were pipetted into wells on one side of the gel and
duplicates containing bacteriophage lambda DNA were
pipetted into wells on the other side of the same gel.
Following electrophoresis, the gel was photographed
under ultraviolet light to visualize the restricted DNA and
verify that all of the lambda DNA had been digested to
completion. A ruler placed alongside the gel was
photographed at the same time to provide a scale of DNA
fragment sizes for later reference. The lanes containing

75
lambda DNA were generally cut away and discarded. The gel
was then exposed to shortwave ultraviolet light for an
additional ten minutes to break the DNA and thereby
facilitate transfer. DNA in the gel was denatured with 0.5
M sodium hydroxide, 0.6 M sodium chloride for one hour,
neutralized in 1 M Tris, pH 7.4, 1.5 M sodium chloride (two
changes of 150 to 200 milliliters for 30 to 45 minutes
each), and transferred to nitrocellulose (Schleicher and
Scheull, Keene, NH) by the method of Southern (1975).
Probes for A-MuLV-related sequences were prepared from
the virus-specific recombinant plasmid pAB3Sub3 (Goff et
al., 1980) by nick translation (Rigby et al., 1977) to a
specific activity of 10 dpm per microgram. The 2p_
labeled sequences were hybridized to the nitrocellulose-
immobilized DNA for twenty hours at 68C by the method of
Wahl (1979). The nitrocellulose blot was then washed
extensively under stringent conditions (0.015 M sodium
chloride, 0.0015 M sodium citrate at 68C). The probed
blots were autoradiographed on XAR-5 film (Eastman-Kodak,
Rochester, NY) with two calcium-tungstate-phosphor
intensifying screens (Cronex Lightning Plus, E.I.DuPont de
Nemours and Co., Wilmington, DE) for two to five days.
Conditioned Media
Embryonic tumor cells were cultured under conditions
similar to those used to generate WEHI-3 conditioned media
(W3CM) (Razin et al., 1984a). Cells from log phase

76
cultures were seeded at 1x10^ per ml in 10P in bacterial-
grade petri dishes (Fisher Scientific Co.). Following four
days (92 to 100 hours) of incubation in humidified five
percent carbon dioxide, the conditioned media were
harvested by centrifugation and filtered through tissue
culture grade 0.2 micron nitrocellulose filters.
Conditioned media were concentrated by stirred-cell
ultrafiltration (Amicon, Danvers, MA), by dialysis against
polyethylene glycol 20,000 (Fisher Scientific), or by
ammonium sulfate (Fisher Scientific Co.) precipitation when
such procedures were desired.
Cell lysates were prepared from cells grown to 7 to
9x10^ per ml as follows: Cell cultures were centrifuged
(ten minutes at 200xg, 4) and the cells were washed twice
in PBS. The cells were resuspended in one milliliter 10P
and subjected to three rounds of alternate freezing (in dry
ice-ethanol) and thawing (at 37C). The lysates, which
contained no viable cells upon microscopic examination,
were cleared centrifuga 1ly at 2000xg for fifteen minutes
(4C) and at 12,000xg for fifteen minutes (4C), and were
filtered through 0.45 micron sterile, disposable filters
(Gelman Sciences, Ann Arbor, MI). Lysates were stored at
-20C prior to use.
Assay for Interleukin 3
Interleukin 3-like activity was analyzed by a
modification of the method of Razin and colleagues (1984a).

77
The proliferation of cell line DA-1 (generously provided by
J.N. Ihle), which requires interleukin 3 for growth, served
as an assay for interleukin 3. DA-1 cells were
centrifuged, washed twice in PBS, and resuspended at 5x10^
per ml in appropriate condititioned media or cell lysates.
One-tenth milliliter aliquots were pipeted in triplicate
into separate compartments of a 96-well microtiter cluster
(Linbro, Flow Laboratories, McLean, VA). The cells were
incubated (37C, five percent carbon dioxide) for sixteen
hours, at which time each well was pulsed with one
microcurie of ^H-TdR (5 Ci per mMol, Amersham Corporation,
Arlington Heights, IL) in ten microliters of EM. Following
six hours additional incubation, the cells were collected
on Whatman glass microfiber filter strips (Whatman Paper
Ltd., Maidstone, U.K.) in water by multiple automated
sample harvester (MASH, Otto Hiller Company, Madison, WI).
The filter strips were air dried. ^H-TdR which was
incorporated into filter-immobilized DNA was counted in
toluene-based scintillation fluid in a liquid scintillation
spectrometer.
Results
Histamine Content of Transformed Placental Cells
Initial light microscopiic examination of some of the
transformed cell lines revealed large mononuclear cells

78
with basophilic granules which stained metachromatically
with acidic toluidine blue (H.R. Katz, personal
communication). Since the cells fit the working definition
of mast cells by these criteria, we began our
characterizations by assaying for intracellular histamine.
All but two of the placental cell lines analyzed contained
histamine as detected by a sensitive, isotopic-enzymatic
microassay (Table II-2). The quantity of histamine detected
in the cells (5 to greater than 500 nanograms per million
cells) was similar to that found in cultured mast cells
derived from mouse spleen (450 to 500 nanograms per million
cells; Razin et al., 1981a), bone marrow (80 to 150
nanograms per million cells; Razin et al., 1982a), and
fetal liver (200 to 1400 nanograms per million cells; Nabel
et al., 1981), and mucosal mast cells (160 to 2000
nanograms per million cells; Befus et al., 1982b;
Bienenstock et al., 1982), but one order of magnitude less
than that found in serosal mast cells (15 micrograms per
million ceils; Bienenstock et al., 1982). Histamine was
not detected in lymphoid or myelomonocytic cell controls,
nor was it detected in the mastocytoma P815, which
reportedly has variants which are devoid of mast cell
granules (Mori et al., 1979). Histamine biosynthesis was
confirmed by chromatographic identification (Galli et al.,
1976) of [^H]-histamine in extracts of f^H]-histidine-
labeled cells (data not shown).

79
Table II-2. Histamine Content of Embryonic
and Control Tumor Cell Lines
Tumor Cell Lines
CELL LINES
HISTAMINE
(ng/lO^ cells)3
PLACENTAL CELL LINES
9P1
>5.00
10P2
2.70
10P6
<0.05
10P8
0.05
10P12
0.06
11P0-1
<0.05
11P62
1.60
CONTROL CELL LINES
WEHI-3
<0.05
RLdl 1
<0.05
18-81
<0.05
FLEI-4
<0.05
P815
<0.05
a: Histamine content was determined as described in
Materials and Methods.

80
Analysis of Hematopoietic Lineage Markers
In view of the morphological and biochemical
similarity of the placental cell lines to mucosal and
culture-derived mast cells, we sought to further confirm
the relationship by analysis of the cell surface
differentiation antigens with an antibody which
discriminated between serosal and culture-derived mast
cells (Katz et al., 1983). This and other cell surface
antigens were analyzed by a simple and sensitive aureus -
antibody sandwich method (Uchanska-Ziegler et al., 1982).
Figure I-1A shows the binding reaction of the cell line
10P12 with bacteria coated with anti-cultured mast cell
antibody B23.1, while Figure II-1B shows that the same cell
line, incubated with bacteria coated with unimmmunized rat
immunoglobulin, resulted in no bacteria bound to the mouse
cell surfaces.
Cell surfaces of the remaining A-MuLV placental
transformants and of a number of control tumor cell lines
were probed with monoclonal antibody B23.1 as well as a
panel of other monoclonal and conventional antibodies
reported to be specific for hematopoietic differentiation
markers (Table II-l). Table II-3 summarizes the results of
the cell surface analyses. Every virus-transformed
embryonic cell line expressed the B23.1 epitope, which is

81
Figure II-l. Detection of Cell Surface Determinants on Abelson
Murine Leukemia Virus-Transformed Embryonic Cells.
Killed Staphylococcus aureus bacteria coated with anti-mast
cell/monocyte B23.1 antibodies (A) and normal rat serum
antibodies (B) were reacted with cells from line 10P12 as
indicated in Materials and Methods.

Table II-3. Analysis of Lineage-Specific Surface Determinants on
A-MuLV-Transformed Embryonic and Control Tumor Cell Lines*
PRE-B MARKERS
¡ PRE-T
MARKERS ¡
T MARKER ¡
A-MuLV
TARGET
MAST CELL
¡ -MONOCYTE
MARKERS
CELL LINE
RA3-3A1
IgM
14.8 RA3-2C2
IgG2b IgM
M6
IgM
Anti-Asialo
GM1
Polyclonal
T24/31.7
IgG
5H1
IgM
B23.1
IgM
9P1 Ab-MuLV
PLACENTA (9D)
_
_
_
PLACENTAL
CELL LINES
+
_
_
+
10P2 Ab-MuLV
PLACENTA (10D)
-
-
-
-
-
-
-
+
10P6 Ab-MuLV
PLACENTA (10D)
-
-
-
-
++
-
-
+
10P8 Ab-MuLV
PLACENTA (10D)
-
-
-
-
-
-
-
++
10P12 Ab-MuLV
PLACENTA (10D)
+
+
++
-
-
-
-
++
11P0-1 Ab-MuLV
PLACENTA (1ID)
++
++
++
+
++
+
11P62 Ab-MuLV
PLACENTA (1ID)

Table II-3. Continued
WEHI-3
MYELOMONOCYTIC
LEUKEMIA
RLd 11
THYMOMA +
B2-4-4
THYMOMA +
YAC-1
THYMOMA ND ND
18-81
Pre-B + ++
FLEI-4
Pre-B ++ ++
P815
MASTOCYTOMA ++ +
CB6ABMC4
MASTOCYTOMA
CONTROL CELL LINES
++
+ ND ND
ND ++ ++
++ ND +
++ +
++
+ ++
++ +
++ ++
++ +
ND ND ND
++
++ £
++
ND +
BALABMC20
MASTOCYTOMA --++++ ND +
* Cell surface determinants were analyzed as described in Materials and Methods.
Scoring: indicates less than 5 bacteria per mouse cell
+ indicates 5 to 50 bacteria per mouse cell
++ indicates more than 50 bacteria per mouse cell
ND indicates reactivity not analyzed

also found on cells of the monocyte lineage (Leblanc et
al.. 1982) as well as on culture-derived mast cells. This
marker was also detected on control mastocytoma (P815) and
myelomonocytic leukemia cells (WEHI-3).
Two of the cell lines expressed three related
antigenic determinants of the 200 to 220 kilodalton surface
glycoprotein family which is found on mouse lymphoid cells.
The cell lines 11P0-1 and 10P12, as well as all of the
control lymphoid and adult-derived mastocytomas which we
examined, expressed the antigens defined by the monoclonal
antibodies 14.8 and RA3-2C2. These epitopes are expressed
on B cells and their surface immunoglobulin-negative
precursors. The progenitor cells react with the monoclonal
antibody RA3-3A1 as well. The latter antibody, however,
did not recognize a 220 kilodalton glycoprotein on tumor
cells of the T lineage which was detected by 14.8 and RA3-
2C2. The selective reactivity of RA3-3A1 has been
confirmed previously (Coffman and Weissman, 1981b). It is
interesting to note, however, the novel expression of this
previously characterized B cell differentiation antigen on
mastocytoma P815 as well as our embryo-derived mast cell
lines.
Placental and control tumor cell lines were also
analyzed for the expression of other primitive lymphoid
markers. The neutral glycolipid asialo-GMl, which is
expressed on the early embryonic, Thy-1-negative thymocytes

85
(Habu et al., 1980) and for the expression of receptors for
the Dolichos biflorus agglutinin, which is expressed on
early fetal thymocytes and on some thymic leukemia cells
(Muramatsu et al., 1980). The monoclonal antibody M6
(Kasai et al., 1983) reportedly recognizes cells bearing
receptors for Dolichos biflorus agglutinin and probably
binds to the receptor itself. Table II-3 shows that only
one of the embryonic cell lines, 11P0-1, expressed the
early thymocyte differentiation marker recognized by anti-
FT-1. The same cell line also bears surface asialo-GMl, as
do two other embryonic cell lines, 9P1 and 10P6. It is
also interesting to note that two A-MuLV-induced adult
mouse mastocytomas, CB6ABMC4 and BALABMC20 were both
positive for FT-1, and that the latter cell line also
expressed the neutral glycolipid asialo-GMl. These
observations will be discussed later in this chapter.
Despite the presence of markers specific for early T
lineage cells on several embryonic cell lines, mature T
lineage markers were only detected on three control cell
lines. Lack of reactivity with monoclonal antibody
T24/31.7 (Dennert et al., 1980) indicated that none of the
cell lines expressed the Thy-1 differentiation marker.
Also conspicuously absent from A-MuLV-transformed embryonic
cell lines was the antigen recognized by the monoclonal
antibody 5H1, which is expressed on Abelson murine leukemia
virus transformation-sensitive targets from mouse bone

86
marrow as well as on thymocytes, fetal liver red blood
cells, and pre-B cells (Shinefeld et al., 1980; E. Siden
and L. Shinefeld, unpublished observations).
Analysis of Receptors for IgE and IgG
High affinity membrane receptors for the Fc regions of
immunoglobulins were detected with a sensitive, isotype-
specific assay developed in this laboratory (Siden and
Siegel, 1986). Coated bacteria were prepared by
derivatizing the bacterial surfaces with the hapten
trinitrophenol (TNP) and then reacting the modified
bacteria with mouse monoclonal anti-TNP antibodies of the
IgE or IgG classes. Experimental and control cell lines
were incubated with the immunoglobulin-coated bacteria at
37C for one hour and processed for cytocentrifugation and
staining. Typical positive and negative reactions are
illustrated in Figure II-2. Observations are summarized in
Table II-4. IgE receptors were detected on all but two of
the embryonic cell lines analyzed, while none of the cell
lines expressed high affinity receptors for IgG. Receptors
for IgG were detected, however, on the myelomonocytic
leukemia cell line WEHI-3 (Table II-4) and on the
macrophage-like tumor cell line P388D1 (data not shown).
The results of the bacterial assay for IgG receptors were
duplicated by the method of Schrader (1981), which used
xenogeneic (rabbit) 7S antibodies immobilized on sheep red

87
B
Figure. II-2. Detection of Surface Receptors for IgE on A-
MuLV-Transformed Embryonic Cells.
Killed Staphylococcus aureus bacteria haptenated with
trinitrophenol (TNP) were incubated with cells from line
10P8 which had previously been incubated with monoclonal
mouse IgE (A) and IgG (B) as described in Materials and Methods.

88
Table II-4. Analysis of Surface Membrane Receptors for IgE
and IgG on A-MuLV-Transformed Embryonic
Cell Lines and on Control Tumor Cell Lines
CELL LINE MEMBRANE RECEPTORS FOR
Mouse IgEa Mouse IgGa Rabbit IgG*3
PLACENTAL
9P1 +
10P2 +
1P6
10P8 +
10P12 +
11P0-1
11P62-4 +
CONTROL
WEHI-3 + +
RL 11 - NDC
18-81
FLEI-4 - ND
P815 - ND
CB6ABMC4 ND
BALABMC20 + ND
a: Membrane receptors for allogeneic (mouse) IgE and IgG were
detected by the TNP-bacteria/anti-TNP method (see Materials
and Methods).
b: Membrane receptors for xenogeneic (rabbit) IgG were detected
by the rabbit anti-sheep RBC/SRBC method of Schrader (1981).
c: ND indicates analysis not performed.

89
blood cells. Rosettes of sheep red blood cells on IgG Fc
receptor-positive cells were detected by phase microscopy
after staining with crystal violet.
Metachromatic Granules in Transformed Cell Lines
Initial examination of several of the embryonic cell
lines, which provided the first evidence for their
relationship to mast cells, was followed by a more detailed
study of the remaining cells. Cells were fixed in Mota's
lead subacetate and stained in acidic toluidine blue to
detect granules rich in basophilic glycosaminoglycan. As
seen in Table II-5, the majority of the original (BALB/c x
B10.BR)F1 embryonic cell lines did not possess
metachromatic granules, although two cell lines with high
histamine content (9P1 and 11P62) did express the
characteristic. Additionally, four of six embryonic cell
lines derived from a different paternal background, and all
long-term culture-derived mast cells (see Chapter III),
stained metachromatically with toluidine blue. Many of the
A-MuLV-transformed embryonic cell lines synthesize and
secrete chondroitin-4,6-disuifate proteoglycan (D. Levitt,
R. Porter, and E. Siden, manuscript in preparation), in
support of our observations.
Analysis of A-MuLV Provirus Integration
Although the embryonic tumor cell lines were derived
from cells infected with Abelson murine Leukemia virus, we
sought to confirm the presence of A-MuLV-specific sequences

90
Table II-5. Metachromatic Granules in A-MuLV-Transformed
Embryonic Cell Lines and Control Tumor Cell Lines.
Metachromatic
Cell Line Granules3
placental
9P1 +
10P2
10P6
10P8
10P12
11P0-1
11P62 +
10PC1 +
11PC14 +
11PC19
11PC20
11PC32 +
12PC1 +
control
WEHI-3
18-81
P815
CB6ABMC4
BALABMC20 +
a: Cytocentrifuged smears of cells were stained with
toluidine blue as indicated in Materials and Methods.
Cells with metachromatic granules (+) and without
metachromatic granules (-) were scored.

91
in cellular DNA by identifying the proviral genome. DNA
isolated from four of the embryonic cell lines was probed
with the Abelson virus-specific recombinant plasmid
pAB3Sub3. The cellular DNA was first digested with
endonuclease BamHI, which recognizes no restriction sites
within the virus-specific sequences of the plasmid. Thus,
each integrated A-MuLV provirus detected was represented by
a single band on the autoradiographed blot. All but one of
the cell lines so analyzed showed two copies of the A-MuLV
provirus integrated into high molecular weight DNA (Figure
II-3); cell line 11P62 showed only one copy. In addition to
integrated viral sequences, the probe also detected two
germ line fragments of the endogenous c-abl gene, which
contains cellular sequences and BamHI sites not present in
the plasmid. The v-abl and c-abl patterns of the control
cell line 160N54, from which the infecting virus was
isolated, are also shown.
Two of the embryonic cell lines, 10P12 and 11P0-1,
were injected into mice to determine whether the cells were
tumorigenic. Both cell lines caused tumors in syngeneic
mice inoculated within two weeks of birth. DNA was
prepared from tumors isolated from the mice (output DNA).
Restriction analysis of the input (original cell line) and
output DNA was performed to determine whether the lesions
were due to cell line proliferation or to subsequent
infection of host cells by shed virus. Tumor cells derived

92
ABODE
r
Figure II-3. Virus-Transformed Cells Contain Abelson Murine
Leukemia Virus-Specific DNA Sequences.
DNA isolated from embryonic cell lines was restricted,
electrophoresed, blotted, and probed with v-abl recombinant
plasmid pAB3Sub3, and the filter was autoradiographed as
described in Materials and Methods. Lanes contain DNA from
control cell line 160N54 (A), 10P8 (B), 10P12 (C), 11P0-1
(D), 11P62 (E). Arrows mark c-abl-containing fragments.

93
from the input cells would have the same sites of provirus
integration, while in vivo virus infection from cells
shedding virus would probably result in the observation of
different sites, due to the random nature of A-MuLV
integration. Both scenarios were observed (Figure II-4).
Cells recovered from lymph nodes of mice which were
injected with 11P0-1, a virus-producing cell line (E.
Siden, personal communication), had viral sequences
integrated into cellular restriction fragments distinct
from the input DNA pattern. The cell line 10P12, however,
which sheds no detectable virus (E. Siden, personal
communication), was reisolated from the liver and had A-
MuLV-specific sequences integrated into cellular
restriction fragments similar in size to the input pattern.
Analysis of Interleukin 3 Production by Embryonic Cell
Lines
The phenotypic similarity of the A-MuLV-transformed
embryonic cell lines and culture-derived mast cells was
sharply contrasted by the growth factor requirements of the
respective populations. The former cells required no
growth factors beyond those provided by medium 10P
(containing fetal bovine serum), while cultured mast cells
required an exogenous source of interleukin 3,
conventionally provided in WEHI-3-conditioned media. We
therefore sought to determine whether the cell lines
produced their own growth factor. Using a sensitive

>
Figure II-4. Tumors Isolated from Mice Injected with Cell Lines
10P12 and 11P0 Contain A-MuLV-Specific DNA Sequences.
DNA was isolated from embryonic cell lines and tumor tissue,
restricted with endonuclease Bam HI, blotted onto nitrocellulose,
and probed with nick-translated pAB3Sub3 as described in
Materials and Methods. Autoradiograph of probed blots is shown.
Lanes contain DNA from input cell line 11P0 (A), cultured lymph
node tumor cells from animals which were injected with 11P0 (B,C),
input cell line 10P12 (D), cultured liver tumor cells from
animals which were injected with 10P12 (E,F). Arrows mark
c-abl-containing fragments.

95
proliferation assay, we were unable to detect interleukin 3
in the supernatants or cell lysates of several of the
embyonic cell lines, even when the samples were
concentrated (Table II-6). Appropriate WEHI-3 conditioned
media and cell lysate controls, however, revealed the assay
was functional.
Discussion
Despite over one hundred years of systematic study,
little is known of the processes of differentiation and
maturation of the pharmacologically-active secretory cell
termed the mast cell (Ehrlich. 1877). In the broadest
sense, mast cells have been associated with a population of
mononuclear cells containing biogenic amines and sulfated
proteoglycans which are stored in cytoplasmic basophilic
granules (Selye, 1965; Metcalfe et al., 1981). The
histochemical signature of these cells is the anomalous,
metachromatic staining of the granules in the presence of
analine dyes (Ehrlich, 1879). Mast cells also have high
affinity membrane receptors for IgE, through which, by
specific immune reactions, the cells elaborate their
biogenic effectors (Austen, 1984).
During the course of the last twenty-five years, a
number of reports have established the existence of two

96
Table II-6. Interleukin 3 Content of Conditioned Media and Cell
Lysates of A-MuLV-Transformed Embryonic Cells and
Control Tumor Cellsa.
Sample
Stimulation
¡ Conditioned Media^
IL3
Standard
lb
Indexc
1.000+/
-0.000
i
i
! 50% W3CM
IL3
Standard
2
0.836
0.048
! lx W3CM (50% SAS ppt)
¡ lx W3CM (80% SAS ppt)
IL3
Standard
3
0.569
0.064
1.8x W3CM (Amicon ret)
¡ 0.9x W3CM (Amicon ret)
IL3
Standard
4
0.319
0.063
¡ lx W3CM (Amicon filtrate)
1
1
IL3
Standard
5
0.146
0.020
¡ 100% 10P12-2 CM
¡ 10% 10P12-2 CM
IL3
Standard
6
0.069
0.006
¡ 5.5x 10P12-2 CM (80% SAS ppt)
¡ 1.4x 10P12-2 CM (80% SAS ppt)
IL3
Standard
7
0.042
0.005
8.3x 10P12-2 CM (Amicon ret)
¡ lx 10P12-2 CM (Amicon ret)
IL3
Standard
8
0.017
0.004
! lx 10P12-2 CM (Amicon fit)
i
i
IL3
Standard
9
0.010
0.003
| 100% 11PC19 CM
¡ 50% 11PC19 CM
i
Blank
0.009
0.002
1
1
1

97
Table II-6. Extended
Stimulation
¡ Cell Lysatese
Stimulation
Index
1
1
1
Index
1.384
WEHI-3 (5.2xl05 C.E.)
0.496
0.061
(2.6xl05 C.E.)
0.321
0.588
(1.3xl05 C.E.)
0.174
1.252
¡ (0.6xl05 C.E.)
0.082
1.242
1
1
0.007
1
1
1
0.007
10P12-2 (7.3xl05 C.E.)
0.005
0.007
(3.6xl05 C.E.)
0.005
0.013
(1.8xl05 C.E.)
0.004
0.008
! (0.9x10s C.E.)
0.004
0.006
1
1
0.011
1
1
0.009
1
1
1
0.007
10P12-2 (7.3xl05 C.E.):
0.009
Standard 1 (1:1)
1
0.764
a. Interleukin 3 content was assayed by a proliferation assay
as described in Materials and Methods.
b. Homogeneous interleukin 3 standard was generously supplied by
Dr. J.N. Ihle as a concentrate in RPMI 1640 and used to his
specifications. Standard 1 was formulated by fifty-fold
dilution of the concentrate. Standards 2 through 9 were
made by serial two-fold dilutions from standard 1. All
dilutions were made in EM, which also served as the blank.
c. Stimulation Index was calculated from raw counts retained on
filters by the formula: Stimulation Index= Mean Sample cpm/
Mean Standard 1 cpm, where mean Standard 1 cpm was 46236
(standard deviation, 16129). Stimulation indices for standards
(expressed as means +/- 1 standard deviation) were compiled from
the results of five experiments of three replicates each, except
for Standard 7 which was compiled from three experiments of three
replicates. All other stimulation indices were compiled from a
minimum of three determinations.
d. Conditioned media were prepared as indicated in Materials and
Methods. Abbreviations: CM (conditioned media), SAS (saturated
ammonium sulfate), Amicon ret (retenate of 10 Kd cutoff Amicon
stirred cell filter), Amicon fit (filtrate of 10 Kd cutoff Amicon
filter).
e. Cell lysates were prepared as indicated in Materials and Methods
from the number of cells noted parenthetically (C.E. is cell
equivalents).

98
subsets of mast cells which have been contrasted on the
basis of morphology, size, thymus-dependent proliferation,
fixation and staining requirements, proteases, proteoglycan
composition, and histamine content and release (reviewed by
Jarrett and Haig, 1984; Shanahan et al., 1984; Katz et al.,
1985a). The original mast cells studied by Ehrlich and his
proteges have been termed connective tissue or serosal mast
cells, while those isolated initially from intestinal
mucosa were termed atypical or mucosal mast cells for their
aberrant fixation and staining properties (Enerback, 1966a,
1966b). The relationship of the two subsets is still
unclear.
A third subset of mast cells was observed with the
development of techniques to propagate hematopoietic cell
in vitro. The culture-derived mast cells are
phenotypically similar to mucosal mast cells by a number of
criteria including morphology, granule number, size, and
staining requirements, histamine content, and proteoglycan
composition. The development of in vitro mast cell
precursor culture (Schrader, 1981; Nakahata et al., 1982b;
Crapper and Schrader, 1983) has permitted the cultivation
of mast cells from a number of adult tissues as well as
from day thirteen fetal mouse liver.
Some of the cell lines resulting from the
transformation of embryonic placental cells bv Abelson
murine leukemia virus are morphologically and

99
histochemically similar to culture-derived mast cells.
Analysis of histamine content of the embryonic cell lines
indicated that most do contain that biogenic amine.
Furthermore, the quantity of histamine detected (5 to more
than 500 nanograms per million cells) were similar to those
reported for mucosal and jji vitro-derived mast cells. The
embryonic cell lines also synthesized chondroitin-4,6-
disulfate proteoglycan, but not heparin (D. Levitt, R.
Porter, and E. Siden, manuscript in preparation).
Chondroitin sulfate is a granule constituent of cultured
and mucosal mast cells, but not serosal mast cells, which
store heparin (Razin et al., 1984a). Based on these two
additional criteria, we have proposed that our embryonic
cell lines are analogous to culture-derived mast cells.
Our analysis of the surface determinants on embryonic
cell lines and control tumor cell lines has supported the
preliminary hypothesis of their lineage. Although we
observed heterogeneity of histamine content and expression
of high affinity receptors for IgE, every embryonic cell
line expressed the differentiation antigen recognized by
the monoclonal antibody B23.1, which is also expressed on
cultured mast cells from mouse bone marrow, spleen, and
blood (Katz et al., 1983). Two of the embryonic cell lines
described in this study (Table II-3), as well as two
embryonic cell lines of different genotype (unpublished
results), also express the Ly5 200 to 220 kiltodalton

100
suface glycoprotein (Omary et al., 1980), which has been
observed on cultured mast cells (Nabel et al., 1981) as
well as on mastocytomas (Scheid and Triglia, 1979). We
have also extended the scope of expression of Ly5 on mast
cell tumors to the methycholanthrene-induced P815 and to
two A-MuLV/pristane in vivo-induced mastocytomas.
Interestingly, the latter three mastocytomas and our Ly5-
positive cell lines express the epitope recognized by the
monoclonal antibody RA3-3A1, which was previously thought
to be B lineage specific (Coffman and Weissman, 1981a,
1981b). Mice homozygous for the mutant lpr gene, which
experience severe early onset autoimmune disease, exhibit
lymphoproliferation of a thymus-dependent Ly 5-positive,
cell population; the proliferating cells, however, lack
other B-lymphoid characteristics and appear to be of the T
lineage (Morse et al., 1982).
Three of the embryonic ceil lines also expressed early
thymocyte antigens. One of the cell lines, 11P0-1,
expressed the fetal thymocyte-specific epitope FT-1 (Kasai
et al., 1983) as well as the neutral giycolipid asialo-GMl,
while two other cell lines (9P1 and 10P6) expressed asialo-
GMl only. The latter differentiation antigen, which is
expressed on twelve to fifteen day fetal thymocytes (Habu
et al., 1980), has also been observed on natural killer
cells (Kasai et al., 1980; Young et al., 1980). However,
none of the embryonic cell lines exhibited natural killer

101
activity (M. Jadus, personal communication). The
expression of B lineage and T lineage markers, as seen on
11P0-1, may be characteristic of one stage of mast cell
differentiation. Alternatively, the expression of B, T,
monocyte, and cultured mast cell markers on the Abelson
virus-transformed embryonic cell lines may be analogous to
that of a yet undefined in vivo multipotent proliferative
stem cell which is present in the midgestation placenta.
The present studies have not further pursued this matter.
We have characterized cell lines, generated by
transformation of embryonic cells by Abelson murine
leukemia virus, which phenotypically resemble culture-
derived mast cells. However, analysis of nontransformed
cells from freshly disaggregated embryonic tissues
indicated that few, if any, mast cells are present in the
midgestational conceptus (Chapter III). Infection may
therefore have transformed the cells prior to stem cell
commitment to the mast cell lineage or before committed
mast cell precursors can be identified. Undifferentiated,
multipotent hematopoietic stem cells, which are responsible
for colonization of other fetal tissues, have been
described in the mouse embryonic yolk sac blood islands at
this stage of development (Moore and Metcalf, 1970).
Abelson murine leukemia virus infection of primary
hematopoietic cells followed by culture in semisolid media
has previously been shown to generate permanent cell lines

102
with B lineage characteristics (Rosenberg and Baltimore,
1976; Siden et al., 1979; Alt et al., 1981), and to induce
terminally differentiated, erythroid colonies from early
placenta and fetal liver (Waneck and Rosenberg, 1981).
Although A-MuLV induces mastocytomas in vivo (Mendoza and
Metzger, 1976; Risser et al., 1978), and can infect and
immortalize cultured mast cells (Pierce et al., 1985;
Chapter III of this work), the proliferation of continuous,
exogenous growth factor-independent mast cells from
midgestation embryonic tissue in the absence of interleukin
3 is unprecedented. Pierce and colleagues (1985) recently
reported the generation of interleukin 3-independent mast
like cell lines from day eighteen fetal liver cells
infected with A-MuLV and subsequently selected in media
containing the growth factor. Those authors speculated
that the omission of beta-mercaptoethanol from their tissue
culture media was probably responsible for the
proliferation of transformed mast cells. Since our cell
lines were produced in mercaptoethanol-containing media, we
suggest rather that the generation of interleukin 3-
independent mast cell lines may be due to unique conditions
of transformation which incoporate the B cell mitogen
dextran sulfate or the specific targets used.
Abeison murine leukemia virus has been shown to
transform, ini vitro, macrophages (Greenberger et al.,
1979), erythroid cells (Waneck and Rosenberg, 1981), and

103
pre-B cells (Boss et al., 1979: Siden et al., 1979; Alt et
al. 1981) as well as interleukin 3-dependent mast cells
(Pierce et al., 1985; Chapter III of this work) and early
myeloid lineage cells (Rapp et al., 1985; Cook et al.,
1985). All of these cells are sensitive to the
proliferative effects of interleukin 3 (Iscove and Roitsch,
1985; Palacios et al., 1984). Our observations, however,
are that A-MuLV-infected mast cells do not synthesize
detectable interleukin 3 (Table II-6) nor do they contain
interleukin 3-specific messenger RNA (E. Siden, personal
communication); the former observation has recently been
corroborated by others studying Abelson virus-infected mast
cells (Pierce et al., 1985) and myeloid cells from more
mature stages of development (Cook et al., 1985).
We are presently uncertain of the apparent
nonautocrine mechanism of interleukin 3 independence in
Abelson virus-infected cells, although several
possibilities, including interaction of the v-abl gene
product with the interleukin 3 receptor (modifying the
activity of the receptor), substitution by the viral gene
product for an interleukin 3 receptor intracellular
function, or short-circuiting of an interleukin 3-induced
proliferation pathway (Pierce et al., 1985) are potentially
satisfying. Farrar and colleagues (1985) recently reported
that interleukin 3 stimulates the transient redistribution
of protein kinase C from the cytosol to the plasma membrane

104
of interleukin 3-dependent FDC-P1 cells. The protein
kinase C translocation kinetics in FDC-P1 cells is
paralleled by the DNA synthesis dose response curve of
these cells, suggesting a relationship between enzyme
association with plasma membranes and cell proliferation.
The Abelson murine leukemia virus transforming gene product
is a transmembrane protein (Witte et al., 1979b) with known
tyrosine protein kinase activity (Witte et al., 1980) and a
normal cellular analogue (Witte et al., 1979a). Cells
transformed by the virus contain a number of cellular
proteins which are phoshorylated on tyrosine residues
(Cooper and Hunter, 1981); Sefton et al., 1981a, 1981b).
We therefore propose that the Abelson murine leukemia virus
transforming protein may act on the substrate of protein
kinase C or one of the other enzymes in the pathways that
are normally activated by extracellular messengers (like
interleukin 3) which generate transmembrane control of
cellular functions (Nishizuka, 1984; Marx, 1984; Michell,
1984). Further studies to determine which, if any, of
these mechanisms is in effect are in order to elucidate the
functional role of the A-MuLV transforming protein.

CHAPTER III
CHARACTERIZATION OF MAST CELLS DERIVED FROM MIDGESTATION
EMBRYONIC TISSUES IN LIQUID CULTURE
Introduction
Mast cells have been the object of innumerable
scientific investigations since their initial description
by Ehrlich (1877). The study of mast cells experienced
renewed impetus in the nineteen-sixties based upon research
in two major areas. First, the development of in vitro
culture techniques confirmed the existence of mast cell
precursors in a variety of tissues and indicated that mast
cell differentiation was dependent on factor(s) elaborated
by stimulated lymphocytes. Secondly, the development of
improved cytological fixation and staining techniques
(Enerback, 1966a, 1966b) permitted the characterization of
a new class of "atypical" mast cells (mucosal mast cells)
which were were distinct from the connective tissue-
associated (serosal) mast cells studied in detail prior to
that time. Within a short period of time, a morphological,
biochemical, and functional relationship between the in
vitro-derived mast cells and mucosal mast cells was noted.
The distinction between the culture-derived and
serosal mast cells was facilitated by the development of a
series of monoclonal antibodies which discriminated between
the two subsets (Katz et al., 1983). The observation of
metachromatic granules in A-MuLV-transformed placental cell
105

106
lines suggested that we probe the cells with B23.1 (Chapter
II), an antibody which recognizes culture-derived mast
cells. These studies led to the observation of other mast
cell markers on the transformed cell lines, namely
histamine and membrane receptors for IgE.
The expression of mast cell characteristics by A-MuLV
transformants derived from murine placenta opened several
related areas for investigation. We first desired to learn
if precursors to culture-derived mast cells exist in
midgestation embryonic tissue. Second, having found
embryonic precursors to these mast cells, we wanted to know
if the markers expressed on the A-MuLV transformants were
also expressed on "normal" cells cultured in interleukin 3.
We therefore conducted a detailed analysis of the
progression of cell populations in mast cell cultures over
the course of several weeks of selection and enrichment.
Third, we studied the effects of Abelson virus infection
and adherent cell cytokines on long-term cultured mast
cells. The scope and significance of our findings are
discussed in this chapter.
Materials and Methods
Procedures for the husbandry of mice, detection of
cell surface determinants and Fc receptors, and cytological
staining were performed as described in Chapter II.

107
WEHI-3 Conditioned Medium (W3CM)
W3CM was prepared following the method of Razin and
colleagues (1984a). WEHI-3 myelomonocytic leukemia cells
from log phase cultures were seeded at 1x10^ per ml into an
enriched medium (EM) consisting of RPMI 1640 (GIBCO, Grand
Island, NY) supplemented with 10% v/v native fetal bovine
serum (Sterile Systems, Logan, UT), 2mM L-glutamine, 0.1 mM
nonessential amino acids, 100 Units per ml penicillin, 100
micrograms per ml streptomycin (GIBCO), and 0.05 mM 2-
mercaptoethanol (Fisher Scientific Co., Medford, MA).
Cultures were maintained in one of two ways: 200 ml
cultures in 17x150 mm dishes (Falcon Plastics, Oxnard, CA)
were incubated at 37C in a humid, five percent carbon
dioxide incubator (Forma Scientific, Marietta, OH);
alternatively, 1000 ml cultures were seeded into pregassed
EM in polycarbonate roller bottles (Corning Glass Works,
Corning, NY) which were placed a horizontal roller
apparatus (New Brunswick Scientific, New Brunswick, NJ) in
a controlled environment room (37C). Following four days
(92-100 hours) of incubation, the conditioned media were
harvested by centrifugation (15 minutes at 2000xg, 4C) and
filtered through tissue culture grade 0.2 micron
nitrocellulose membranes (Nalgene, Rochester, NY). Media
harvested from small cultures were pooled into 1000 ml lots
prior to filtration. W3CM was aliquoted in smaller volumes
(100, 250, 500 ml) in sterile bottles and stored for up to

108
six months at -20C; volumes for immediate use were stored
for up to one month at 4C. Fresh glutamine was added to
all media over one month of age. Unless otherwise noted,
W3CM was diluted with an equal volume of EM to yield 50%
W3CM.
Preparation of Cell Suspensions for In Vitro Culture of
Mast Cells
Bone marrow
Two to three month-old BALB/cAn mice were killed by
cervical dislocation. The skinned hind legs were
disarticulated at the pelvis and tarsals and were placed in
EM. The limbs were transferred to a fresh dish of EM in
which most of the flesh was removed from the femorae and
tibiae by cutting and teasing with sterile dissection
tools. Following transfer of the bones to a third dish of
EM, the bones were disarticulated and the ends sheared off.
Bone marrow was then harvested by flushing the contents of
all four bones with EM (1 ml injected through each end with
a 25 gauge needle) through 110 micron mesh nylon screens
(Tetko, Inc., Elmsford, NY) into a 50 ml conical
polypropylene centrifuge tube (Corning Glass Works) (Siegel
et al., 1985) .
Spleen
Two to three month-old BALB/cAn mice were killed by
cervical dislocation, after which the spleens were removed
by careful, asceptic dissection and placed in a dish of EM.

109
The spleens were transferred to fresh medium and residual
fat and membranes were removed. The trimmed spleens were
minced with sterile scissors onto nylon screens and
dissociated through the screens by massaging the tissue
with the rubber end of a plunger of a disposable 3cc
syringe. Disaggregated cells were washed through the
screen into the underlying centrifuge tube with ten to
twenty milliliters of EM.
Embryonic tissues
Embryonic tissues were isolated at the indicated days
of gestation after vaginal plugs were observed on day 0.
Pregnant mice were killed by cervical dislocation and the
gravid uteri were surgically removed and placed in a petri
dish containing EM. After two to five minutes (to allow
the tissues to bleed into the media) each uterus was
transferred to a second dish of EM and the residual
mesentery and fat were trimmed off. The uteri were
transferred to third dish of EM and concepti were dissected
away from the maternal tissues and placed in a fresh dish
of EM. Concepti of ten or more days of age were teased
into placental and nonplacental embryonic tissue (NPET)
components. The ectoplacental cones were dissected free of
attached membranes and placed in a fresh dish of EM.
Concepti of eight and nine days were processed without
further dissection. All tissues were dissociated through
nylon screens as previously described.

110
Establishment of Liquid Cultures of Factor-Dependent
Mast Cells
Mast cells were cultured from fresh tissues following
modification of a previously published protocol Razin et
al., 1984a). Except for variations in the methods used to
dissociate the cells from their native structures, as noted
above, the procedure for propagating mast cells from each
source was the same.
Disaggregated cell suspensions were allowed to settle
by gravity at room temperature in conical centrifuge tubes
for five to ten minutes. Cells remaining in suspension
were transferred to a clean tube and were washed three
times in EM by centrifugation. Viable, nucleated cells
were enumerated by trypan blue exclusion and the cells were
resuspended at a concentration of 1x10^ per ml in 50% W3CM.
Cultures were maintained in a humidified atmosphere of five
percent carbon dioxide in air at 37C. Mast cells were
enriched in liquid culture and selected at weekly intervals
by gently swirling the dishes and transferring the
suspended cells with a pipet to a centrifuge tube;
following centrifugation, the cells were resuspended in 50%
W3CM at 1 to 2x10^ per ml.
Infection of In Vitro-Derived, Factor-Dependent Mast Cells
with Abelson Murine Leukemia Virus
Cultures of mast cells derived from adult and
embryonic tissues were infected at various times during the

Ill
enrichment-selection period and thereafter. Transforming
virus stocks, prepared as previously described (Siegel et
al., 1985) by superinfection of Abelson P160 nonproducer
cell line 160N54, contained 1 to 2x10^ PFU of Moloney
murine leukemia virus (M-MuLV) and 0.5 to 1.0x10^ of A-MuLV
per ml and were stored at -70C prior to rapid thawing
immediately before use. Mast cells were pelleted at 200xg
at room temperature for ten minutes and resuspended in A-
MuLV virus stock with 4 micrograms Polybrene (Aldrich
Chemical Co., Milwaukee, WI) per ml at 0.5 to 4x10^ cells
per ml. Cells were incubated with virus for two and one-
half hours at 37C (Rosenberg and Baltimore, 1976a) in
capped 12x75 mm polypropylene culture tubes (Fisher
Scientific Co.) with gentle, end-over-end rotation.
Following the adsorption period, the suspension was diluted
to a final cell concentration of 1x10^ per ml with EM or
W3CM (final concentration of 50% W3CM, plus virus stock and
EM) and cultured in a humidified atmosphere of five percent
carbon dioxide in air. Cells in culture were counted at
two to five day intervals and fed weekly by centrifuging
the cells and resuspending them at 1 to 2x10^ per ml in 50%
W3CM or EM, as appropriate. Disaggregated cells from fetal
livers dissected from day 18 embryos were infected with A-
MuLV and cultured as above.

112
Immunoprecipitation of Viral Proteins
Viral proteins in A-MuLV-infected cells were
identified by immunoprecipitation as previously described
(Siden et al., 1979). Virus-infected culture-derived mast
cells and control cells were pelleted at 200xg for ten
minutes at room temperature and washed once with balanced
salts solution (BSS). The cells (1.5 to 2x10^) were
resuspended at 2x10^ per ml in labeling media consisting of
RPMI 1640 media without methionine (Flow Laboratories,
Rockville, MD), 2 mM glutamine, lx RPMI vitamins (GIBCO),
100 units penicillin and 100 micrograms streptomycin per ml
(GIBCO), 10 mM HEPES (Sigma Chemical Company), pH 7.35,
0.05 mM 2-mercaptoethanol (Sigma Chemical Company), and 78
microcuries of 35s_met]1onne (H26 Ci per mMol, New England
Nuclear, Boston, MA). Cells were incubated in labeling
media for two hours at 37C with gentle rocking. The
labeled cells were centrifuged for five minutes at 400xg,
washed once with BSS and lysed in 1 ml of phosphate lysis
buffer (PLB, 10 mM sodium phosphate (Fisher Scientific
Co.), pH 7.5, 100 mM sodium chloride (Fisher Scientific
Co.), 1 percent v/v Triton X-100 (Fisher Scientific Co.),
0.5 percent w/v sodium deoxycholate (Fisher Scientific
Co.), and 0.1 percent w/v sodium dodecyl sulfate (SDS, BDH
Chemicals, Ltd., Poole, U.K.)) which was supplemented with
0.098 percent w/v bovine serum albumin (Sigma Chemical
Co.), 1 mM EDTA (Fisher Scientific Co.), 1 mM TAME (Sigma

113
Chemical Co.), 1 mM PMSF (Sigma Chemical Co.), and 0.0002
percent w/v Aprotinin (Boehringer Mannheim GmbH,
Indianapolis, IN). The lysates were cleared at 4C for ten
minutes at 2,000xg and sixty minutes at 45,000 revolutions
per minute in a Ti50 rotor of a Beckman L5-50
ultracentrifuge (Beckman, Palo Alto, CA).
Lysates were analyzed for incorporated radioisotope by
spotting five microliter volumes on Whatman 3MM paper
(Fisher Scientific Co.). The filters were washed in five
percent w/v trichloroacetic acid, dried, and counted in a
Beckman liquid scintillation spectrometer with toluene-
based scintillation fluid. Volumes of labeled lysates,
normalized to equivalent numbers of counts, were
immunoprecipitated with five microliters of either goat
anti-M-MuLV or normal goat serum by incubation over night
at 4C on a rotator, followed by incubation for two to three
hours with prewashed, heat-killed S. aureus bacteria (50
microliters at 10 percent w/v) in 1.5 ml microfuge tubes.
The contents of the tubes were centrifuged at 12,000xg for
thirty seconds and the pellets were washed three times with
PLB and resuspended in fifty microliters of sample buffer
containing 62.5 mM Tris, pH 6.8, one percent w/v SDS, 50 mM
dithiothreitol (Sigma Chemical Co.), 5 mM EDTA, and
bromophenol blue (Fisher Scientific Co.).
Immunoprecipitated proteins were heated for thirty minutes
at 68C and microfuged for three minutes at room temperature

114
to pellet the bacteria prior to electrophoresis on a 7.0
percent SDS-polyacrylamide gel with a 5.0 percent stacking
gel. Running buffer of 0.6 percent w/v Tris, 2.9 percent
w/v glycine, 0.1 percent w/v SDS and 0.114 percent w/v
sodium thioglycollate (Sigma Chemical Co.) was used.
Following electrophoresis at 150 volts (DC), the gel was
stained for fifteen minutes with 0.25 percent w/v Coumassie
Brilliant Blue R in aqueous 45 percent (v/v) methanol, ten
percent (v/v) glacial acetic acid. The gel was destained
over night against several changes of ten percent (v/v)
acetic acid with two "Adsorptors" destaining sponges,
rehydrated with three, ten minute changes of water, and
soaked for forty minutes in 1M sodium salicylate (Fisher
Scientific Co.) in water. The gel was sandwiched between a
clean sheet of 3MM filter paper and plastic wrap prior to
drying under vacuum (BIO-RAD gel drier, Oakland, CA). The
dried, fluorographed gel was exposed to XAR-5 film
(Eastman-Kodak, Rochester, NY) at -70C for the time
indicated.
Coculture of Long Term Mast Cells with Adherent Cell
Underlayers and Adherent Cell Conditioned Media
Bone marrow cells were prepared from normal BALB/cAN
tissue isolated as previously described (see Preparation of
Cell Suspensions for In Vitro Culture of Mast Cells).
Washed cells were resuspended at 1x10^ per ml in 50% W3CM
and 2.5 ml were aliquoted in each well of a six-well tissue

115
culture cluster (Costar, Broadway, MA). After six days in
culture (five percent carbon dioxide in air at 37C), the
supernatants, with nonadherent cells, were removed and 2.5
ml of fresh 50% W3CM were added. Two days later, the bone
marrow-derived adherent cell-conditioned media (bmadhCM)
were harvested by centrifugation (200xg, fifteen minutes)
and filtration through a PBS-washed, 0.2 micron disposable
filter (Gelman Sciences, Inc., Ann Arbor, MI). The
adherent cell monolayers were washed twice with cold PBS to
remove any residual nonadherent cells and conditioned media
and then overlayered with 2 ml of 0.3 percent agar (Difco,
Detroit, MI) in 50% W3CM.
P388D1 monocytic tumor cells were grown in RPMI 1640
with 10 percent v/v heat-inactivated fetal bovine serum,
2 mM glutamine and 0.05 mM 2-mercaptoethanol (10P) to a
density of 2x10^ to 1x10^ per ml. Cells were pelleted by
centrifugation (200xg for ten minutes at room temperature)
and resuspended at lxlO6 per ml in 10P made 20 micrograms
per ml with Escherichia coli serotype B5:055
lipopolysaccharide (LPS, Sigma Chemical Co.) (10P/LPS)
after the method of Lachman and colleagues (1979). Within
three hours, most of the cells, which were aliquoted at
1x10^, 0.5x10^ and 0.25x10^ per well in a six well cluster
(in 1 ml of 10P/LPS), had become adherent. P388/LPS-
conditioned media (P388/LPSCM) were harvested after two
additional days in culture in a manner identical to that

116
described for bmadhCM. Cells from the healthiest, most
confluent monolayers (seeded at 1x10^ per ml) were washed
twice with cold PBS and overlayered with 2 ml of 0.3
percent agar containing 10P/LPS or 50% W3CM.
Exponentially growing cultures of WEHI-3
myelomonocytic cells at 4x10-5 per Were centrifuged for
ten minutes at 200xg and resuspended at 1x10^ per ml in
fresh 10P/LPS. Individual compartments of a twelve-well
tissue culture cluster (Costar) received 1x10^, 0.5x10^ or
O.lxlO^ cells; all volumes were normalized to one ml with
10P/LPS. Cells were cultured for two days under these
conditions, at which time the conditioned media were
harvested and processed as previously described. Only
those wells seeded with 0.1x10^ cells had healthy adherent
layers and were selected for coculture experiments. The
latter cells were washed twice with cold PBS prior to
overlayering with 1 ml 0.3 percent agar in either 50% W3CM
or 10P/LPS.
Mast cells derived from (BALB/cAN x CBA/J)F1 placenta
and BALB/cAN bone marrow were selected and enriched to a
homogeneous population as previously described. At day 37
of culture, the cells were prepared for the experiment by
centrifugation at 200xg for ten minutes at room
temperature. The cell pellet was resuspended at a
concentration of 2x10^ per ml in 100% W3CM and one ml of
the suspension was pipeted into each well of the six-well

117
clusters used in the adherent bone marrow and P388D1
experiments; alternatively, 0.5 ml of suspension was
pipeted into each well of the twelve-well cluster used in
the WEHI-3 experiments. Mast cells were cocultured with
adherent monolayers prepared as previously described and
with control underlayers of 0.3 percent agar in appropriate
media but without adherent cells. All suspensions were
made 50% W3CM by addition of an equal volume of EM.
The effects of adherent cell-derived factors were
investigated by making the mast cell suspensions 50% W3CM
by addition of an equal volume of bmadhCM, P388/LPSCM or
W3/LPSCM. Control cultures of mast cells were similarly
made by addition of equal volumes of EM or 10P/LPS as
appropriate. Cultures were fed weekly for three weeks by
addition of an equal volume of homologous media and
analyzed for surface markers as previously described.
Results
Progression of Lineage Markers in Mast Cell Cultures in
Liquid Media
Previous studies have dealt with the expression of
surface and cytochemical markers in the cells alternatively
called cultured mast cells and P cells, but only after the
populations had become homogeneous by successive selection

118
and enrichment, a process requiring at least four weeks in
culture. Characteristics such as IgE receptors,
metachromatic cytoplasmic granules (Schrader, 1981) and a
surface determinant recognized by the monoclonal antibody
B23.1 (Katz et al., 1983) have been ascribed to such cells.
We chose to analyze the expression of these markers, as
well as markers found on A-MuLV-transformed embryonic mast
cells, on the heterogeneous populations of cells from which
mast cells evolve to better understand the evolution of
homogeneous mast cell cultures.
Embryonic tissues
The first significant finding of this study was that
mast cells or their precursors exist in the midgestation
embryonic placenta and nonplacental embryonic tissues
(NPET). The existence of mast cells in such tissues was
hypothesized after homologous A-MuLV transformants were
characterized (CHAPTER II). As seen in Figure III-l, the
number of liquid culture cells with cytoplasmic
metachromatic granules (Figure III-2) rose from less than
one percent in freshly disaggregated tissues to nearly
eighty percent of the cells after four weeks in culture.
Concurrently, the number of cells expressing high affinity
receptors for IgE rose from less than one percent to
approximately fifty percent. A similar time course was
followed by the determinant recognized by monoclonal
antibody B23.1, which was expressed by nearly all cells in

119
Figure III-l. Progression of Hematopoietic Lineage Markers in Long-Term
Mast Cell Cultures Derived From Embryonic Tissues
Cultures were maintained and analyzed as described in Materials
and Methods. Cultures were derived from nonplacental embryonic
tissue (NPET, panels A, C, E) and from placenta (P, panels B, D, F)
isolated from (BALB/c x B10.BR)F1 (panels A, B, C, D) and (BALB/c
x CBA)F1 (panels E,F) concepti at 10(panels A,B) and ll(panels C,
D,E,F) days post coitum. Percent cells expressing determinants
recognized by monoclonal antibodies RA3-3A1 (A) and B23.1(B),
receptors for IgE(E) and IgG(G), and cells with metachromatic
granules (M) are indicated.

100
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
0
120
C.dll(BALB/c x B 10. BR)F I NPET
B B B B
D.dIKBALB/c x B 10. BR)FI P
E. d II (BALB/c x CBA) FI NPET
F.d INBALB/c x CBA) F I P
1234567 1234567
TIME IN CULTURE (WEEKS)

121
/
Figure III-2. Metachromatic Granules of Long-Term, Culture-
Derived Embryonic Mast Cells.
Mast cells were propagated, cytocentrifuge smears were prepared,
and slides were fixed and stained with 0.1 percent acidic
toluidine blue as described in Materials and Methods.

122
culture by the end of the third week. The latter
determinant, however, was expressed at significant levels
(25 to 25 percent) on freshly dissociated placental cells
of (BALB/c x BI0.BR)F1 origin, but not on syngeneic embryo
(NPET) cells or on cells derived from (BALB/c x CBA/J)
concepti (Figure III-2).
The expression of high affinity receptors for IgG and
the lymphoid B-220 determinant recognized by the antibody
RA3-3A1 remained relatively insignificant during the course
of culture. Expression of markers on mast cells derived
from days ten and eleven placenta and NPET were similar;
furthermore, there were no qualitative differences between
mast cells isolated from (BALB/c x B10.BR)F1 and (BALB/c x
CBA)F1 embryonic tissues.
After four weeks in culture, no adherent cells were
observed in the dishes and essentially all of the cells
contained metachromatic granules as assessed by toluidine
blue staining. In addition to expression of the surface
determinant recognized by monoclonal antibody B23.1,
membrane receptors for IgE (but not IgG), and metachromatic
granules, mast cells derived from midgestation murine
placenta also expressed the paternal Class I
histocompatability antigen (k haplotype), confirming that
the precursors to the cells were embryonic, rather than

123
maternal (data not shown). The expression of Class I
markers on P cells was previously reported by Schrader
(1981).
Bone marrow
The progression of cell surface and cytochemical
markers on bone marrow-derived mast cells was similar to
that observed in embryonic-derived tissues (Figure III-3).
The number of cells expressing the B23.1 determinant and
staining metachromatically with toluidine blue rose
dramatically from basal levels in fresh tissue to
essentially all of the cells at four weeks, while the
number of cells expressing receptors for IgE rose more
gradually to approximately fifty percent of the population.
At the same time, cells expressing receptors for IgG and
the B-220 lymphoid determinant were an insignificant
portion of the total cells in culture by two weeks.
Expression of Cell Surface Antigens on Cultured Mast Cells
Infected with Abelson Murine Leukemia Virus
Long term liquid cultures of mast cells are
characterized by a relatively homogeneous population of
cells which express the determinant recognized by the
monoclonal antibody B23.1, have receptors for IgE and
possess metachromatic granules in their cytoplasm. They
are, however, devoid of the B-lymphoid marker recognized by
the monoclonal antibody RA3-3A1, which is expressed on some
of the A-MuLV transformed placental mast-like cell lines.

1.24
Figure III-3. Progression of Hematopoietic Lineage Markers in
Long-Term Mast CeLl Cultures Derived From Adult
Rone Marrow
Cultures were maintained and analyzed as described in Materials and
Methods. Data shown are for BALB/c bone marrow-derived mast cell
cultures characterized for expression of determinants recognized by
monoclonal antibodies RA3-3A1 (A), B23.HB), receptors for IgE (E)
and IgG (G), and cells with metachromatic granules (M).

125
We were therefore interested in determining whether A-MuLV
infection of culture-derived mast cells could induce the
expression of the lymphoid B-220 antigen.
Initial experiments were designed to infect bone
marrow-derived mast cells throughout the course of their
maturation and to analyze the surface marker distribution
of the population. Cells infected one, two, and three
weeks after initial isolation and culture were considered
"transitional" in that the target population was
heterogeneous with respect to the surface and cytological
markers assayed; similarly, cells four weeks or later in
the culture period were relatively homogeneous (Figure III-
3) .
Following A-MuLV infection, mast cell cultures
experienced a decrease in population size attributed to
virus-related killing of some infected cells (Figure III-4).
The effects were more dramatic in virus-infected cultures
which were grown in the absence of growth factor and were
attributed to the inability of factor-dependent cells to
proliferate in EM alone, as demonstrated by the short half-
life of uninfected mast cells which were grown in parallel
cultures without W3CM (Figure III-4). The period of
attrition peaked within the first four days of culture
post-infection and was followed by an equally dramatic
period of proliferation of surviving cells; parallel

Number of Cells Per Milliliter
126
Days In Culture Post Infection
Figure III-4. Population Dynamics of Bone Marrow-Derived Mast Cells
Infected with A-MuLV
Long-term (at least 28 days) bone marrow-derived mast cells were
infected with A-MuLV or sham-infected and subsequently grown in
2.5 ml cultures with or without W3CM.

127
cultures infected with M-MuLV or sham infected (incubated
with fresh DME with 4 micrograms Polybrene per ml) did not
proliferate if deprived of growth factors.
The expression of the lymphoid B-220 antigen was
analyzed as a function of primary culture age. Initially,
populations of cells which had been in culture for one,
two, three, or four weeks prior to infection were probed
twenty-one days post infection. The data from these
experiments are summarized in Figure III-5. Populations of
cells infected at early time points during the development
of mast cell cultures displayed similar proportions of
cells expressing the B-220 determinant as uninfected
populations, while at late times the cells retain the B-
220-negative phenotype of the cells in liquid culture
(Figure III-3). The cell culture systems, however, were
different by one important criterion. In the standard
procedure used to propagate culture-derived mast cells,
only nonadherent cells are passaged each week, thereby
selecting against the adherent population. In the virus-
and sham-infected cell cultures, however, the nonadherent
cells were maintained in the presence of the adherent cells
present in the original culture (and their progeny). Thus,
more adherent cells were present in cultures infected at
one week than at later times. Adherent cells supported the
growth of lymphoid cells as well as culture-derived mast
cells. The RA3-3Al-positive population is probably

128
CO
LlI
o
L
>
H
CO
O
Q.
WEEKS IN CULTURE PRIOR TO INFECTION
Figure III-5. Expression of Lv5 Antigen on A-MuLV-Infected
Mast Cells
Bone marrow cells were cultured under conditions favorable for
selection and enrichment of mast cells and infected with A-MuLV
at the times noted above. Uninfected cells were assayed at the
time of infection (unshaded bars) and parallel cultures of
infected (shaded bars) and sham-infected (stippled bars) were
assayed twenty-one days post infection for the presence of Lv5
with monoclonal antibody RA3-3A1 as described in Materials
and Methods.

129
transformed bone marrow pre-B cells which are present in
the early cultures. This was substantiated by the
morphology of the cells in the cultures. Figure III-6 shows
both RA3-3Al-positive agranular, lymphoid cells and RA3-
3Al-negative granular cells in the same day 28 culture.
Subsequent infections of mast cells with A-MuLV were
performed with long-term cultures which were deficient in
both adherent cells and RA3-3Al-positive cells. When
assayed as described above, neither A-MuLV- nor sham-
infected culture-derived mast cells had constituent
subpopulations expressing the lymphoid marker (data not
shown).
The presence of A-MuLV in cultures of cells treated
with the virus was assayed to confirm that the cells were
indeed productively infected with the same transforming
virus which provided us with the placental tumor cell lines
described in Chapter II. Biosynthetically -^^S-methionine-
labeled cells were lysed in detergent with proteinase
inhibitors and the cleared lysates were specifically
precipitated with goat anti-M-MuLV. The latter antiserum
precipitates all major M-MuLV proteins and will likewise
bind to the A-MuLV gag-abl transforming protein by nature
of its M-MuLV gag epitopes. The immunoprecipitated
proteins were separated electrophoretically on a 7.0
percent, SDS polyacrylamide gel. The autoradiograph of the
gel (Figure III-7) shows virus specific bands of
i

130
Figure III-6. Mixed population of RA3-3A1-Positive Lymphoid
Cells and RA3-3A1-Negative Cultured Mast
Cells in Long-Term Bone Marrow Cultures.
Bone marrow cells were cultivated for one week and infected
with A-MuLV (A) or sham infected (B) as described in
Materials and Methods. After three additional weeks of
culture, cells were probed with aureus-RA3-3A1 as
previously described.

131
Figure III-7. Abelson Murine Leukemia Virus-Infected Mast
Cells Express v-abl Gene Product.
Proteins in A-MuLV-infected cells and controls were
biosynthetically labeled and immunoprecipitated with goat
anti-Moloney MuLV prior to SDS-polyacrylamide gel electrophoresis,
fluorography, and autoradiography as described in Materials and
Methods. Electrophoresis standards used were (bars on right,
from top to bottom) myosin (205 kilodaltons, Kd), beta-
galactosidase (116 Kd), phosphorylase B (97.4 Kd), and bovine
serum albumin (66 Kd). Lanes 1, 3, and 5 were loaded with immune
precipitated cell lysates; lanes 2 and 4 were loaded with lysates
adsorbed with normal goat serum. Lanes 1 and 2, A-MuLV-transformed
placental cell line 10P12; lanes 3 and 4, A-MuLV-transformed
culture-derived mast cells; lane 5, sham-infected culture-derived
mast cells. Upper arrow indicates v-abl gene product; lower
arrow indicates major Moloney-MuLV gene product (Pr65).

132
approximately 160 and 65 kilodaltons (Kd) in lanes loaded
with immune precipitated lysates from the placental tumor
cell line 10P12 (lane 1), from A-MuLV infected fetal liver
(lane 3) and from A-MuLV infected embryonic mast cells
(lane 5). The same bands are absent from the lanes loaded
with nonimmune (normal goat) serum-treated lysates of the
same cells (lanes 2, 4 and 6, respectively) and from a lane
loaded with immune precipitated lysate from sham-infected
mast cells (lane 7). We conclude, therefore, that the mast
cells were productively infected with A-MuLV. Furthermore,
the infection of culture-derived mast cells with A-MuLV did
not induce the de novo expression of the lymphoid B-220
marker in those cells.
Effects of Adherent Cells and Adherent Cell-Derived Factors
on Populations of Cultured Mast Cells
Because conditions which involved coculture with
adherent cells generated mast cells expressing Ly 5, we
tried to recreate this milieu using bone marrow adherent
cells, cell lines, or conditioned media. Expression of B-
220 was assayed with RA3-3A1 antibody as previously
described. As seen in Table III-l, neither the
cocultivation with adherent cells nor their conditioned
media with homogeneous populations of mast cells was
sufficient to induce the expression of B-220. The
expression of the antigen recognized by the B23.1 antibody,
receptors for immunoglobulin IgE and the presence of

Table III-l. Effect of Cocultivation of Culture-Derived Mast Cells
with Adherent Cells and Their Conditioned Media.
Mast Cell Treatment3 Percent Cells Expressing^
RA3-3A1
B23.1
IgE
Metachromat
Epitope
Epitope
Receptors
Granules
day 11 Placenta-derived mast
cellsc cocultured with:
50% W3CMd only
8
95
55
72
LPS-induced P388 cell underlayer
with 10P/LPS in agar
4
93
52
70
with 50% W3CM in agar
<1
99
51
66
Agar only underlayer
with 10P/LPS in agar
2
98
54
69
with 50% W3CM in agar
<1
92
49
68
No underlayer
+50% W3CM + 50% 10P/LPS
3
99
47
69
+50% W3CM + 50% P388CM
4
97
45
67
50% W3CM only
2
96
55
72
LPS-induced WEHI-3 underlayer
with 10P/LPS in agar
<1
94
ND
ND
with 50%W3CM in agar
3
90
46
73
Agar only underlayer
with 10P/LPS in agar
2
92
45
80
with 50% W3CM in agar
<1
89
42
71
No underlayer
+50% W3CM + 50% 10P/LPS
<1
94
47
72
+50% W3CM + 50% W3/LPSCM
2
96
48
74
50% W3CM
2
100
52
67
Adherent bm cell underlaver
with 50% W3CM
8
97
48
53
No underlayer
+50% W3CM + 50% bmadh CM
5
98
49
67
133

Table III-l. Continued
Mast Cell Treatment Percent Cells Expressing
bone marrow-derived mast cellse
cocultured with:
RA-3A1
Epitope
B23.1
Epitope
IgE
Receptors
Metachromatic
Granules
50% W3CM only
Adherent bone marrow cells underlayer
with bmadh CM in agar
<1
99
56
100
+50% W3CM + 50% bmadh CM
Adherent bone marrow cell underlayer
<1
99
50
93
with 50% W3CM in agar
Agar underlayer
<1
100
52
96
with bmadh CM
No underlayer
2
93
ND
+
+ bmadh CM
<1
95
60
98
a. Long term (four weeks and older) mast cell cultures were cocultured with adherent cells
in agar underlayers, with cell-free agar underlayers and with no underlayers. In some
experiments, media other than 50% W3CM was used in formulating the agar (these are
prefixed by "with"). In some experiments, the liquid media in which the mast cells were
grown was supplemented with adherent cell conditioned media (these are prefixed by "+").
For details see Materials and Methods.
b. Following coculture under the indicated conditions,mast cells were analyzed as described
in Materials and Methods. One hundred to two hundred cells were assessed microscopically
for each characteristic and the number of characteristic-positive cells was calculated
as a percentage of the entire population. In some instances, insufficient cell numbers
precluded assay (ND); in one instance, less than one hundred cells were counted, and no
percentage was determined of the marker-positive cells (+).
c. Mast cell cultures were derived from embryonic placenta of (BALB/c x CBA)F1 concepti
isolated at 11 days post coitum as described in Materials and Methods.
d. Abbreviations used: W3CM (WEHI-3 conditioned media), LPS (bacterial lipopolysaccharide),
10P/LPS (standard medium 10P supplemented with LPS, 20 micrograms per ml), P388CM
(conditioned media of cell line P388), W3/LPSCM (conditioned media of cell line WEHI-3
grown in media supplemented with LPS), bm (bone marrow), bmadh CM (conditioned media of
adherent bone marrow cells).
e. Mast cells cultures were derived from BALB/cAN bone marrow as described in Materials and
Methods.
134

135
metachromatic granules remained unchanged as well in mast
cells derived from adult bone marrow or embryonic (day
eleven) placenta.
Discussion
The observation of mast-like cells in lines derived by
the transformation of midgestation embryonic placenta by
Abelson murine leukemia virus (Chapter II) prompted a number
of questions which were addressed in this chapter: Are
there mast cells in the placental and nonplacental
embryonic tissues at days ten and eleven of gestation? Are
there mast cell precursors in these tissues? Is A-MuLV
responsible for the expression of lymphocyte
characteristics on mast cells? The results of our
experiments have provided new and significant insight to
our understanding of mast cell differentiation.
Analysis of dissociated, fresh bone marrow, placenta
and nonembryonic placental embryonic tissues indicated few
or no cells with toluidine blue-stained metachromatic
granules indicative of mast cells. Mast cells, therefore,
comprise at most a minor, and perhaps insignificant,
portion of the cells in the tissues examined.
We have also documented, for the first time, the
presence of mast cell precursors in both embryonic placenta
and nonplacental embryonic tissues. Mast cell precursors

136
are present in significant numbers in both types of tissues
derived ten and eleven days post coitum. Previously,
Ginsburg and colleagues (1982) reported that mast cells
occassionally arose in control cultures of day ten to day
thirteen adherent embryonic cells which were generally used
for feeder layers in cultures of mouse thymus-derived mast
cells. The authors, however, did not provide detailed
description of the phenotype of such cells.
Numerous authors have reported that mast cells derived
from a variety of adult tissues can be maintained in
culture for prolonged periods of time in the presence of
required growth factors. Although no attempts were made to
establish permanent mast cell lines like those previously
reported (Nabel et al., 1981; Nagao et al., 1981; Razin et
al., 1981a; Schrader et al., 1981; Tertian et al., 1981), we
were able to maintain bone marrow-derived mast cells in
vitro for more than seven weeks without appreciable loss of
viability (determined by trypan blue exclusion). In
contrast, mast cells derived from placenta seldom survived
more than five weeks before catastrophic decline in viable
cells. Similar results were reported by Ginsburg and
colleagues (1982) for culture of adult lymphoid tissue-
derived mast cells on embryonic feeder layers. Those
authors, however, ascribed the decline in cell number to
loss of growth factor, a variable which we have controlled
throughout culture. We propose that the embryo-derived

137
mast cells, in contrast to adult bone marrow-derived mast
cells, prematurely become insensitive to the proliferative
effects of interleukin 3. These cells may be impoverished
of interleukin 3 receptors, perhaps by a capping mechanism
similar to that observed in B cells, in which anti-mouse mu
chain antibodies induced the disappearance of cell surface
IgM in 14 day mouse fetal liver explant and dissociated
adult lymphoid tissue cultures (Raff et al., 1975) and
inhibited the mitogenic effect of lipopolysaccharide in the
absence of 2-mercaptoethanol (Sidman and Unanue, 1978).
Alternatively, the embryonic culture-derived mast cells may
be defective in a post-receptor molecular mechanism, the
nature of which is unknown. We have not further
investigated this interesting phenomenon, which requires
more detailed examination.
Within one week of initiation of culture, over fifty
percent of bone marrow-derived cells express the B23.1
differentiation marker. The proportion of cells
expressing this marker continues to increase until, at four
weeks, almost all cells are B23.1-positive. Similar
population dynamics were observed when the cells were
stained with acidic toluidine blue. The proportion of
cells which expressed membrane receptors for IgE increased
more gradually from background levels in fresh bone marrow
to approximately fifty percent of the cells at week four.
The latter data are in contrast to a previous report

138
(Tertian et al., 1981) that most of the cells in long-term
cultures of mast cells have IgE receptors. This
discrepancy may be the result of differences in receptor
assay technique or may result from other experimental
variances such as cell cycling which could temporally
affect the expression of membrane receptors. We have
observed on one A-MuLV-transformed embryonic cell line that
the rosetting assay used by Tertian and colleagues, detects
more cells with membrane receptors for IgE than the S.
aureus assay (data not shown).
The progression of surface markers on cultured
embryonic cells (Figure III-l) follows a pattern similar to
those observed for bone marrow-derived mast cells (Figure
III-3). Freshly disaggregated placenta contains a
population of cells which have receptors for normal rat IgG
and are recognized by monoclonal antibody B23.1, but few or
no such cells bind IgE or have metachromatic granules.
Based on previous reports (Katz et al., 1983), these cells
are probably related to the monocyte-mononuclear phagocyte
lineage and may be the cells responsible for the binding
and degradation of anti-paternal antibodies (Raghupathy et
al., 1984). Fewer cells expressing the B23.1 epitope are
found in the nonplacental embryonic tissues of (BALB/c x
B10.BR)F1 concepti. The development of metachromatic
granules and IgE receptors in cultures of cells derived
from embryonic tissues was delayed (perhaps because the

139
precursors were more immature) in contrast to bone marrow-
derived mast cells cultures (Figures III-l and III-3) for
the first week of in vitro growth but subsequently reached
similar proportions by week four. Expression of the B23.1
epitope, however, demonstrated similar population dynamics
for both adult and embryonic tissue-derived cells following
the first week in culture.
Reports of the progression of markers and cell types
in mast cell cultures have previously lacked quantitative
data of the kind presented here. In a series of studies
spanning two decades, Ginsburg (Ginsburg, 1963; Ginsburg
and Sachs, 1963; Ginsburg and Lagunoff, 1967; Davidson et
al., 1983) presented observations of sequential changes in
population morphology, size, granule content, nucleus-to-
cytoplasm ratio, and mitotic activity of cultures of mouse
thymocytes on feeder layers. The four stages of In vitro
mast cell development began with small colonies of
primitive mast cells and progressed to large lymphocytes
(or mastoblasts), young mast cells with mitotic figures,
and amitotic mature mast cells. Ishizaka et al. (1976)
reported that although less than 0.05 percent of similarly
prepared rat thymocytes initially possessed receptors for
IgE, most of the cells at the end of one week in culture
were blasts which had variable numbers of granules but were
still incapable of binding IgE. IgE receptors were not
detected in the Ishizaka cultures before day fourteen.

140
Having established the presence of mast cell
precursors in tissues identical to those used for A-MuLV
transformation, we infected "differentiated, long-term
culture-derived mast cells with A-MuLV to determine whether
such cells would be induced for the expression of the RA3-
3A1 determinant. The infected cells were phenotypically
identical to uninfected cells grown in the presence of
interleukin 3 with respect to metachromatic granules and
expression of the B23.1 epitope. The B lymphoid Ly5 marker
found on two of the original A-MuLV-transformed placental
cell lines (Chapter II) was not detected on either infected
or uninfected mast cells, although it was detected on
nongranular lymphoid cells in heterogeneous early cultures
of bone marrow cells which had been infected with A-MuLV
(Figures III-5, III-6). The RA3-3Al-positive cells were
probably Abelson virus-transformed pre-B cells, the
precursors of which are absent in long-term, homogeneous
mast cell cultures.
Our observations that Abelson murine leukemia virus
can infect mature cultured mast cells and abrogate their
requirement for interleukin 3 have recently been
corroborated in the literature. Pierce and colleagues
(1985) reported that fetal liver-derived mast cells,
infected with A-MuLV and maintained in media containing
interleukin 3 for three weeks, subsequently formed colonies
at high efficiency in the absence of the growth factor.

The A-MuLV-infected, fetal liver-derived mast cells were
phenotypically similar to uninfected cells with respect to
morphology, presence of metachromatic granules, 20-alpha-
hydroxysteroid dehydrogenase, and high affinity receptors
for IgE. Attempts to establish factor independent mast
cell lines with BALB-murine sarcoma virus (MSV), Harvey-
MSV, and Moloney-MSV have been unsuccessful (Pierce et al.,
1985). Abelson virus has also been shown to abrogate the
interleukin 3-dependence of the early myeloid cell line
FDP-1 and to similarly release the interleukin 2-dependent
cytotoxic T cell line CTB6 (Rapp et al., 1985; Cook et al.,
1985). The mechanism by which the Abelson transforming
protein releases our cell lines, and those reported
elsewhere, from growth factor requirement is not
understood; it would appear, however, to be independent of
autocrine effects (Cook et al., 1985; Pierce et al., 1985;
Rapp et al., 1985).
Although the expression of the Ly5 differentiation
marker on cultured mast cells has been previously reported
(Nabel et al., 1981; Tertian et al., 1981; Wong et al.,
1982), the detection of the B lymphoid variant of Ly5 on A-
MuLV-transformed mast cells (Chapter II) and on mast cells
picked from agar colonies (Chapter IV), but not on liquid
culture-derived mast cells (Chapter III) in this laboratory
was enigmatic. The cells which expressed the RA3-3A1
epitope had, in common, maturation in agar in association

142
with adherent cell monolayers which grew in the presence
or absence of WEHI-3 conditioned media. Early reports of
mouse mast cell culture noted the absolute requirement of
such monolayers for mast cell differentiation and
proliferation (Ginsburg, 1963; Ginsburg and Sachs, 1963;
Ginsburg and Lagunoff, 1967). Ishizaka and colleagues
(1976) observed that rat thymus-derived mast cells grew
more rapidly in the presence of a feeder layer, while
Davidson and colleagues (1983) reported that fibroblasts
were required for mast cell granule synthesis.
In conclusion, we have demonstrated the presence of
mast cell precursors in the murine placenta and
nonplacental embryonic tissues during days ten and eleven
of gestation. The mast cells generated in liquid culture
are phenotypically similar to some Abelson virus-
transformed cell lines derived from the same tissue as well
as cultured mast cells derived from other tissues. We have
described the evolution of homogeneous mast cell cultures
from heterogeneous uninfected tissues in terms of
histochemical and surface marker expression. Finally, we
have demonstrated that while A-MuLV can infect mature mast
cells and abrogate their requirement for interleukin 3, the
virus does not induce the ectopic expression of the Ly 5
differentiation antigen which is expressed on some of the
Abelson virus-transformed mast-like cell lines.

143
Neither cocultivation with adherent cells nor their
conditioned media resulted in the expression of the B220
antigen on long-term cultured mast cells (Table III-l),
while the expression of receptors for IgE, the epitope of
monoclonal antibody B23.1, and the presence of
metachromatic granules was unchanged with respect to
untreated mast cells. We therefore conclude that neither
the adherent cells used nor their cytokines are responsible
for the expression of Ly5 on the surface of mature mast
cells. Further studies are required to clarify the Ly5
expression enigma. It seems likely that Ly5 may only be
transiently expressed on mast cells during the course of
their differentiation. We propose that the expression of
the determinant is perhaps fixed when such cells are
transformed with Abelson murine leukemia virus and
protracted when immature mast cells are maintained in agar
culture. Alternatively, the B-220 variant of the Ly 5
antigen may be expressed on a more differentiated form of
mast cell which is not present in standard liquid cultures.

CHAPTER IV
ISOLATION, ENUMERATION, AND CHARACTERIZATION OF IN VITRO
MAST CELL PRECURSORS DERIVED FROM MIDGESTATION EMBRYONIC
PLACENTA
Introduction
The understanding of the relationships between the
various components of the hematopoietic system has greatly
benefited from the development of clonal assay systems
during the last quarter century. From the seminal work of
Till and McCulloch (1961), the concept of a pluripotent
hematopoietic stem cell (CFU-S), capable of clonally
reconstituting the spleen and bone marrow of lethally
irradiated mice, led to the paradigm that all of the
cellular elements of blood were related by a single
progenitor. The rapid evolution of in vitro clonal culture
techniques has further contributed to our understanding of
normal and pathogenic hematopoiesis. Techniques are
presently available for quantitation of pluripotent
hematopoietic precursors from a variety of sources (Johnson
and Metcalf, 1977; Hara and Ogawa, 1978; Fauser and
Messner, 1979). In addition, methods for isolation,
differentiation, and enumeration of committed, multipotent
hematopoietic precursors have been described for a number
of lineages.
The in vitro quantitation of mast cell precursors in a
variety of tissues has thus been accomplished by limiting
dilution in liquid culture (Crapper and Schrader, 1983;
144

145
Guy-Grand et al., 1984) as well as clonal cultures in
semisolid media (Schrader et al., 1981; Zucker-Franklin et
al., 1981; Nakahata et al., 1982b). All of these methods
exploit the ability of mast cell precursors to proliferate
and differentiate in the presence of a factor now termed
interleukin 3 (Ihle et al., 1981) which has been isolated
from media conditioned by lectin- or antigen-stimulated T
lymphocytes as well as several permanent cell lines
(reviewed by Clark-Lewis et al., 1985; Ihle, 1985; Iscove
and Roitsch, 1985; Schrader et al., 1985; Yung and Moore,
1985). Under such conditions, mast cell precursors have
been found in a variety of tissues including adult bone
marrow, spleen, thymus, lymph nodes, gastric and intestinal
mucosa, blood, neonatal cord blood, and fetal liver
(reviewed by Katz et al, 1985a; Austen, 1984; Jarrett and
Haig, 1984).
In previous chapters we have characterized the mast
like, A-MuLV-transformed murine cell lines derived from
midgestation embryonic placenta (Chapter II) and the
subsequent detection of precursors of mast cells from
similar uninfected tissues (Chapter III). We now report the
isolation and quantitation of mast cell precursors from
embryonic tissues of mid- and late gestation. A number of
significant and novel findings are described. First, we
have described mast cell precursors at the earliest
reported time in embryonic mouse development. Second, the

146
midgestation placenta and nonplacental embryonic tissues
(NPET) are a rich reservoir of mast cell precursors,
containing proportionately at least as many such cells as
adult bone marrow. Third, in characterizing the cells
in mast cell colonies, we have found that they
express a variant of the Ly5 differentiation antigen
previously reported to be specific for B lymphocytes.
Finally, we describe preliminary experiments which
differentiate mast cell precursors from other bone marrow
elements on the basis of surface membrane determinants.
These findings provide significant new information about
mast cell differentiation and ontogeny. Based upon our
findings, we discuss the role of mast cells and their
precursors in terms of murine embryonic hematopoiesis and
the immunobiology of the maternal-fetal interface.
Materials and Methods
Procedures for the husbandry of mice, detection of
cell surface determinants and Fc receptors, and cytological
staining were performed as described in Chapter II.
The procedure for the preparation of WEHI-3 conditioned media
(W3CM) was performed as described in Chapter III.
Cultivation of Mast Cell Precursors in Semisolid Media
Mast cell precursors were cultivated from fresh

147
tissues following a modification of the method used to
propagate culture-derived mast cells in liquid suspension
culture. Disaggregated cells were prepared from tissues as
described in Chapter III. Washed, enumerated cell
suspensions from various tissues were resuspended at twice
the concentration desired for the most concentrated
innoculum in 50% W3CM. The cells were then aliquoted into
12 well tissue culture clusters (Costar, Broadway, MA) in
volumes necessary to provide the total number of cells
desired per culture, and the total volume of each well was
brought to 0.5 ml with 50%W3CM. BACTO agar (DIFCO
Laboratories, Detroit, MI) was prepared in sterile water
(Travenol Laboratories Inc., Deerfield, IL) at 3.0% w/v,
autoclaved for twenty minutes and cooled to 42 to 45
degrees in a water bath. The agar was added to prewarmed
(37C) 50% W3CM at one part agar to four parts medium and
mixed thoroughly by pipeting. One-half ml of the 0.6% agar
was added to each well of cell suspension and the contents
of each well were mixed well by pipeting. The cultures
were then allowed to gel at room temperature for ten to
twenty minutes before being transferred to a five percent
carbon dioxide incubator.
Cultures were fed by diluting freshly prepared, 42 to
45C autoclaved agar in prewarmed 50% W3CM at one volume
of agar per nine volumes of media. One ml of the 0.3% agar
was overlayered onto each well and allowed to gel at room

148
temperature as described above. Cultures were fed once
weekly until the colonies were enumerated and analyzed.
Surface Markers on CFU-MC: Sorting of Mast Cell
Precursors with Monoclonal Antibodies
Mast cell precursors (CFU-MC) in adult BALB/cAN bone
marrow were screened for the presence of determinants
recognized by the monoclonal antibodies B23.1 (Katz et al.,
1983) and RA3-3A1 (Coffman and Weissman, 1981b) and for
expression of receptors for IgE. Initial experiments
designed to define the optimum method for selection of
cells with the desired phenotype, when performed with tumor
cell controls, indicated that neither panning (Wysocki and
Sato, 1978; Kay et al., 1977) nor complement-mediated
cytolysis were effective. A procedure for selective
depletion of rosetted cells (D. Levitt, personal
communication), however, proved effective in achieving the
desired goal.
B23.1 and RA3-3Al-positive cell selection
Hybridoma tissue culture fluids were obtained from
high cell density cultures, cleared by centrifugation at
2000xg for fifteen minutes, filtered through 0.2 micron
membranes, and were stored at 4C after addition of 0.01 to
0.02 percent w/v sodium azide. The hybridoma supernatants
were recleared centrifugally, warmed to room temperature
and placed in a beaker with a magnetic stir bar. Sodium
sulfate (Fisher Scientific Co.) was added slowly (over the

149
course of one to one and one-half hours) to the stirring
liquid to a final concentration of 9g per 50 ml. The
slurry was poured into large polyethylene tubes and
centrifuged at room temperature in a J-21C centrifuge with
a JA-20 rotor (Beckman, Palo Alto, CA) for fifteen minutes
at 5000 RPM. The supernatant was discarded and the pellet
was dissolved in a small volume of phosphate-buffered
saline (PBS) and dialyzed extensively (two changes of 400
to 1000 volumes) against 0.1M sodium bicarbonate (Fisher
Scientific Co.) at 4C. The protein concentration of the
dialysate was estimated by absorbance at 280 nm in a SP8-
100 ultraviolet spectrophotometer (Pye Unicam, Ltd.,
Cambridge, UK) prior to further processing. N-
hydroxysuccinyl biotin (NHS-biotin, Sigma Chemical Co.) was
dissolved in dimethylsulfoxide (Sigma Chemical Co.) at 1 mg
per ml. The sodium bicarbonate dialysate and NHS-biotin
were mixed vigorously at 200 micrograms of derivatized
biotin per milligram of protein, then incubated for four
hours at room temperature on a rotator. The modified
antibody preparation was dialyzed extensively against PBS
at 4C and was filtered through a 0.2 micron disposable
nitrocellulose filter (Gelman Sciences, Inc., Ann Arbor,
MI) into a sterile polypropylene tube (Fisher Scientific
Co.) prior to storage at 4C.
Sheep red blood cells (SRBC, from blood freshly drawn
at the J. Hillis Miller Health Center Animal Resources

150
facilities and diluted with an equal volume of sterile
Alsever's solution (Mishell and Shiigi, 1980)) were washed
four times by centrifugation (ten minutes, 400xg, room
temperature) with sterile 0.15 M saline. The pellet of the
final wash was resuspended by flicking the tube and an
equal volume of filter-sterilized 0.5 mg per ml egg avidin
(Sigma Chemical Co.) was added. Two ml of filter-
sterilized 0.1 mg per ml chromic chloride (Fisher
Scientific Co.) was added dropwise to the red blood cell-
avidin mixture with constant, low speed vortexing over two
to three minutes at room temperature in a biological
containment hood. The reactants were held an additional
five to ten minutes at room temperature and then the
avidin-modified sheep red blood cells were washed four
times by centrifugation with balanced salts solution (BSS).
The cells were resuspended to 5 percent v/v in BSS and
stored for up to one week at 4C.
Bone marrow cells were prepared from two to four month
old BALB/cAN female mice as previously described. After
counting the washed cells, 1x10^ to 3x10^ cells were
pelleted by centrifugation and washed once in cold PBS.
The pelleted cells were resuspended in cold, biotin-
modified antibody. Pilot studies, using tumor cells rich
in the surface markers (11P62-4 for B23.1, 18-81 for RA3-
3A1), indicated 0.1 ml of antibody preparation per 1x10^
cells provided optimum labeling; the same volume to cell

151
number ratio was used in the depletion experiments.
Following a thirty minute incubation at 4C, the cells were
washed three times by centrifugation in cold BSS and
resuspended in avidin-modified SRBC at a ratio of 100 SRBC
per bone marrow cell. The cells were pelleted for one
minute at 200xg (4C) and then incubated on ice for twenty
minutes. The cells were then centrifuged for an additional
five minutes, the supernatant was removed and the pellet
resuspended by flicking. The cells were overlayered on 3
ml of isotonic Ficoll-Hypaque (Lympholyte M, Accurate
Chemical and Scientific Corp., Hicksville, NY) and
centrifuged for twenty minutes at 800xg (room temperature).
The separation medium and its contained cells were
carefully transferred to a second tube, the pelleted
rosettes and free SRBC were flicked, and both were washed
twice in cold PBS prior to lysis with 0.17M ammonium
chloride, 0.01M HEPES, pH 7.35 (Hall, 1981).
Alternatively, the density-separated populations were
depleted of red blood cells by hypotonic shock (Mishell and
Shiigi, 1980) after two washes in room temperature BSS.
The red blood cell-depleted, sorted bone marrow cells were
washed two times with cold PBS or room temperature BSS (as
appropriate) and resuspended in EM for counting. Counted
cells were resuspended in 50% W3CM for plating in 0.3
percent agar as previously described.

152
IgE receptor selection
Media from high density cultures of hybridoma IgELa2
(American Type Culture Collection, Rockville, MD) were
cleared by centrifugation and preserved with sodium azide
as previously described. The processed supernatants were
dialyzed for two days against PBS (three changes of 200
volumes) at 4C to remove the sodium azide, filter
sterilized and stored at 4C in a polypropylene tube.
Sheep red blood cells in Alsever's solution (see
above) were washed four times by centrifugation with PBS/
one percent w/v glucose. Haptenating reagent was prepared
by dissolution of trinitrobenzene sulfonic acid (TNBS,
Sigma Chemical Co.) in 0.28M cacodylate buffer, pH 6.9 at
18.75 mg TNBS per ml and sterile filtration (Mishell and
Shiigi, 1980). The sterile TNP-SRBC were prepared by
dropwise addition of one volume of the flicked cell pellet
into seven volumes of haptenating reagent with constant,
gentle mixing. The reagents were pipeted back into the
tube previously emptied of the SRBC and then incubated on a
rotator for thirty minutes at room temperature. Following
centrifugation and removal of the supernatant, the TNP-SRBC
were washed in PBS/one percent w/v glucose/one percent v/v
heat inactivated fetal bovine serum four to six times,
until the supernatants were colorless, resupended to 5
percent by volume and stored at 4C.

153
Washed and counted bone marrow cells were incubated
with PBS-dialyzed, filter-sterilized IgE anti-DNP2
hybridoma supernatants; again, 0.1 ml of antibody
preparation was used per 1x10^ cells (empirically
determined to be optimum) and 1x10^ to 3x10^ cells were
sorted at one time. The tubes containing the cells were
incubated for one hour at 37C, centrifuged (ten minutes at
200xg, 4C) and washed twice with cold BSS. The pelleted
cells were flicked, resuspended in heavily-modified TNP-
SRBC (in 100-fold excess) and centrifuged one minute at
375xg (4C). Following a twenty-minute incubation on ice,
the tubes were centrifuged at 375xg for an additional five
minutes, the supernatants removed and the resuspended
pellets carefully overlayered on 3 ml of Lympholyte M.
Remaining procedures were identical to those performed on
B23.1 and RA3-3Al-sorted cells, with the notable exception
that only hypotonic shock was found to be effective at
lysing TNP-SRBC.
Multiple sorting experiments were performed as
described for single sorting, with the following
exceptions: Bone marrow cells were incubated with one
antibody preparation, washed three times with cold PBS and
then resuspended in the second antibody preparation for an
additional thirty minute incubation. Following washes as
noted before, the bone marrow cells were mixed with either
200 fold excess of avidin-SRBC or 100 fold excess each of

154
avidin-SRBC and TNP-SRBC, as appropriate. Further
processing was identical to that noted above, with
hypotonic shock used to deplete the reactants of red blood
cells.
Results
Frequency of Mast Cell Precursors in Midgestational
Embryonic Tissues
Following cultivation of disaggregated placental- or
nonplacental embryonic tissue (NPET)-derived cells in 0.3
percent w/v agar, colonies were observed at the time of the
first feeding (one week in culture) and at each subsequent
weekly feeding. No attempt was made to quantitate the
number of mast cell colonies prior to three weeks in
culture because previous experience with liquid culture-
derived mast cells indicated that early cultures were
heterogeneous in morphology and cell surface phenotype
(CHAPTER III). Reports of other cell lineages in similar
semisolid cultures of less than three weeks duration
confirmed our observations (Nakahata et al., 1982b; Pharr
et al., 1984). All observations were therefore made
between twenty-one and twenty-eight days of culture. Mast
cell colonies, which predominate in long-term cultures,
were identified by distinctive colony and cell morphology
(Figure IV-1A, -IB). Non-mast cell colonies, consisting

155
Figure IV-1. Colonies in Long-Term Agar Cultures of Embryonic
Cells in Conditioned Media.
Colonies of (BALB/c x CBA)F1 placental cells (day 12)
were photographed at constant magnification after four
weeks of growth in 0.3% agar with 50% W3CM.
(A) Small to medium mast cell colony;
(B) large mast cell colony;
(C) adherent, non-mast cells;
(D) diffuse non-mast cells;
(E) small granulocyte colony;
(F) mast cells were picked from the colony in A and
stained with toluidine blue to visualize metachromatic
granules.

156

157
of several distinguishable cell types (Figure IV-1C, -ID,
-IE) were not enumerated in these studies. In addition to
the colonial populations, long-term agar cultures also
contained a large number of adherent cells. Although the
identity of mast cells was not verified for each colony
counted, staining of cells (picked from agar) with
toluidine blue indicated that more than ninety percent of
the colonies enumerated contained cells with metachromatic
granules indicative of mast cells (Figure IV-IF).
Cells for culture were prepared from embryonic tissues
of the two FI crosses (BALB/cAN x CBA/J and BALB/cAN x
B10.BR/SgSn) used in the Abelson murine leukemia virus
infection experiments and from BALB/cAN homozygous embryos.
As seen in Figure IV-2, the number of mast cell precursors
in embryonic tissues is greatest in midgestation, peaking
at eleven or twelve days post coitum and following the same
distribution in both placenta and nonplacental embryonic
tissues. This tendency was most evident in the
heterozygous tissues, notably (BALB/cAN x CBA/J)F1, in
which the number of mast cell colonies per one million
inoculating cells increase twenty- to thirty-fold between
days eight and twelve post coitum (Figure IV-2A). The
increase in mast cell precursors during midgestation was
less pronounced, but still significant, in the cells
derived from (BALB/cAN x BIO.BR/SgSn)Fl embryonic tissues,
peaking between days ten and twelve with a ten- to twenty-

8 9 10 II 12 8 9 10 II 12 8 9 10 II 12
DAYS OF GESTATION
Figure IV-2. Frequency ot Mast Cell Precursors in Midgestation Embryonic Tissues
Bars represent weighted means ot 2 to 11 samples; error bars represent weighted
standard deviations. Tissues analyzed: TC: cotal conceptus; P: placenta;
E: nonplacental embryonic tissues.
158

159
fold increase over the levels observed at day eight (Figure
IV-2B). In cells derived from homozygous tissues,
however, the tendency toward increased numbers of mast cell
precursors as embryonic development proceeded was much more
subtle. Although the numbers of embryonic precursors were
similar to those observed in heterozygous crosses earlier
in gestation (day eight), the number of mast cell colonies
in BALB/cAN tissues increased only three- to five-fold
(Figure IV-2C).
Frequency of Mast Cell Precursors in Embryonic Tissues of
the Third Trimester of Gestation
The increase in mast cell precursors in midgestation
embryonic tissues, most dramatically demonstrated in the
(BALB/cAN x CBA/J)F1 crosses, prompted us to ask whether
the numbers were maintained in the placenta throughout the
remaining course of gestation. As seen in Figure IV-3,
the number of embryonic mast cell precursors in the
placenta decreased seven- to eight-fold between days twelve
and thirteen and then continued to fall rapidly to the
lowest levels observed in our experiments by day fifteen.
Frequency of Mast Cell Precursors in Adult Bone Marrow
The number of mast cell precursors in the bone marrow
of adult mice has been analyzed by a number of other
investigators, thus providing a methodological control and
standard to which the frequency of mast cells in embryonic
tissue could be compared. It was additionally of interest

160
DAYS OF GESTATION
Figure 1V-3. Frequency of Placental Mast Cell Precursors in the
Third Trimester of Gestation.
Mast cells were cultured from (BALB/cAN x CBA/J)F1 placenta as
indicated in Materials and Methods. Bars represent weighted
means (Bahm,1972) of 5 to 13 samples; error bars represent
standard deviations.

161
to determine whether the elevated number of mast cell
precursors observed in heterozygous midgestation embryonic
tissues, when compared to homozygous embryonic tissues, was
maintained in the adult. As seen in Table IV-1, the
number of precursors in BALB/cAN bone marrow was determined
to be one per 2500 nucleated cells. These numbers are
similar to those previously reported for other agar assays
(Schrader et al., 1981; Sredni et al., 1983), as well as
methylcellulose assays (Nakahata et al., 1982b; Pharr et
al., 1984; Suda et al., 1985), and limiting dilution assays
(Crapper and Schrader, 1983; Guy-Grand et al., 1984). In
addition to the bone marrow data, we found that adult mouse
spleen contained 30 mast cell precursors per million input
cells, again consistent with previously published results
(Nakahata et al., 1982b; Crapper and Schrader, 1983; Guy-
Grand et al., 1984; Pharr et al., 1984).
Analysis of bone marrow derived from both of the
heterozygous crosses demonstrated that these tissues are
equally rich reservoirs of mast cell precursors. No
quantitative differences in mast cell precursor frequency
in (BALB/cAN x CBA/J)F1 and (BALB/cAN x BIO.BR/SgSn)Fl bone
marrows were found, nor was there a tendency toward
elevated precursor numbers in the heterozygotes when
compared to homozygotes. The differences in precursor

Table IV-1. Frequency of Mast Cell Precursors in Adult Bone Marrow from Homozygous and Heterozygous Mice
Number of Colonies per Million Bone Marrow Cells3
Strain
Experiment 1
Experiment 2
Experiment 3
Experiment 4
All Experiments
BALB/cAN
450+/-150(2)
378+/-25.4(4)
371+/-20.9(3)
391+/-51.6(9)
BIO.BR/SgSn
352+/-34.7(3)
308+/-8.5(3)
290+/-10(2)
320+/-18.7(8)
CBA/J
435+/-18.7(3)
398+/-13.1(3)
408+/-16.5(3)
420+/-8.2(3)
415+/-14.1(12)
(BALB/c x BIO.BR)F1
301+/-13.4(4)
304+/-28.1(4)
377+/-28.7(3)
382+/-6.2(3)
336+/-19.3(14)
(BALB/c x CBA)F1
338+/-22.8(4)
350+/-36(4)
403+/-20.5(3)
402+/-19.3(3)
369+/-25.3(14)
a: Mast cell cultures in agar were prepared as described in Materials and Methods. Numbers represent
statistical means +/- one standard deviation (number of cultures). Data for all experiments were
calculated as weighted means and standard deviations (Bahn,1972).
162

163
frequency observed between heterozygous and homozygous
tissues, therefore, are only limited temporally to a short
period during midgestation.
Analysis of Surface Markers and Metachromasia of Colony
Cells
Mast cell colonies are the predominant colony type
between three and four weeks of in vitro growth in agar;
other cell types, however, do persist in the presence of
WEHI-3 conditioned media. We have frequently observed
partially or confluent monolayers of adherent cells,
sometimes associated with colonies of mast cells, as seen
in Figure IV-1C. Also evident are colonies of cells both
larger and smaller than mast cells which have distinctive
morphologies (Figure IV-ID, -IE). The lineages of the
non-mast cell types were not investigated in these studies,
as they have been extensively described in the past
(Nakahata et al., 1982b; Pharr et al., 1984).
Although it would be technically impossible to verify
the identity of the cells comprising each enumerated mast
cell colony, we picked a number of representative colonies
to that end. The presence of metachromatic granules is a
hallmark of mast cells of both the connective tissue and
mucosal/cultured mast cell types (Jarrett and Haig, 1984).
Macroscopic colonies of mast cells were identified
initially at 100X magnification on an inverted, phase
contrast microscope (Swift Instruments International,

164
Tokyo, Japan) and were picked under sterile conditions by
gentle aspiration with a Pasteur pipet. The colonies were
expelled from the pipet into a well of a 96-well tissue
culture cluster (Costar, Broadway, MA) and the cells were
disaggregated by gentle trituration with 0.1 ml of 50%
W3CM. Some cells were analyzed immediately, while others
were incubated in a humidified atmosphere of five percent
carbon dioxide in air for one to two days prior to
analysis. Approximately half of the cells were prepared
for toluidine blue staining by cytocentrifugation and air
drying. Following fixation in Mota's fixative (Yam et al.,
1971), or acidic ethanol (Mota's fixative without lead
subacetate), the slides were stained with 0.1 percent w/v
toluidine blue, ph 0.5. Most (at least 90%) of the cells
from each colony contained metachromatic granules
indicative of mast cells (Figure IV-IF, Table IV-2).
Similarly identified and picked cells were also
analyzed for surface markers as previously described. The
cells had receptors for IgE but not for IgG, further
confirming their identity as mast cells. Most of the cells
also expressed the surface determinant recognized by
monoclonal antibody B23.1. As previously noted, the latter
marker is expressed by both mast cells and mononuclear
phagocytes.
The expression of the B220 lymphoid marker recognized
by the monoclonal antibody RA3-3A1 on A-MuLV-transformed

165
Table IV-2. Expression of Surface and Cytochemical
Markers on Colony-Derived Mast Cells3.
Percent cells expressing marker^
Colony
Source
RA3-3A1
B23.1
IgE
Metachromatic
granules
dlO embryo
67
84
56
100
dlO placenta
55
65
48
89
dll embryo
36
83
51
100
dll placenta
47
78
53
96
dl2 placenta
37
89
55
99
bone marrow
43
94
52
94
bone marrow
47
91
54
97
3 cells were cultured in 0.3% agar, 50% W3CM, for three to
four weeks before analysis,
k cells were analyzed for cell surface markers and
toluidine blue metachromasia as indicated in Materials
and Methods.

166
placental cells prompted us to assay for its expression on
colony-derived mast cells. Most of the colonies analyzed
contained a significant number of cells which expressed the
antigen recognized by the RA3-3A1 antibody (Table IV-2),
in contrast to the results of analyses of numerous liquid
culture-derived mast cells from similar sources (Chapter
III) .
Surface Markers on Mast Cell Precursors: Sorting of Cells
with Monoclonal Antibodies
The presence of the determinants recognized by
monoclonal antibodies B23.1 and RA3-3A1, and well as
receptors for IgE, on the surface of both transformed and
agar-cultured mast cells led us to ask whether these
antigens were also expressed on the agar-colony mast cell
precursors. Conditions for the sorting experiments were
initially optimized on tumor cell lines previously shown to
express high levels of the markers of interest. As seen in
Table IV-3, biotinylated antibody plus avidin-modified
sheep red blood treatment, followed by density gradient
separation, was effective in depleting the control
populations of most marker-positive cells. Rosetted cells,
observed in suspension after staining nucleated cells with
crystal violet (0.2 percent w/v), accounted for virtually
all of the cells in the marker-positive, Lympholyte M
pellets. On the other hand, few of the cells in the marker
negative gradient supernatant were associated with SRBC in

167
Table
IV-3. Sorting of
Control Cells
by Rosetting
Percent Rosetted Cells
Cell
Rosetting Phase of Separation Crystal
Cyto-
Line
Agents Medium Analyzed
Violet
Centrifuge
Method3
Method^
10P8
IgE/TNP-SRBC
supernatant
7
ND
pellet
96
82
PBS/TNP-SRBC
supernatant
0
ND
pellet
0
0
11P62
B23.1-Biotin
supernatant
11
ND
/Avidin-SRBC
pellet
98
0
PBS/Avidin-
supernatant
0
ND
SRBC
pellet
0
0
18-81
RA3-3A1-
supernatant
8
ND
Biotin/
Avidin-SRBC
pellet
100
95
PBS/Avidin-
supernatant
0
ND
SRBC
pellet
0
0
a. Following sorting of cells, an aliquot was made 0.2%
crystal violet and the number of rosetted and
unrosetted cells per 10-t ml hemocytometer field
were counted on an inverted phase microscope.
b. Following sorting of cells, an aliquot was cyto-
centrifuged and stained with Wright's Giemsa.
Coverslips were affixed with Permount and at least
one hundred cells were microscopically analyzed for
association with three or more sheep red blood cells.

168
suspension. Cytocentrifuged samples of the preparations
showed somewhat lower numbers of rosetted cells, presumably
due to mechanical shearing and lysis of SRBC during the
processing. The latter data, however, are similar to those
generated by the S. aureus-antibody sandwich technique.
The data for the sorting of BALB/cAN bone marrow are
summarized in Table IV-4. Several significant
observations are evident. First, if bone marrow cells
(unfractionated) were incubated on ice but not subjected to
antibody treatment, rosetting, gradient separation, lysis
and numerous centrifugations in the time-consuming (eight
to twelve hour) procedure, the number of mast cell colonies
was reduced approximately forty percent compared to
previous, unsorted experiments. This decrement was
presumably due to the extended processing time in the
absence of growth factor, which was two to three times that
required to do the initial experiments. Second, the
manipulation of cells by incubation with antibody and sheep
red blood cells, separation on a density gradient medium,
incubation with ammonium chloride or hypotonic salts
solutions, and numerous centrifugations resulted in no
significant further depletion of mast cell precursors, as
evident from the similar number of colonies in sorted and
unsorted marrow populations. Finally, the method of lysis
of SRBC did not significantly alter the results of the
assay. This last observation is surprising in light of the

Table IV-4. Sorting of Bone Marrow Cells by Surface Determinants
Number of Colonies per 10^ Cells Analyzed0
Experiment3
Rosetting
Agents^
Unfractionated
Control1^
Marker
Positivee
Marker
Negative^
Reconstructed
Population
1A
B23.1-Biotin/
Avidin-SRBC
212+/
-13(3)
185+/-23(3)
ND§
178+/
-2(2)
IB
RA3-3Al-Biotin/
Avidin-SRBC
212
10(3)
ND
190+/
-22(3)
185
5(2)
1C
IgE/TNP-SRBC
240
14(3)
ND
205
5(2)
148
8(2)
2A
IgE/TNP-SRBC
232
26(3)
2 2(3)
230
22(3)
175h
2B
B23.1-Biotin/
Avidin-SRBC
220
20(2)
222 20(2)
ND
215
15(2)
2C
RA3-3A1-Biotin/
Avidin-SRBC
ND
0(1)
215
55(2)
232
8(2)
a: Experiments 1A, IB, 1C were performed with 3 to 6 x 10^ cells prior to sorting and
with ammonium chloride-mediated SRBC lysis. Experiments 2A, 2B, 2C were performed with
0.7 to 2.8 x 10? cells prior to sorting and with hypotonic shock-mediated SRBC lysis,
b: Sorting was performed with the antibody/SRBC combination as indicated in Materials and
Methods.
c: Numbers represent mean number of colonies (standardized per 10^ cells per well)
+/- 1 standard deviation (number of wells analyzed),
d: Unfractionate controls were kept on ice without rosetting agents for the duration of the
sorting experiment and plated in agar at the same time as the sorted cells,
e: The "marker positive" fraction consisted of sorted cells from the separation medium
pellet which were subsequently depleted of sheep red blood cells,
f: The "marker negative" fraction consisted of sorted cells from the separation medium
supernatant which were subsequently depleted of sheep red blood cells,
g: ND: experiment not done due to insufficient cells in the indicated fractions,
h: Only one well plated due to insufficient cells.
169

170
remaining TNP-SRBC in the cultures of IgE-sorted bone
marrow cells; these cells apparently had no effect on the
proliferation of mast cell precursors in agar.
Sorting of bone marrow with B23.1-biotin and avidin-
modified SRBC resulted in the segregation of all of the
mast cell precursors to the marker-positive fraction.
Ninety to one hundred percent of the precursors were
recovered from the rosetted pellet when compared to
unsorted cells. Dilutions of sorted cells cultured in agar
showed linearity of inoculum versus colony number,
indicating that the assay was a reliable measure of
precursor frequency. If cells from each fraction were
combined in proportion to the number which partitioned with
the rosettes (pellets) or free cells (supernatants), the
frequency of colonies enumerated was the same as that in
unfractionated bone marrow. This last observation
indicated that the minute B23.1 marker-negative population
contributes neither stimulatory nor inhibitory factors
required for the efficient seeding of mast cell precursors.
When bone marrow cells were sorted with biotinylated-
RA3-3A1 antibody and avidin-modified SRBC, all of the mast
cell precursors were found in the marker-negative
(supernatant) fraction. As seen with the B23.1 sorting,
essentially all (ninety percent) of the mast cell
precursors were recovered after RA3-3A1 sorting, and the
plot of the cells seeded versus mast cell colonies was

171
linear in two experiments. The number of mast cell
colonies observed after reconstitution of both fractions at
the correct ratio was very close to the anticipated value
in one experiment and slightly greater than (140 percent)
the anticipated value in the second experiment, perhaps
indicating synergism between the partitioned cell types in
the formation of colonies. Further experiments to test the
cooperative hypothesis would be required before the latter
could be concluded.
Bone marrow sorted with IgE anti-TNP and TNP-SRBC
exhibited partition characteristics similar to that sorted
with RA3-3A1. Eighty-five to ninety-nine percent of the
mast cell precursors were recovered following sorting in
two independent experiments using two different methods to
lyse the SRBC. Cells seeded versus colonies enumerated
were linear in both experiments, and the number of colonies
counted in reconstitution experiments was similar to those
anticipated if only cells from the marker-negative
(supernatant) population were seeded, indicating that the
mast cell precursors found in the IgE receptor-negative
fraction were independent of IgE receptor-positive cells in
their ability to form mast cell colonies.

172
Discussion
The development of mast cells from embryonic tissues
in liquid cultures led us to quantitate the number of mast
cell precursors in those tissues. The frequency of such
precursors has historically been analyzed by several
methods. Nakahata et al. (1982b) developed a culture assay
for mouse mast cell colonies in methylcellulose. The assay
was adapted from previous work (Parmley et al., 1976) which
enumerated multipotent hematopoietic progenitors, but
selected for mast cell growth with lectin-stimulated
conditioned media. Subsequent use of the technique by
others in the same laboratory (Pharr et al., 1984; Suda et
al., 1985) has established the reliabilty of this method.
Concurrent to the development of the methlcellulose assay,
Schrader et al. (1981) and Zucker-Franklin et al. (1981)
reported the development of a similar clonal assay using
agar-based semisolid media with results similar to those
obtained with methylcellulose. In addition to the
semisolid medium techniques, limiting dilution analysis in
liquid culture has been used to quantitate the cultured
mast cell precursor frequency in a variety of tissues
(Crapper and Schrader, 1983; Guy-Grand et al, 1984).
Based on experience, supplies, and the similar results
obtained in independent studies (above), we chose to
analyze the frequency of mast cells in embryonic tissues by

173
a clonal agar assay (Schrader et al., 1981). We modified
the assay, however, to be consistent with our previous
liquid culture experiments (Chapter III), by substituting
WEHI-3 condidtioned media (Razin et al., 1984a) for the Con
A- spleen-conditioned media of those authors. We verified
the reliability of our assay by quantitation of bone marrow
and spleen-derived mast cell precursors and found that our
data were consistent with those previously published.
The results of our analyses of mast cell precursors in
midgestation and late gestation embryonic tissues present
several significant observations. First, we have described
the earliest mast cell precursors reported in the mouse,
being first detected at eight days post coitum. Previous
to this report, the earliest reported mast cell precursors
were derived from disaggregated day ten or eleven embryos
which had been cultured in the presence of T cell-derived
growth factors (Ginsburg et al, 1982). In the mouse,
embryonic mast cell precursors have also been identified in
the fetal liver as early as day thirteen post coitum
(Kitamura et al., 1979c; Nabel et al., 1981). Similar
observations have been reported in the embryonic rat
(Ishizaka et al., 1976) and in the embryonic human (Razin
et al., 1981b).
A second significant observation concerns the
frequency of embryonic mast cell precursors as gestation
progresses. As seen in Figure IV-2, the number of mast

17 b
cell precursors increases between days eight and twelve.
In the most dramatic case studied, this augmentation
represented a twenty- to thirty-fold increase in mast cell
colonies for (BALB/cAN x CBA/J)F1 embryos (reaching levels
similar to those detected in adult bone marrow), while
smaller increases were observed for (BALB/cAN x B10.BR)F1
and homozygous BALB/cAN embryos. The discrepancies between
heterozygous and homozygous embryonic mast cell precursor
frequencies were not observed in adult bone marrow, leading
us to speculate that allogeneic differences may be
responsible for mast cell precursor proliferation at this
critical period of fetal development.
A third significant observation of this study is that
the number of mast cell precursors in the murine embryonic
placenta drops rapidly from its peak at twelve days to the
lowest levels noted in that tissue during the course of the
third trimester of pregnancy. Our observation that day
eleven embryonic tissue contained fewer mast cell
precursors than those of day thirteen were supported by
similar reports in the literature (Ginsburg et al., 1982).
Although precursor frequency in the embryo proper was not
investigated during the same period of time, we have noted
that Kitamura et al. (1979c) reported the isolation of mast
cell precursors in the fetal liver of day thirteen and day
fourteen mouse embryos; the former observation was
substantiated by Nabel et al. (1981).

175
We propose that mast cell precursors in the
midgestation placenta either perish (due to deprivation of
interleukin 3 in their microenvironment or programmed
annihilation) or migrate around day thirteen to a new
microenvironment in the fetal liver, mucosal sites, or
perhaps the thymus. The fetal liver is the major
hematopoietic organ of late gestation (Metcalf and Moore,
1971). In support of the model of mast cell precursor
annihilation in the third trimester placenta is a recent
report of "waves" of pluripotent hematopoietic stem cells
(CFU-S) which respond to interleukin 3 (Spivak et al.,
1985). Like mast cells, CFU-S require interleukin 3 for
survival as well as proliferation in vitro; in the absence
of interleukin 3, CFU-S concentration decreased
precipitously. Even in the presence of interleukin 3, the
number of proliferating cells exhibited cyclic
fluctuations, which may be an inherent property of all
hematopoietic cells (King-Smith and Morley, 1970). If such
processes were in force in vivo as well as in vitro, we
would propose the loss of interleukin 3 responsiveness in
placenta-associated mast cell precursors beginning at day
thirteen (following at least three days of extensive
proliferation) and the concurrent increase in
responsiveness of fetal liver-associated mast cell
precursors (Kitamura et al., 1981) represent these waves.

176
A fourth significant observation of this work concerns
the expression of mast cell-specific markers on bone
marrow-derived cells picked from colonies between three and
four weeks in culture. Initial attempts to propagate mouse
mast cells in agar were met with complete failure (McCarthy
et al., 1980). Colonies, which were observed after one
week in culture, yielded no cells with astra blue-staining
granules characteristic of mast cells. Later investigators
(Schrader et al., 1981; Zucker-Franklin, 1981; Nakahata et
al., 1982b) established that a minimum of two weeks in
culture was required for mouse mast cell maturation.
Longer incubation periods were, in fact, preferable, since
mast cell colonies are less transient than those composed
of other lineages (Pharr et al., 1984). The presence of
mast cells in macroscopic colonies was first confirmed in
situ by microscopic observation of round cells with clearly
delineated, refractile edges and slight dark hue (Nakahata
et al., 1982b). More than ninety percent of the cells
picked from such colonies contained metachromatic granules
when stained with acidic toluidine blue. These cells also
had surface receptors for IgE (but not IgG) and most
expressed the surface determinant recognized by the
monoclonal antibody B23.1, and thus, by three criteria,
could be identified as mast cells.
The colony-derived mast cells also expressed the B-
lineage variant of the Ly5 surface antigen. Ly5 has

177
previously been observed on cultured mast cells (Nabel et
al., 1981; Wong et al., 1982) and mast cell tumors (Scheid
and Triglia, 1979); in addition, it was detected on two A-
MuLV-transformed placental mast cell lines and on the
murine mastocytoma P815 and two A-MuLV/pristane-induced
mastocytomas (Siegel et al., 1985; Chapter II). The
expression of Ly5 on the surface of mast cells may
represent a discreet stage in the maturation which is not
attained under the conditions of our liquid culture system
(Chapter III) .
The final aspect of our studies described in this
chapter dealt with the determination of cell surface
markers on mouse bone marrow-derived mast cell precursors.
Although the literature contains numerous references to
surface markers on liquid culture-derived mast cells
(reviewed by Katz et al., 1985a), we were only able to find
a single reference to expression of such markers on mast
cell precursors (Yung et al., 1983). Treatment of mouse
bone marrow cells with anti-la and rabbit complement
resulted in the loss of fifty percent of the interleukin 3-
responsive (proliferating) population; the same treatment
also depleted granulocyte-macrophage colony-forming units
(CFU-GM) by the same factor.
Three surface determinants detected on A-MuLV-
transformed embryonic mast-like cell lines were selected
for sorting mast cell precursors. The sorting methodology

178
was optimized and proven effective on previously
characterized mouse cell lines (Table IV-3). The time-
consuming technique in the loss of approximately forty
percent of the mast cell precursors, presumably due to the
extended processing time in the absence of interleukin 3
and not numerous manipulations. By combining the sorting
procedure with the cultivation of cells in semisolid agar
media, we were able to effectively deplete all of the mast
cell precursors with B23.1 antibody; in contrast, neither
IgE nor RA3-3A1 significantly depleted bone marrow of mast
cell precursors. These preliminary results, however, were
questionable in light of previous experience with the same
antibodies in the system used to detect surface markers on
liquid culture cell populations (see Chapter III). Under
the latter conditions, fresh bone marrow contained twenty
two percent RA3-3A1 reactive cells and six percent B23.1
positive cells. In the sorting procedure, bone marrow
contained three to twenty-four percent RA3-3A1 positive
cells and ninety-six to ninety-nine percent B23.1 positive
cells. The reason for this discrepancy is unknown, since
all experimental controls gave predicted values. Despite
the intertechnique inconsistencies, we are confident that
few or no mast cell precursors express receptors for IgE or
the determinant recognized by monoclonal antibody RA3-3A1,
while it would appear that most, if not all, may express
the determinant recognized by B23.1. Further studies, with

179
efforts to minimize nonspecific depletion of cell
populations, will be required to better define the surface
phenotype of precursors to cultured mast cells.
The midgestation murine conceptus is the site of a
number of interesting and perhaps time-related phenomena
which may be associated with the presence of culture-
derived mast cell precursors. The pluripotent
hematopoietic stem cell, which gives rise to all of the
hematopoietic lineages and is quantitated in the in vivo
CFU-S assay (Till and McCulloch, 1961), has been isolated
during embryogenisis from the blood islands of the yolk sac
between days eight and ten and from the fetal liver
throughout the remainder of gestation (Moore and Metcalf,
1970). Mast cells have been shown to arise from CFU-S
(Kitamura et al., 1981) as well as multipotent in vitro
colony forming cells (Schrader et al., 1981; Sonoda et al.,
1983; Pharr et al., 1984). Furthermore, CFU-S and their in
vitro correlates are responsive to the proliferative
effects of interleukin 3 (Goldwasser et al., 1983; Garland
and Crompton, 1983; Spivak et al., 1985; Rennick et al.,
1985), the mast cell growth factor prevalent in WEHI-3
conditioned media. The presence of mast cell precursors
in embryonic tissues as early as eight days of gestation
may thus be an indication of multipotent hematopoietic stem
cells or their interleukin 3-responsive progeny which
differentiate into mast cells in the continued presence of

180
that growth factor. The absence of mature mast cells from
the same tissues may be indicative of in vivo control
mechanisms which are abrogated by in vitro propagation.
Abelson murine leukemia virus targets are most
frequent in embryonic tissues at day ten of gestation,
while the number of culture-derived mast cell precursors
peaks at day twelve. A-MuLV is capable of transforming
cells of most of the hematopoietic lineages, among them
mast cells (Chapter III; Pierce et al., 1985). We propose
that the cells detected in the transformation assay are
precursors to the untransformed cells which proliferate in
response to interleukin 3. The difference between the two
populations may be one of yet undescribed differentiation
markers: Cells which are targets for A-MuLV, not yet
responsive to interleukin 3, may further differentiate into
cells which are responsive to interleukin 3 but no longer
possess receptors for the transforming virus. This model
remains untested and its mechanisms demand further
elucidation. We have previously proposed that the A-MuLV
oncogene product may substitute for interleukin 3, thus
inducing the mast cell-like characteristics of our
transformed placental cell lines (Siegel et al., 1985).
The expression of c-abl messenger RNA, which also peaks in
the embryo at ten days of gestation (Muller et al., 1982)
and may encode a growth factor activity which normally acts

181
on the virus transformation-sensitive target cells which
may then differentiate into interleukin 3- responsive
culture-derived mast cell precursors.
The studies described in this chapter may also
contribute to the understanding of the immunobiology of the
maternal-fetal interface. The allogeneically foreign
conceptus first presents immunogenic antigens from the
cytotrophoblast to its mother between days nine and eleven
of gestation (Roe and Bell, 1982). The spectrum of
maternal-fetal immunological reactions have been
extensively reviewed in the literature (Bell and
Billington, 1983; Chaouat et al., 1983; Lala et al., 1983).
The mother, recognizing the fetal graft as nonself,
responds with both humoral and cell-mediated arms of the
immune system. The placenta, which has been described as
an immunological barrier and an immunoadsorbent (Wegmann et
al., 1979), also has been reported to play an active
immunoregulatory role (Chaouat et al., 1980; Remacle-Bonnet
et al., 1983; Chaouat and Kolb, 1985), although the
cellular source(s) of immunomodulatory factors is not
defined. Histamine, which is reported to play an important
role in blastocyst implantation (Dey et al., 1979; Dey and
Johnson, 1980a, 1980b; Nalbandov, 1971), has also been
shown to inhibit cytotoxic T lymphocyte effector functions
(Plaut et al., 1973; Schwartz et al., 1980; Chaouat and
Kolb, 1985), to inhibit the production of macrophage

182
inhibitory factor (Rocklin, 1976) and to have other
immunomodulatory effects (Askenase et al., 1981) as well as
promoting tissue growth and repair (Kahlson and Rosengren,
1968). Prostaglandins and leukotrienes, related mast cell
products, may play similar immunomodulatory roles (Lala et
al., 1983). Although our studies of freshly dissociated
tissues have indicated that mast cells are, at most, a
minor fraction of the total conceptus between days ten and
twelve post coitum, a small but significant population of
cells at the maternal-fetal interface could indeed
contribute to the maintenance of the fetal graft.

CHAPTER V
SUMMARY AND CONCLUSIONS
The experimental procedures described in the preceding
pages have enabled us to report a number of novel and
significant observations which contribute to the existing
body of knowledge concerning mast cells. We began our
studies following the observation of cells with basophilic
and metachromatic granules in lines of Abelson murine
leukemia virus-transformed cells which were derived from in
vitro-infected midgestation mouse embryonic placentae. The
relationship of the A-MuLV-transformed cells to culture-
derived mast cells was further substantiated by the
observation that both expressed the epitope recognized by
the B23.1 monoclonal antibody, which binds to a determinant
on culture-derived, but not peritoneal, mouse mast cells.
Most of the cell lines also contained histamine in
quantities similar to those found in culture-derived mast
cells. Furthermore, there was a direct correlation between
the histamine content and the expression of surface IgE
receptors, another mast cell phenotypic marker, in the cell
lines which were analyzed. In contrast to culture-derived
mast cells, however, the A-MuLV-transformed mast-like cells
proliferated in the absence of exogenous interleukin 3.
Unlike other autogenous mast-like cells (Schrader and
Crapper, 1983) but similar to recently reported A-MuLV
fetal liver transformants (Pierce et al., 1985), the cell
lines produced no detectable interleukin 3.
183

184
In the course of characterizing the Abelson virus-
transformed embryonic cell lines, we developed a sensitive,
nonisotopic, nonfluorometric assay for membrane receptors
for cytophillic immunoglobulin. The method utilizes
hapten-specific monoclonal antibodies and homologous
hapten-derivatized bacteria which form rosettes with cells
bearing the appropriate receptors. The marker positive
cells are easily identified by light microscopy of
cytocentrifuged, fixed, and stained preparations. The
technique has the advantage over previously described
isotopic (Mendoza and Metzger, 1976) and nonisotopic
(Schrader, 1981) methods of allowing morphological
characterization of the reactive cells. This methodology
is applicable to the characterization of other
hematopoietic lineages and can be modified to identify two
specificities on the same cell (Siden and Siegel, 1986).
The identification of mast cell characteristics in
lines derived by the transformation of midgestation
enbryonic placenta by Abelson murine leukemia virus led us
to analyze homologous, untransformed embryonic tissues for
mast cells and their precursors. Similar to the
observations of Kitamura and colleagues (1979c) on mouse
fetal liver as early as day thirteen post coitum, we
detected no mast cells in embryonic tissues at days ten and
eleven of gestation. We were, however, able to culture
mast cells from those tissues, thus providing the novel

185
observation that mast cell precursors exist in the mouse
embryo at least five days before the first mast cells are
detectable (Kitamura et al., 1979c). Subsequent analysis
of mast cell precursors in embryonic tissues between days
eight and nineteen of gestation indicated that the
precursors to culture-derived mast cells exist in very low
numbers prior to day nine of gestation, increasing by day
twelve to levels similar to those found in adult bone
marrow. The number of embryonic placental precursors to
culture-derived mast cells then falls precipitously between
days thirteen and nineteen of gestation. The results are
particularly interesting in light of the previously noted
observation that jm vivo mast cell precursors are abundant
in the fetal liver at day thirteen (Kitamura et al.,
1979c). It is enticing to speculate that mast cell
precursors either migrate through the placenta to the fetal
liver between days twelve and thirteen, or an independent
second "wave" of precursors develops in the fetal liver at
that time; however, there is no definitive evidence that
the embryonic in vitro and in vivo mast cell precursors are
identical.
In the course of our investigations, we studied the
progression of five hematopoietic markers in mast cell
cultures of embryonic and adult tissues over the course of
four weeks. Previous reports of phenotypic changes in mast
cell cultures were limited to morphological and

186
cytochemical analysis performed at the microscopic level,
with surface determinant characterization performed on
mature culture-derived mast cells. In contrast, our
investigations documented the sequential selection and
enrichment of cells expressing both membrane receptors for
IgE and the culture-derived mast cell determinant
recognized by the monoclonal antibody B23.1 (Katz et al.,
1983), as well as metachromatic granules, from a
heterogeneous population of cells relatively impoverished
of the markers. We feel that our observations complement
those of Ginsburg (1963) and later investigators and add a
further level of analytical sophistication to their
historical contributions.
Two features of the Abelson virus-transformed mast
cell-like lines, interleukin 3 independence and expression
of the B lineage variant of the Ly 5 differentiation
marker, were notably absent from cultured mast cells
derived from the same tissues. Culture-derived mast cells
were infected with Abelson virus to investigate the role of
that agent in the induction of those characteristics. The
virus-infected cells exhibited factor-independent growth,
but were still devoid of the lymphoid marker. Observation
of nonadherent cells expressing the same lymphoid
determinant in cultures of unselected (adherent and
nonadherent) uninfected cells grown in the presence of
interleukin 3 prompted us to investigate the role of

187
adherent cells and their factors on culture-derived mast
cells. Long-term culture-derived mast cells did not
express the B lineage variant of Ly 5 before or after
coculture with adherent cells of several sources. We have
therefore concluded that neither Abelson murine leukemia
virus nor adherent cell monolayers were responsible for the
expression of a B lymphoid determinant on the mast-like A-
MuLV-transformed cell lines.
In the final chapter of this dissertation, we
enumerated and characterized mast cell precursors in
embryonic tissues and in adult bone marrow. Mast cells
derived from semisolid agar cultures expressed the B
lymphoid Ly 5 variant previously noted on the surfaces of
A-MuLV-transformed mast-like cells. Based on these and
previous observations, we have proposed that the expression
of such surface markers on agar culture-derived mast cells
represents a discreet stage of mast cell differentiation
which is not observed in liquid culture.
The ability to ascribe surface determinants to
cultured mast cell precursors may provide us with a better
understanding of the differentiation of all mast cells. We
have sorted primary bone marrow cells with monoclonal
antibodies which recognize surface determinants previously
noted on culture-derived mast cells and on A-MuLV-
transformed mast-like cells. Cells lacking the B
lymphoid variant of Ly 5 and receptors for IgE were

188
observed to provide a rich source of mast cell precursors,
while cells expressing the same markers produced few mast
cell colonies in semisolid agar. In contrast, cells
rosetted by red blood cells bearing monoclonal antibody
B23.1 were as rich in mast cell precursors as untreated
cells. These preliminary studies have significantly
contributed to the meager body of information which
describes the phenotype of the culture-derived mast cell
precursor (Yung et al., 1983).
In conclusion, the investigations presented in this
dissertation have demonstrated the existence of culture-
derived mast cell precursors in the earliest reported
stages of murine development. Although its role in
hematopoiesis is yet undefined, the embryonic mouse
placenta is a rich source of culture-derived mast cell
precursors for a brief period of fetal development. These
findings may serve as an impetus for further studies which
will better define the role of interleukin 3-responsive
cells in embryogenesis and mast cell differentiation.

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BIOGRAPHICAL SKETCH
Michael L. Siegel was born on June 15, 1949, in New
York City, the younger of two sons of Frances and Seymour
Siegel and the brother of Victor (who sometimes claimed to
be an only child). After a relatively uneventful childhood
and adolescence in Punta Gorda, Florida, Bronx, New York,
and Spring Valley, New York, Michael attended Cornell
University and managed to achieve a bachelor of science
degree in animal science while attending Woodstock,
protesting social injustice and the American way, and
locking in to age seventeen. Late in his senior year at
Cornell, Michael abandoned his lifelong goal of becoming a
veterinarian and decided instead to pursue an academic and
research career at the University of Florida. His new
pursuit was soon postponed, however, as financial problems
forced him to withdraw from classes.
Serendipidously, Union Carbide Corporation was looking
for someone with Michael's background (so he convinced
them), and thus began another era in his life. Michael
worked for Union Carbide for five years developing
radioimmunoassays, and then accepted an offer from Meloy
Laboratories to manage its immunoreagents production unit.
Living in Manassas, Virginia, Michael met his wife,
Jeremie, and her children, David and Rebecca. In June of
219

220
1980, Meloy Laboratories reduced its middle management
staff, allowing Michael the opportunity to return to
graduate studies at the University of Florida. Following
completion of his doctoral studies, Michael will study
retroviral oncogenesis and hematopoiesis in the laboratory
of Carlo Moscovici, continue his attempts to find
renaissance, and, as always, stay seventeen.

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of.Doctor of Philosophy.
Edward J. Siden, Chairperson
Assistant Professor of Immunology
and Medical Microbiology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
u /d'T ll /l
Ja>s B. Flanegan/
Associate ProfqaVor of Immunology
and Medical Microbiology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree pf Doctor of Philosophy.
Carlo Mocovici
Professor of Pathology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of l^ilosophy.
O.X"
Stephen W. Russell
Professor of Pathology and
Veterinary Medicine
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Weiner
Professor of Immunology and
Medical Microbiology

This dissertation was submitted to the Graduate Faculty
of the College of Medicine and to the Graduate School and
was accepted as partial hu-Hsi 1 Iment of the requirements for
the degree of Doctor of Phi/l())pophy.
May, 1986
Den, Gradd
late SchopJ



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FL.


9
implantation (Nalbandov, 1971; Dey and Johnson, 1980a,
1980b; Dey, 1981), as were prostaglandins (Kennedy, 1977),
but the number of detectable mast cells in the gravid
uterus was shown to decrease after implantation
(Shelesnyak, 1960; Brandon and Bibby, 1979). Mast cell
association with nerve tissue was first noted in 1878
(cited in Selye, 1965) and intimate contact between nerve
endings and mast cell plasma membranes was documented by
Weisner-Menzel and colleagues (1981) and Newson and
colleagues (1983). Histamine release has been attributed
to stimulation of cutaneous nerves (Kiernan, 1972), thus
establishing a neuroendocrine-mast cell axis. Mast cells
in alimentary tract mucosa have been implicated in
promotion of gut mobility (Erjavek et al., 1981; Fjellner
and Hagermark, 1981); histamine-induced gastric secretion
was shown to be blocked by histamine H2 receptor agonists
(Soli et ai., 1981) and enhanced by glucocorticoids
(Sathiamoorthy et al., 1976). The latter observation is
contradictory to published reports of glucocorticoid-
induced suppression of intestinal anaphylaxis (King et al.,
1985), and may indicate different modes of action of
steroids on sensitized and nonsensitized mast cells. Thus,
Daeron and colleagues (1982) noted that glucocorticoids
inhibited antigen-induced, but not calcium ionophore
A23187-induced, histamine release from mast cells.


131
Figure III-7. Abelson Murine Leukemia Virus-Infected Mast
Cells Express v-abl Gene Product.
Proteins in A-MuLV-infected cells and controls were
biosynthetically labeled and immunoprecipitated with goat
anti-Moloney MuLV prior to SDS-polyacrylamide gel electrophoresis,
fluorography, and autoradiography as described in Materials and
Methods. Electrophoresis standards used were (bars on right,
from top to bottom) myosin (205 kilodaltons, Kd), beta-
galactosidase (116 Kd), phosphorylase B (97.4 Kd), and bovine
serum albumin (66 Kd). Lanes 1, 3, and 5 were loaded with immune
precipitated cell lysates; lanes 2 and 4 were loaded with lysates
adsorbed with normal goat serum. Lanes 1 and 2, A-MuLV-transformed
placental cell line 10P12; lanes 3 and 4, A-MuLV-transformed
culture-derived mast cells; lane 5, sham-infected culture-derived
mast cells. Upper arrow indicates v-abl gene product; lower
arrow indicates major Moloney-MuLV gene product (Pr65).


163
frequency observed between heterozygous and homozygous
tissues, therefore, are only limited temporally to a short
period during midgestation.
Analysis of Surface Markers and Metachromasia of Colony
Cells
Mast cell colonies are the predominant colony type
between three and four weeks of in vitro growth in agar;
other cell types, however, do persist in the presence of
WEHI-3 conditioned media. We have frequently observed
partially or confluent monolayers of adherent cells,
sometimes associated with colonies of mast cells, as seen
in Figure IV-1C. Also evident are colonies of cells both
larger and smaller than mast cells which have distinctive
morphologies (Figure IV-ID, -IE). The lineages of the
non-mast cell types were not investigated in these studies,
as they have been extensively described in the past
(Nakahata et al., 1982b; Pharr et al., 1984).
Although it would be technically impossible to verify
the identity of the cells comprising each enumerated mast
cell colony, we picked a number of representative colonies
to that end. The presence of metachromatic granules is a
hallmark of mast cells of both the connective tissue and
mucosal/cultured mast cell types (Jarrett and Haig, 1984).
Macroscopic colonies of mast cells were identified
initially at 100X magnification on an inverted, phase
contrast microscope (Swift Instruments International,


118
and enrichment, a process requiring at least four weeks in
culture. Characteristics such as IgE receptors,
metachromatic cytoplasmic granules (Schrader, 1981) and a
surface determinant recognized by the monoclonal antibody
B23.1 (Katz et al., 1983) have been ascribed to such cells.
We chose to analyze the expression of these markers, as
well as markers found on A-MuLV-transformed embryonic mast
cells, on the heterogeneous populations of cells from which
mast cells evolve to better understand the evolution of
homogeneous mast cell cultures.
Embryonic tissues
The first significant finding of this study was that
mast cells or their precursors exist in the midgestation
embryonic placenta and nonplacental embryonic tissues
(NPET). The existence of mast cells in such tissues was
hypothesized after homologous A-MuLV transformants were
characterized (CHAPTER II). As seen in Figure III-l, the
number of liquid culture cells with cytoplasmic
metachromatic granules (Figure III-2) rose from less than
one percent in freshly disaggregated tissues to nearly
eighty percent of the cells after four weeks in culture.
Concurrently, the number of cells expressing high affinity
receptors for IgE rose from less than one percent to
approximately fifty percent. A similar time course was
followed by the determinant recognized by monoclonal
antibody B23.1, which was expressed by nearly all cells in


59
by berberine sulfate. Mast cell phenotype, therefore, may
be functionally regulated at the level of the tissue
microenvironment in which a multipotent mast cell precursor
or intermediate develops.
Epilogue
Despite an apparent wealth of literature available on
the subject, the potential still exists for scholarly,
significant contributions to the body of knowledge which
describes mast cells. The lineage relationship between the
mast cells found ini vivo (mucosal and serosal) is still
poorly defined, ond only recently have preliminary studies
approached the relationship between the aforementioned
cells and their putative correlate, the culture-derived
mast cell. Little is known of the phenotype of the cells
which give rise to mast cells in culture, and the ontogeny
of the mast cell in early embryonic tissues is documented
in scant and unsystematic reports.
In the course of the remaining chapters of this
dissertation, we describe our recent contributions to the
study of the mast cell. Beginning with the observation of
cell lines with basophilic granules, we have characterized
Abelson murine leukemia virus-transformed mast cell-like
lines of midgestational, embryonic origin using panels of
monoclonal antibodies as well as biochemical and molecular
biological techniques (Chapter II). Although mast cells
were not detected in homologous, uninfected tissues,


197
Garland, J.M., and S. Crompton. 1983. A preliminary
report: Preparations containing interleukin-3 (IL-3)
promote proliferation of multipotential stem cells
(CFU-S) in the mouse. Exp. Hematol. 11: 757-761.
Ghiara, P., D. Boraschi, L. Villa, G. Scapigliati, C. Taddei,
and A Tagliabue. 1985. In vitro generated mast
cells express natural cytotoxicity against tumors.
Immunology 55: 317-324.
Ginsburg, H. 1963. The in vitro differentiation and
culture of normal mast cells from the mouse thymus.
Ann. N.Y. Acad. Sci. 103: 20-39.
Ginsburg, H., D. Ben-Shahar, and E. Ben-David. 1982. Mast
cell growth on fibroblast monolayers: Two-cell entities.
Immunology 45: 371-380.
Ginsburg. H., and D. Lagunoff. 1967. The in vitro
differentiation of mast cells. Culture of cells from
immunized mouse lymph nodes and thoracic duct lymph on
fibroblast monolayers. J. Cell. Biol. 35: 685-697.
Ginsburg, H., I. Nir, I. Hammel, R. Eren, B.-A. Weissman, and
Y. Naot. 1978. Differentiation and activity of mast
cells following immunization in cultures of lymph-node
cells. Immunology 35: 485-502.
Ginsburg, H., E.C. Olson, T.F. Huff, H. Okudaira, and T.
Ishizaka. 1981. Enhancement of mast cell
differentiation in vitro by T cell factor(s). Int.
Archs. Allergy Appl. Immunol. 66: 447-458.
Ginsburg, H., and L. Sachs. 1963. Formation of pure
suspensions of mast cells in tissue culture by
differentiation of lymphoid cells from the mouse thymus.
J. Natl. Cancer Inst. 31: 1-39.
Goff, S.P., E. Gilboa, O.N. Witte, and D. Baltimore. 1980.
Structure of the Abelson murine leukemia virus genome
and the homologous cellular gene: Studies with cloned
viral DNA. Cell 22: 777-785.
Goldwasser, E., J.N. Ihle, M.B. Prystowsky, I. Rich, and G.
VanZant. 1983. The effect of interleukin-3 on
hematopoietic precursor cells. In: D.W. Golde and P.A.
Marks (eds.), Normal and Neoplastic Hematopoiesis. Alan
R. Liss, New York, New York.
Gonzales-Molina, A. and H.L. Spiegelberg. 1978. A
subpopulation of normal human peripheral B lymphocytes
that binds IgE. J. Clin. Invest. 59: 616-624.


206
Ogawa, M., T. Nakahata, A.G. Leary, A.R. Sterk, K. Ishizaka,
and T. Ishizaka. 1983. Suspension culture of human
mast cells/basophils from umbilical cord blood
mononuclear cells. Proc. Natl. Acad. Sci. USA 80:
4494-4498.
Olson, E.C., and D.A. Levy. 1976. Thymus-dependency of
the mast cell response to Nippostrongylus brasiliensis
in mice. Fed. Proc. 35: 491.
Olsson, I., B. Berg, L.A. Fransson, and A. Norden. 1970.
The identity of the metachromatic substance of
basophilic leucocytes. Scand. J. Haematol. 7: 440-444.
Omary, M.B., I.S. Trowbridge, and M.P. Scheid. 1980. T200
cell surface glycoprotein of the mouse. Polymorphism
defined by the Ly-5 system of alloantigens. J. Exp. Med
151: 1311-1316.
Orenstein, N.S., S.J. Galli, A.M. Dvorak, J.E. Silbert, and
H.F. Dvorak. 1978. Sulfated glycosaminoglycans of
guinea pig basophilic leukocytes. J. Immunol. 121:
586-592.
Padawer, J. 1974. Mast cells: Extended life span and lack
of granule turnover under normal rn vivo conditions.
Exp. Mol. Pathol. 20: 269-280.
Palacios, R., G. Henson, M. Steinmetz, and J.P. McKearn.
1984. Interleukin-3 supports the growth of mouse pre-B
clones in vitro. Nature 309: 126-131.
Parmley, R.T., M. Ogawa, S.S. Spicer, and N.J. Wright. 1976.
Ultrastructure and cytochemistry of bone marrow
granulocytes in culture. Exp. Hematol. 4: 75-89.
Pearce, F.L. 1982. Functional heterogeneity of mast cells
from different species and tissues. Klin. Wschr. 60:
954-957.
Pearce, F.L. 1983. Mast cell heterogeneity. Trends
Pharmacol. Sci. 4: 165-167.
Pearce, F.L., H. Ali, K.E. Barrett, A.D. Befus, J.
Bienenstock, J. Brostoff, M. Ennis, K.C. Flint, B.
Hudspith, N.M. Johnson, K.B.P. Leung, and P.T. Peachell.
1985. Functional characteristics of mucosal and
connective tissue mast cells of man, the rat and other
animals. Int. Archs. Allergy Appl. Immunol. 77: 274-276


The A-MuLV-infected, fetal liver-derived mast cells were
phenotypically similar to uninfected cells with respect to
morphology, presence of metachromatic granules, 20-alpha-
hydroxysteroid dehydrogenase, and high affinity receptors
for IgE. Attempts to establish factor independent mast
cell lines with BALB-murine sarcoma virus (MSV), Harvey-
MSV, and Moloney-MSV have been unsuccessful (Pierce et al.,
1985). Abelson virus has also been shown to abrogate the
interleukin 3-dependence of the early myeloid cell line
FDP-1 and to similarly release the interleukin 2-dependent
cytotoxic T cell line CTB6 (Rapp et al., 1985; Cook et al.,
1985). The mechanism by which the Abelson transforming
protein releases our cell lines, and those reported
elsewhere, from growth factor requirement is not
understood; it would appear, however, to be independent of
autocrine effects (Cook et al., 1985; Pierce et al., 1985;
Rapp et al., 1985).
Although the expression of the Ly5 differentiation
marker on cultured mast cells has been previously reported
(Nabel et al., 1981; Tertian et al., 1981; Wong et al.,
1982), the detection of the B lymphoid variant of Ly5 on A-
MuLV-transformed mast cells (Chapter II) and on mast cells
picked from agar colonies (Chapter IV), but not on liquid
culture-derived mast cells (Chapter III) in this laboratory
was enigmatic. The cells which expressed the RA3-3A1
epitope had, in common, maturation in agar in association


108
six months at -20C; volumes for immediate use were stored
for up to one month at 4C. Fresh glutamine was added to
all media over one month of age. Unless otherwise noted,
W3CM was diluted with an equal volume of EM to yield 50%
W3CM.
Preparation of Cell Suspensions for In Vitro Culture of
Mast Cells
Bone marrow
Two to three month-old BALB/cAn mice were killed by
cervical dislocation. The skinned hind legs were
disarticulated at the pelvis and tarsals and were placed in
EM. The limbs were transferred to a fresh dish of EM in
which most of the flesh was removed from the femorae and
tibiae by cutting and teasing with sterile dissection
tools. Following transfer of the bones to a third dish of
EM, the bones were disarticulated and the ends sheared off.
Bone marrow was then harvested by flushing the contents of
all four bones with EM (1 ml injected through each end with
a 25 gauge needle) through 110 micron mesh nylon screens
(Tetko, Inc., Elmsford, NY) into a 50 ml conical
polypropylene centrifuge tube (Corning Glass Works) (Siegel
et al., 1985) .
Spleen
Two to three month-old BALB/cAn mice were killed by
cervical dislocation, after which the spleens were removed
by careful, asceptic dissection and placed in a dish of EM.


176
A fourth significant observation of this work concerns
the expression of mast cell-specific markers on bone
marrow-derived cells picked from colonies between three and
four weeks in culture. Initial attempts to propagate mouse
mast cells in agar were met with complete failure (McCarthy
et al., 1980). Colonies, which were observed after one
week in culture, yielded no cells with astra blue-staining
granules characteristic of mast cells. Later investigators
(Schrader et al., 1981; Zucker-Franklin, 1981; Nakahata et
al., 1982b) established that a minimum of two weeks in
culture was required for mouse mast cell maturation.
Longer incubation periods were, in fact, preferable, since
mast cell colonies are less transient than those composed
of other lineages (Pharr et al., 1984). The presence of
mast cells in macroscopic colonies was first confirmed in
situ by microscopic observation of round cells with clearly
delineated, refractile edges and slight dark hue (Nakahata
et al., 1982b). More than ninety percent of the cells
picked from such colonies contained metachromatic granules
when stained with acidic toluidine blue. These cells also
had surface receptors for IgE (but not IgG) and most
expressed the surface determinant recognized by the
monoclonal antibody B23.1, and thus, by three criteria,
could be identified as mast cells.
The colony-derived mast cells also expressed the B-
lineage variant of the Ly5 surface antigen. Ly5 has


IV ISOLATION, ENUMERATION, AND CHARACTERIZATION OF
IN VITRO MAST CELL PRECURSORS DERIVED FROM
MIDGESTATION EMBRYONIC PLACENTA 144
Introduction 144
Materials and Methods 146
Results 154
Discussion 172
V SUMMARY AND CONCLUSIONS 183
REFERENCES 189
BIOGRAPHICAL SKETCH 219
v


110
Establishment of Liquid Cultures of Factor-Dependent
Mast Cells
Mast cells were cultured from fresh tissues following
modification of a previously published protocol Razin et
al., 1984a). Except for variations in the methods used to
dissociate the cells from their native structures, as noted
above, the procedure for propagating mast cells from each
source was the same.
Disaggregated cell suspensions were allowed to settle
by gravity at room temperature in conical centrifuge tubes
for five to ten minutes. Cells remaining in suspension
were transferred to a clean tube and were washed three
times in EM by centrifugation. Viable, nucleated cells
were enumerated by trypan blue exclusion and the cells were
resuspended at a concentration of 1x10^ per ml in 50% W3CM.
Cultures were maintained in a humidified atmosphere of five
percent carbon dioxide in air at 37C. Mast cells were
enriched in liquid culture and selected at weekly intervals
by gently swirling the dishes and transferring the
suspended cells with a pipet to a centrifuge tube;
following centrifugation, the cells were resuspended in 50%
W3CM at 1 to 2x10^ per ml.
Infection of In Vitro-Derived, Factor-Dependent Mast Cells
with Abelson Murine Leukemia Virus
Cultures of mast cells derived from adult and
embryonic tissues were infected at various times during the


55
than mast cells. This conclusion, however, directly
disagreed with Zucker-Franklin (1980), who contended that
human mast cells and basophils share common ultrastrucural
organization.
Tadokoro and colleagues (1983) cultured cells with
metachromatic granules and lobulated nuclei from normal
human bone marrow in conditioned media from lectin-
stimulated blood lymphocytes. The culture-derived cells
contained 500 to 2000 nanograms of histamine per million
cells and were responsive to IgE-anti-IgE- and calcium
ionophore-mediated histamine release but were refractory to
the effects of compound 48/80. The authors concluded that
their conditioned media contained a basophil-promoting
activity which furthermore had a molecular weight of 25 to
40 kilodaltons and was distinct from interleukin 2. The
same group recently reported that media conditioned by
phytohemagglutinin A- and concanavalin A-stimulated human
blood lymphocytes could support the interleukin 3-dependent
mouse cell line 32Dcl as well as promote the growth of
human culture-derived basophils (Stadler et al., 1985).
Furthermore, the interleukin 3 and basophil-promoting
activities, which were also found in media conditioned by
the growth of E-rosetting T lymphocytes and the MoT cell
line, were biochemically distinct by at least five
different criteria. The isolated human interleukin 3 was
shown to promote the growth of mast cells which were unable


This dissertation was submitted to the Graduate Faculty
of the College of Medicine and to the Graduate School and
was accepted as partial hu-Hsi 1 Iment of the requirements for
the degree of Doctor of Phi/l())pophy.
May, 1986
Den, Gradd
late SchopJ


180
that growth factor. The absence of mature mast cells from
the same tissues may be indicative of in vivo control
mechanisms which are abrogated by in vitro propagation.
Abelson murine leukemia virus targets are most
frequent in embryonic tissues at day ten of gestation,
while the number of culture-derived mast cell precursors
peaks at day twelve. A-MuLV is capable of transforming
cells of most of the hematopoietic lineages, among them
mast cells (Chapter III; Pierce et al., 1985). We propose
that the cells detected in the transformation assay are
precursors to the untransformed cells which proliferate in
response to interleukin 3. The difference between the two
populations may be one of yet undescribed differentiation
markers: Cells which are targets for A-MuLV, not yet
responsive to interleukin 3, may further differentiate into
cells which are responsive to interleukin 3 but no longer
possess receptors for the transforming virus. This model
remains untested and its mechanisms demand further
elucidation. We have previously proposed that the A-MuLV
oncogene product may substitute for interleukin 3, thus
inducing the mast cell-like characteristics of our
transformed placental cell lines (Siegel et al., 1985).
The expression of c-abl messenger RNA, which also peaks in
the embryo at ten days of gestation (Muller et al., 1982)
and may encode a growth factor activity which normally acts


211
Scher, C.D. and R. Siegler. 1975. Direct transformation of
3T3 cells by Abelson murine leukemia virus. Nature 253:
729-731.
Schindler, R., M. Day, and G.A. Fisher. 1959. Culture of
neoplastic mast cells and their synthesis of 5-
hydroxytryptamine and histamine synthesis j_n vitro.
Cancer Res. 19: 47-51.
Schrader, J.W. 1981. The in vitro production and
cloning of the P cell, a bone marrow-derived null cell
that expresses H-2 and la antigens, has mast cell-like
granules, and is regulated by a factor released by
activated T cells. J. Immunol. 126: 452-458.
Schrader, J.W., F. Battye, and R. Scollay. 1982. Expression
of Thy-1 antigen is not limited to T cells in cultures
of mouse hemopoietic cells. Proc. Natl. Acad. Sci. USA
79: 4161-4165.
Schrader, J.W., I. Clark-Lewis, and P. Bartlett. 1980.
Lymphoid stem cells. In R.P. Gale and C.F.Fox (eds.),
ICN-UCLA Symposia on Molecular and Cellular Biology,
Vol. 17. Academic Press, New York.
Schrader, J.W., I. Clark-Lewis, R.M. Crapper, and G.W.
Wong. 1983a. P-cell stimulating factor:
Characterization, action on multiple lineages of bone
marrow-derived cells and role in oncogenesis.
Immunol. Rev. 76: 79-104.
Schrader, J.W., I. Clark-Lewis, R.M. Crapper, G.H.W. Wong,
and S. Schrader. 1985. P cell stimulating factor:
biochemistry, biology, and role in oncogenisis.
Contemp. Top. Mol Immunol. 10: 121-146.
Schrader, J.W., and R.M. Crapper. 1983. Autogenous
production of a hemopoietic growth factor, persisting-
cell-stimuiating factor, as a mechanism for
transformation of bone marrow-derived cells. Proc.
Natl. Acad. Sci. USA 80: 6892-6896.
Schrader, J.W., S.J. Lewis, I. Clark-Lewis, and J.G. Culvenor.
1981. The persisting (P) cell: Histamine content,
regulation by a T-cell derived factor, origin from a
bone marrow precursor, and relationship to mast cells.
Proc. Natl. Acad. Sci. USA 78: 323-327.
Schrader, J.W., and G.J.V. Nossal. 1980. Strategies for the
analysis of accessory cell function: The in vitro
cloning and characterization of the P cell. Immunol.
Rev. 53: 61-85.


extensively against the same buffer. Normal embryonic
tissue, prepared in an identical manner, provided control
DNA. All DNA was quantitated spectrophotometrically by the
OD260/OD28O methd (Maniatis et al., 1982).
Ten micrograms of DNA were incubated with 10 units of
restriction endonuclease BAM HI (New England Biolabs,
Beverly, MA) in TA buffer (O'Farrell et al., 1980);
completeness of digestion was monitored by the addition of
one microgram of bacteriophage lambda DNA to a duplicate
sample. The digested DNA was mixed with sample buffer (50 mM
Tris, pH 7.5, 5 mM EDTA, 25% w/v Ficoll, 0.05% w/v
bromophenol blue, at 5X concentration) and electrophoresed
for eighteen hours at 40 volts D.C. on an 0.8% agarose gel
in TEA buffer (40 mM Tris-acetate, 2 mM EDTA, pH 7.8).
Both the gel and the electrophoresis buffer contained 0.5
micrograms of ethidium bromide per milliliter. Samples
were organized such that a set of digested cellular DNAs
were pipetted into wells on one side of the gel and
duplicates containing bacteriophage lambda DNA were
pipetted into wells on the other side of the same gel.
Following electrophoresis, the gel was photographed
under ultraviolet light to visualize the restricted DNA and
verify that all of the lambda DNA had been digested to
completion. A ruler placed alongside the gel was
photographed at the same time to provide a scale of DNA
fragment sizes for later reference. The lanes containing


166
placental cells prompted us to assay for its expression on
colony-derived mast cells. Most of the colonies analyzed
contained a significant number of cells which expressed the
antigen recognized by the RA3-3A1 antibody (Table IV-2),
in contrast to the results of analyses of numerous liquid
culture-derived mast cells from similar sources (Chapter
III) .
Surface Markers on Mast Cell Precursors: Sorting of Cells
with Monoclonal Antibodies
The presence of the determinants recognized by
monoclonal antibodies B23.1 and RA3-3A1, and well as
receptors for IgE, on the surface of both transformed and
agar-cultured mast cells led us to ask whether these
antigens were also expressed on the agar-colony mast cell
precursors. Conditions for the sorting experiments were
initially optimized on tumor cell lines previously shown to
express high levels of the markers of interest. As seen in
Table IV-3, biotinylated antibody plus avidin-modified
sheep red blood treatment, followed by density gradient
separation, was effective in depleting the control
populations of most marker-positive cells. Rosetted cells,
observed in suspension after staining nucleated cells with
crystal violet (0.2 percent w/v), accounted for virtually
all of the cells in the marker-positive, Lympholyte M
pellets. On the other hand, few of the cells in the marker
negative gradient supernatant were associated with SRBC in


62
Boss et al., 1979; Siden et al., 1979; Alt et al., 1981).
Abelson virus can also induce agar colony-forming cells
which express erythroid characteristics (Waneck and
Rosenberg, 1981); the latter cells, however, fail to
proliferate as permanent cell lines in liquid culture.
Based upon Abelson virus's propensity to immortalize B
lineage precursors jji vitro, experiments were designed to
study early embryonic lymphoid precursors. The
midgestation embryonic placenta has been reported to be the
earliest source of B cell precursors (Melchers and
Abramczuk, 1980; Melchers, 1979) in the mouse. The
successsful development of permanent cell lines from
midgestation embryonic placenta transformed iua vitro by A-
MuLV has recently been reported (Siegel et al., 1985).
Primary agar colony counts indicated that the frequency of
A-MuLV targets is highest at ten days of gestation. Unlike
previously reported A-MuLV-transformed embryonic cell
lines, the genomes of the placental cells contain a
germline immunoglobulin heavy chain locus characteristic of
nonlvmphoid cells and perhaps very immature lymphoid cell
precursors.
We proceeded to analyze this novel group of A-MuLV
embryonic transformants to better ascribe them to cells of
a particular lineage. This chapter summarizes our efforts
to characterize the A-MuLV transformants derived from
midgestation embryonic tissues and presents several


CHAPTER I
INTRODUCTION AND
REVIEW OF THE LITERATURE
Introduction: Hematopoietic Cell Differentiation
and Tumor Models
The ontogeny of the hematopoietic system of the mouse
can be viewed as a progression of finite, genetically
programmed stages in the maturation of pluripotent stem
cells into the terminally differentiated state of each of
the various blood lineages. Pluripotent hematopoietic stem
cells, defined bv their ability to reconstitute lethaliy
irradiated recipients (Till and McCulloch, 1961), are first
detected in the murine yolk sac between eight and twelve
days of gestation (Tyan, 1968). Beginning with day ten and
throughout the remainder of gestation, ceils with the same
differentiative capacity are found in the fetal blood and
liver (Moore and Metcalf, 1970). In the adult, pluripotent
hematopoietic stem cells are found in the bone marrow (Till
and McCulloch, 1961) and spleen (Nakahata and Ogawa, 1982).
The mechanisms involved in the differentiation of
pluripotent hematopoietic stem cells into mature,
functional blood elements are, for the large part, unknown.
These pathways may involve the interaction of pluripotent
or committed progeny stem cells with other cells or
macromolecular products in their inductive environment
(Kincade et al., 1981a), resulting in the cell's commitment
to one of several genetically programmed, phenotypically
distinguishable chains of events; alternately, random
1


16
dense, homogeneous spheres which dissociate into fibrillar
structures in hypertonic salt solution (Lagunoff, 1972).
The initial observation of intraspecies mast ceil
heterogeneity is generally attributed to Maximow (1906),
who reported that the rat intestine was replete with mast
cells which differed from other rat mast cells in
morphology and stain affinity. These differences were
reinvestigated by Enerback, who, sixty years after
Maximow's observations, published a series of reports which
described in detail the differences in morphology, stain
affinities (Enerback, 1966a, 1966b), and sensitivity to
degranulating agents (Enerback, 1966c, 1966d) between
dermal mast cells (representative of the connective tissue
or serosal subset) and intestinal mast cells
(representative of mucosal or atypical mast cells). Thus,
mucosal mast cells were shown to be smaller, possess uni-
or bilobed nuclei, and be less granulated than serosal mast
cells, and the granules of the former population were far
more heterogeneous in size than those of the latter.
Enerback also observed that the mucosal mast cells stained
red with acidic toluidine blue, while serosal mast cells
stained purple. He noted that standard formaldehyde
fixatives used to preserve serosal mast cell granules were
ineffective on mucosal mast cells, and selected and
adapted several fixatives (such as Carnoy's and Mota's
preparations) to more adequately preserve the more labile


43
mast cell precursors from various tissues was reported by
Crapper and Schrader (1983). Using limiting dilution
analysis of cells in liquid culture containing WEHI-3
conditioned media, The authors were able to enumerate mast
cell precursors in bone marrow, spleen, mononuclear
peripheral blood cells, and lymph nodes. All of the data
recorded in the latter experiments concurred with the
previously cited results of Schrader and colleagues (1981)
as well as those reported by the Nakahata group (Nakahata
et al, 1982b; Pharr et al., 1984). Furthermore, Crapper
and Schrader were able to substantiate the findings of Suda
and colleagues that mast cell deficient mice had similar
numbers of culture-derived mast cell precursors (in bone
marrow and spleen) when compared to appropriate wild-type
controls, although the former authors used W^/W^ and the
latter authors used W/Wv.
The development of techniques for the propagation of
culture-derived mast ceils has also permitted the
characterization of such cells at various stages of
differentiation. Thus, Ginsburg and colleagues (Ginsburg,
1963; Ginsburg and Sachs, 1963; Ginsburg and Lagunoff,
1967; Ginsburg et al., 1982; Davidson et al., 1983)
reported a progression of characteristics of cultured mast
cells, starting with large, mononuclear "stem" cells.
After six to ten days in culture, large, lymphocvte-like
"mastoblasts" with round and bilobed nuclei and a narrow


220
1980, Meloy Laboratories reduced its middle management
staff, allowing Michael the opportunity to return to
graduate studies at the University of Florida. Following
completion of his doctoral studies, Michael will study
retroviral oncogenesis and hematopoiesis in the laboratory
of Carlo Moscovici, continue his attempts to find
renaissance, and, as always, stay seventeen.


132
approximately 160 and 65 kilodaltons (Kd) in lanes loaded
with immune precipitated lysates from the placental tumor
cell line 10P12 (lane 1), from A-MuLV infected fetal liver
(lane 3) and from A-MuLV infected embryonic mast cells
(lane 5). The same bands are absent from the lanes loaded
with nonimmune (normal goat) serum-treated lysates of the
same cells (lanes 2, 4 and 6, respectively) and from a lane
loaded with immune precipitated lysate from sham-infected
mast cells (lane 7). We conclude, therefore, that the mast
cells were productively infected with A-MuLV. Furthermore,
the infection of culture-derived mast cells with A-MuLV did
not induce the de novo expression of the lymphoid B-220
marker in those cells.
Effects of Adherent Cells and Adherent Cell-Derived Factors
on Populations of Cultured Mast Cells
Because conditions which involved coculture with
adherent cells generated mast cells expressing Ly 5, we
tried to recreate this milieu using bone marrow adherent
cells, cell lines, or conditioned media. Expression of B-
220 was assayed with RA3-3A1 antibody as previously
described. As seen in Table III-l, neither the
cocultivation with adherent cells nor their conditioned
media with homogeneous populations of mast cells was
sufficient to induce the expression of B-220. The
expression of the antigen recognized by the B23.1 antibody,
receptors for immunoglobulin IgE and the presence of


19
cells in its connective tissues (Wlodarski, 1976; Reed et
al., 1982). Aldenborg and Enerback (1985) recently reported
that congenitally athymic rnu/rnu rats have at least as many
(or more) peritoneal mast cells as normal controls for the
first fourteen weeks of life; adult rnu/rnu rats, however,
have fewer peritoneal mast cells than their wild type
counterparts. These results mav indicate that peritoneal
mast cell populations may be subject to thymic influences
later in life or may simply reflect a separate, thymus-
independent defect inherent in the athymic rat. Further
studies will be necessary to elucidate the apparent
contradiction.
The proteoglycan composition of serosal mast cell
granules has been the subject of study since the initial
discovery of heparin in the canine liver by Jorpes in 1937
(cited in Selye, 1965). Subsequently, heparin has been
identified in rat peritoneal mast cells (Tas and
Berndsen, 1977; Yurt et al., 1977; Stevens and Austen,
1982), human lung mast cells (Metcalfe et al., 1979), and
mouse peritoneal mast cells (Razin et al., 1982c).
Mucosal Mast Cells
Following the development of improved methods for
their fixation and staining by Enerback (1966a, 1966b), the
study of the atypical, or mucosal, mast cell accelerated
significantly. Early reports of similarities between
cultured, thymus-derived mast cells of the mouse (Ginsburg,


57
Thy 1-/Lyt 1-/ Lyt 2- phenotype, which are demonstrably
cytotoxic, are sensitive to the proliferative activities of
interleukin 3 (Djeu et al., 1983; Lattime et al., 1983).
Evidence of more direct relationships between in
vijtro-derived mast cells and their in vivo correlates has
been elusive. Several investigators have associated the
high incidence of culture-derived mast cell precursors and
mucosal mast cells in the intestine of normal mice (Crapper
and Schrader, 1983; Guy-Grand et al., 1984). The role of
antigenic stimulation and T cell function in the
proliferation of mucosal mast cells per se has been
thoroughly described in the literature (for reviews see
Jarrett and Haig, 1984; Shanahan et al., 1984; Bienenstock
et al., 1983). Guy-Grand and colleagues (1984) also showed
that the number of mast cells which could be cultured from
intestinal mucosa increased with antigenic stimulation and
WEHI-3 tumor burden, implicating the role of interleukin 3
in the m vivo proliferation of mast cell precursors. The
results, however, associated the iji vivo and in vitro mast
cell precursors by existence in the same tissue, and did
not directly show that the populations involved were
identical.
The most suggestive evidence to date of the
relationship between in vivo- and jji vitro-derived mast
cells involves the demonstration that culture-derived mast
cells, when injected into mast cell-deficient mice,


216
Wegmann, T.G., B. Singh, and G.A. Carlson. 1979. Allogeneic
placenta is a paternal strain immunoadsorbent. J.
Immunol. 122: 270-274.
Weisner-Menzel, L., B. Schulz, F. Vakilzadeh, and B.M.
Czarnetzki. 1981. Electron microscopal evidence for a
direct contact between nerve fibres and mast cells. Acta
Derm. Venereol. 61: 465.
Whetton, A.D., G.W. Bazill, and T.M. Dexter. 1985.
Hemopoietic growth factor: Characterization and mode of
action. In C. Sorg and A. Schimpl (eds.), Cellular
and Molecular Biology of Lymphokines. Academic Press,
Orlando, FL.
Whitlock, C.A., S.F. Zeigler, L.J. Treiman, J.I. Stafford,
and O.N. Witte. 1983. Differentiation of cloned
populations of immature B cells after transformation
with Abelson murine leukemia virus. Cell 32: 903-911.
Wingren, U., and L. Enerback. 1983. Mucosal mast cells of
the rat intestine: A re-evaluation of fixation and
staining properties, with special reference to protein
blocking and solubility of the granular
glycosaminoglycan. Histochem. J. 15: 571-582.
Witte, O.N., A. Dasgupta, and D. Baltimore. 1980. Abelson
murine leukemia virus is phosphorylated in vitro to form
phosphotyrosine. Nature 283: 826-831.
Witte, O.N., N. Rosenberg, and D. Baltimore. 1979a.
Identification of a normal cell protein cross-reactive to
the major Abelson murine leukemia virus gene product.
Nature 281: 396-398.
Witte, O.N., N. Rosenberg, and D. Baltimore. 1979b.
Preparation of syngeneic tumor regressor serum reactive
with the unique determinants of the Abelson MuLV encoded
P120 protein at the cell surface. J. Virol. 31: 776-784.
Wlodarski, K. 1976. Mast cells in the pinna of BALB/c
"nude" (nu/nu) and heterozygous (nu/+) mice.
Experientia 32: 1591-1592.
Wong, G.H.W., I. Clark-Lewis, J.L. Me Kimm-Breschkin, and
J.W. Schrader. 1982. Interferon gamma-like molecule
induces la antigens on cultured mast cell progenitors.
Proc. Natl. Acad. Sci. USA 79: 6989-6993.
Woodbury, R.G., and H. Neurath. 1980. Mast cell proteases.
In H. Holber (ed.), Metabolic Interconversions of
Enzymes. Springer-Verlag, New York, NY.


99
histochemically similar to culture-derived mast cells.
Analysis of histamine content of the embryonic cell lines
indicated that most do contain that biogenic amine.
Furthermore, the quantity of histamine detected (5 to more
than 500 nanograms per million cells) were similar to those
reported for mucosal and jji vitro-derived mast cells. The
embryonic cell lines also synthesized chondroitin-4,6-
disulfate proteoglycan, but not heparin (D. Levitt, R.
Porter, and E. Siden, manuscript in preparation).
Chondroitin sulfate is a granule constituent of cultured
and mucosal mast cells, but not serosal mast cells, which
store heparin (Razin et al., 1984a). Based on these two
additional criteria, we have proposed that our embryonic
cell lines are analogous to culture-derived mast cells.
Our analysis of the surface determinants on embryonic
cell lines and control tumor cell lines has supported the
preliminary hypothesis of their lineage. Although we
observed heterogeneity of histamine content and expression
of high affinity receptors for IgE, every embryonic cell
line expressed the differentiation antigen recognized by
the monoclonal antibody B23.1, which is also expressed on
cultured mast cells from mouse bone marrow, spleen, and
blood (Katz et al., 1983). Two of the embryonic cell lines
described in this study (Table II-3), as well as two
embryonic cell lines of different genotype (unpublished
results), also express the Ly5 200 to 220 kiltodalton


188
observed to provide a rich source of mast cell precursors,
while cells expressing the same markers produced few mast
cell colonies in semisolid agar. In contrast, cells
rosetted by red blood cells bearing monoclonal antibody
B23.1 were as rich in mast cell precursors as untreated
cells. These preliminary studies have significantly
contributed to the meager body of information which
describes the phenotype of the culture-derived mast cell
precursor (Yung et al., 1983).
In conclusion, the investigations presented in this
dissertation have demonstrated the existence of culture-
derived mast cell precursors in the earliest reported
stages of murine development. Although its role in
hematopoiesis is yet undefined, the embryonic mouse
placenta is a rich source of culture-derived mast cell
precursors for a brief period of fetal development. These
findings may serve as an impetus for further studies which
will better define the role of interleukin 3-responsive
cells in embryogenesis and mast cell differentiation.


Table II-3. Analysis of Lineage-Specific Surface Determinants on
A-MuLV-Transformed Embryonic and Control Tumor Cell Lines*
PRE-B MARKERS
¡ PRE-T
MARKERS ¡
T MARKER ¡
A-MuLV
TARGET
MAST CELL
¡ -MONOCYTE
MARKERS
CELL LINE
RA3-3A1
IgM
14.8 RA3-2C2
IgG2b IgM
M6
IgM
Anti-Asialo
GM1
Polyclonal
T24/31.7
IgG
5H1
IgM
B23.1
IgM
9P1 Ab-MuLV
PLACENTA (9D)
_
_
_
PLACENTAL
CELL LINES
+
_
_
+
10P2 Ab-MuLV
PLACENTA (10D)
-
-
-
-
-
-
-
+
10P6 Ab-MuLV
PLACENTA (10D)
-
-
-
-
++
-
-
+
10P8 Ab-MuLV
PLACENTA (10D)
-
-
-
-
-
-
-
++
10P12 Ab-MuLV
PLACENTA (10D)
+
+
++
-
-
-
-
++
11P0-1 Ab-MuLV
PLACENTA (1ID)
++
++
++
+
++
+
11P62 Ab-MuLV
PLACENTA (1ID)


85
(Habu et al., 1980) and for the expression of receptors for
the Dolichos biflorus agglutinin, which is expressed on
early fetal thymocytes and on some thymic leukemia cells
(Muramatsu et al., 1980). The monoclonal antibody M6
(Kasai et al., 1983) reportedly recognizes cells bearing
receptors for Dolichos biflorus agglutinin and probably
binds to the receptor itself. Table II-3 shows that only
one of the embryonic cell lines, 11P0-1, expressed the
early thymocyte differentiation marker recognized by anti-
FT-1. The same cell line also bears surface asialo-GMl, as
do two other embryonic cell lines, 9P1 and 10P6. It is
also interesting to note that two A-MuLV-induced adult
mouse mastocytomas, CB6ABMC4 and BALABMC20 were both
positive for FT-1, and that the latter cell line also
expressed the neutral glycolipid asialo-GMl. These
observations will be discussed later in this chapter.
Despite the presence of markers specific for early T
lineage cells on several embryonic cell lines, mature T
lineage markers were only detected on three control cell
lines. Lack of reactivity with monoclonal antibody
T24/31.7 (Dennert et al., 1980) indicated that none of the
cell lines expressed the Thy-1 differentiation marker.
Also conspicuously absent from A-MuLV-transformed embryonic
cell lines was the antigen recognized by the monoclonal
antibody 5H1, which is expressed on Abelson murine leukemia
virus transformation-sensitive targets from mouse bone


also found on cells of the monocyte lineage (Leblanc et
al.. 1982) as well as on culture-derived mast cells. This
marker was also detected on control mastocytoma (P815) and
myelomonocytic leukemia cells (WEHI-3).
Two of the cell lines expressed three related
antigenic determinants of the 200 to 220 kilodalton surface
glycoprotein family which is found on mouse lymphoid cells.
The cell lines 11P0-1 and 10P12, as well as all of the
control lymphoid and adult-derived mastocytomas which we
examined, expressed the antigens defined by the monoclonal
antibodies 14.8 and RA3-2C2. These epitopes are expressed
on B cells and their surface immunoglobulin-negative
precursors. The progenitor cells react with the monoclonal
antibody RA3-3A1 as well. The latter antibody, however,
did not recognize a 220 kilodalton glycoprotein on tumor
cells of the T lineage which was detected by 14.8 and RA3-
2C2. The selective reactivity of RA3-3A1 has been
confirmed previously (Coffman and Weissman, 1981b). It is
interesting to note, however, the novel expression of this
previously characterized B cell differentiation antigen on
mastocytoma P815 as well as our embryo-derived mast cell
lines.
Placental and control tumor cell lines were also
analyzed for the expression of other primitive lymphoid
markers. The neutral glycolipid asialo-GMl, which is
expressed on the early embryonic, Thy-1-negative thymocytes


33
Conditioned Media-Dependent Mast Cells
The development of techniques for culturing mast cells
from hematopoietic and lymphoid tissues has led to an
exponential increase in mast cell research and literature
citations. With the burst of scientific activity, however,
has come a concurrent increase in the number of terms used
to describe culture-derived mast cells, including P
(persisting) cells (Schrader and Nossal, 1980), histamine-
containing granular cells (Sredni et al., 1983), mucosal
mast cells, basophil/mast cells, and atypical mast cells.
Despite the discrepancy of terms, however, the long term
suspension cultures of mast cells appear to be strikingly
similar. Mast cells have thus been derived from mouse bone
marrow (Tertian et al., 1980, 1981; Nagao et al., 1981;
Razin et al., 1981a, 1982a,b,c; Schrader, 1981; Schrader et
al., 1981; Galli et al., 1982b; Crapper and Schrader, 1983;
Sredni et al., 1983; Wedling et al., 1983, 1985; Yung et
al., 1983; Suda et al., 1985), spleen (Hasthorpe, 1980;
Schrader and Nossal, 1980; Schrader et al., 1980, 1981;
Schrader, 1981; Tertian et al., 1981; Crapper and Schrader,
1983; Sredni et al., 1983; Pharr et al., 1984), fetal liver
(Nabel et al., 1981; Razin et al., 1984b), peripheral blood
(Crapper and Schrader, 1983; Suda et al., 1985), thymus
(Tertian et al., 1981; Schrader, 1981; Davidson et al.,
1983), lymph nodes (Ginsburg et al., 1978; Crapper and
Schrader, 1983), and intestinal mucosa (Schrader et al.,


I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of.Doctor of Philosophy.
Edward J. Siden, Chairperson
Assistant Professor of Immunology
and Medical Microbiology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
u /d'T ll /l
Ja>s B. Flanegan/
Associate ProfqaVor of Immunology
and Medical Microbiology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree pf Doctor of Philosophy.
Carlo Mocovici
Professor of Pathology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of l^ilosophy.
O.X"
Stephen W. Russell
Professor of Pathology and
Veterinary Medicine
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Weiner
Professor of Immunology and
Medical Microbiology


161
to determine whether the elevated number of mast cell
precursors observed in heterozygous midgestation embryonic
tissues, when compared to homozygous embryonic tissues, was
maintained in the adult. As seen in Table IV-1, the
number of precursors in BALB/cAN bone marrow was determined
to be one per 2500 nucleated cells. These numbers are
similar to those previously reported for other agar assays
(Schrader et al., 1981; Sredni et al., 1983), as well as
methylcellulose assays (Nakahata et al., 1982b; Pharr et
al., 1984; Suda et al., 1985), and limiting dilution assays
(Crapper and Schrader, 1983; Guy-Grand et al., 1984). In
addition to the bone marrow data, we found that adult mouse
spleen contained 30 mast cell precursors per million input
cells, again consistent with previously published results
(Nakahata et al., 1982b; Crapper and Schrader, 1983; Guy-
Grand et al., 1984; Pharr et al., 1984).
Analysis of bone marrow derived from both of the
heterozygous crosses demonstrated that these tissues are
equally rich reservoirs of mast cell precursors. No
quantitative differences in mast cell precursor frequency
in (BALB/cAN x CBA/J)F1 and (BALB/cAN x BIO.BR/SgSn)Fl bone
marrows were found, nor was there a tendency toward
elevated precursor numbers in the heterozygotes when
compared to homozygotes. The differences in precursor


71
volume (5 microliters) of S.aureus-RAMIgG-monoclonal
antibody sandwiches. Following a thirty-minute incubation
on ice, the contents of each well were washed six times and
a sample was prepared as a cytocentrifuge mount. Slides
were stained with May-Gruenwald Giemsa or Wright's Giemsa.
Fc Receptor Assays
Receptors for the Fc domain of mouse igE and IgG were
detected by a novel modification of the S. aureus method
used to detect other surface antigens (Siden and Siegel,
1986). Indicator bacteria were prepared by incubating 25
microliters of packed S. aureus or Escherichia col i (E.
coli) with 0.175 ml of 2,4,6-trinitrobenzene sulfonic acid
(3.7 mg per ml in 0.28M cacodylate buffer, pH6.9) for 10
minutes at room temperature. The 1.5 ml microfuge tube
containing the reactants was wrapped in foil to retard
photodecomposition and taped to a rotator during
incubation. After four washes with 0.01 M phosphate-
buffered 0.15 M sodium chloride, pH 7.3 (PBS; Mishell and
Shiigi, 1980), the TNP-S. aureus or TNP-E. coli were
resuspended to their orginal volume in balanced salt
solution (BSS; Mishell and Shiigi, 1980) with 10 mM HEPES
(pH 7.35), 0.1% w/v sodium azide, 5x10^ M 2-
mercaptoethanol (Sigma Chemical Co.) and 1% v/v fetal
bovine serum (H10BNF1).
Hybridoma supernatants containing mouse IgE anti-
(DNP)2 (IgELa2, American Type Culture Collection) and


24
biochemically undefined), less histamine, and serotonin
(Bienenstock et al., 1983).
The response of mucosal mast cells to a variety of
secretagogues has been the subject of a number of studies.
Similar to serosal mast cells, mucosal mast cells are
responsive to the degranulation effects of IgE and antigen,
IgE and anti-IgE, concanavalin A, ionomycin, and compounds
23187 and Br-X537A, albeit with the release of less of
their total histamine content (Befus et al., 1982a, 1982b;
Pearce et al., 1982). Enerback's early observations on the
insensitivity of rat mucosal mast cells to degranulation by
compound 48/80 jui vivo were confirmed by other
investigators in canine (Lorenz et al., 1969; Rees et al.,
1981) and murine (Enerback, 1981) models. This
unresponsiveness was confirmed in isolated mucosal mast
cells and extended to the secretagogue Bee Venom Peptide
401 (Befus et al., 1982a, 1982b), for which membrane
receptors were found to be absent on mucosal, but not
serosal, mast cells (Pearce et al., 1982). The same group
also reported that, unlike serosal mast cells, mucosal mast
cells were unresponsive to enhanced, antigen-induced
secretion of histamine mediated by phosphatidyl serine.
Mucosal mast cells were also shown to be distinct from
serosal mast cells in their responsiveness to secretory
antagonists disodium chromoglycate, theophylline, and
AH9679, while both subsets were equally sensitive to the


210
Rocklin, R.E. 1976. Modulation of cellular immune responses
in vivo and in vitro by histamine receptor-bearing
lymphocytes. J. Clin. Invest. 57: 1051-1058.
Roe, R., and S.C. Bell. 1982. Humoral immune responses in
murine pregnancy. II. Kinetics and nature of the
response in females pre-immunized against paternal
antigens. Immunology 46: 23-30.
Rose, M.L., D.V.M. Parrott, and R.G. Bruce. 1976.
Migration of lymphoblasts to the small intestine. I.
Effect of Trichinella spiralis infection on the
migration of mesenteric lymphoblasts and mesenteric T
lymphoblasts in syngeneic mice. Immunology 31: 723-730.
Rosenberg, N., and D. Baltimore. 1976a. A quantitative
assay for transformation of bone marrow cells by Abelson
murine leukemia virus. J. Exp. Med. 145: 1453-1463.
Rosenberg, N., and D. Baltimore. 1976b. In vitro
lymphoid cell transformation by Abelson murine leukemia
virus. In D. Baltimore, A.S. Huang, and C.F. Fox
(eds). Animal Virology. Academic Press, New York, NY.
Rosenberg, N., and D. Baltimore. 1980. Abelson virus. In
G. Klein (ed.). Viral Oncology. Raven Press, New York,
NY.
Rosenberg, N., D. Baltimore, and C.D. Scher. 1975. In
vitro transformation of lymphoid cells by Abelson
murine leukemia virus. Proc. Natl. Acad. Sci. USA 72:
1932-1936.
Ruitenberg, E.J., and A. Elgersma. 1976. Absence of
intestinal mast cell response in congenitally athymic
mice during Trichinella spiralis infection. Nature
264: 258-260.
Sathiamoorthy, S.S., A.K. Ganguly, and O.P. Bhatnagar. 1976.
Effect of bilateral adrenalectomy and parenteral
betamethasone on gastric mucosal mast cell population in
albino rats. Experientia 32: 1300-1301.
Schalm, O.W., N.C. Jain, and E.J. Carroll. 1975. Materials
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Scheid, M.P., and D. Triglia. 1979. Further description of
the Ly-5 system. Immunogenetics 9: 423-433.


CHAPTER V
SUMMARY AND CONCLUSIONS
The experimental procedures described in the preceding
pages have enabled us to report a number of novel and
significant observations which contribute to the existing
body of knowledge concerning mast cells. We began our
studies following the observation of cells with basophilic
and metachromatic granules in lines of Abelson murine
leukemia virus-transformed cells which were derived from in
vitro-infected midgestation mouse embryonic placentae. The
relationship of the A-MuLV-transformed cells to culture-
derived mast cells was further substantiated by the
observation that both expressed the epitope recognized by
the B23.1 monoclonal antibody, which binds to a determinant
on culture-derived, but not peritoneal, mouse mast cells.
Most of the cell lines also contained histamine in
quantities similar to those found in culture-derived mast
cells. Furthermore, there was a direct correlation between
the histamine content and the expression of surface IgE
receptors, another mast cell phenotypic marker, in the cell
lines which were analyzed. In contrast to culture-derived
mast cells, however, the A-MuLV-transformed mast-like cells
proliferated in the absence of exogenous interleukin 3.
Unlike other autogenous mast-like cells (Schrader and
Crapper, 1983) but similar to recently reported A-MuLV
fetal liver transformants (Pierce et al., 1985), the cell
lines produced no detectable interleukin 3.
183


46
(Schrader et al., 1983b; Guy-Grand et al., 1984). Like
their precursors, more differentiated culture-derived mast
cells are deficient in Thy 1, Lyt 1, and Lyt 2 ( Ginsburg
et al., 1981; Nabel et al., 1981; Schrader, 1981; Schrader
et al., 1981; Tertian et al., 1981; Davidson et al., 1983;
Sredni et al., 1983; Ghiara et al., 1985), although
Schrader and colleagues (1982) reported that Thy 1 may be
transiently expressed on these cells. Similarly, culture-
derived mast cells lack surface immunoglobulin, a B lineage
marker (Ginsburg et al., 1981; Schrader et al., 1981;
Tertian et al., 1981; Sredni et al., 1983), NK-1, a marker
of natural killer cells (Nabel et al., 1981), complement
receptors and MAC-1, a differentiation antigen of
mononuclear phagocytes which is also expressed by some
natural killer and T lymphoma cells (Tertian et al., 1981).
Like their ini vivo correlates, murine culture-derived
mast cells express surface receptors for IgE (Ginsburg et
al., 1978; Nagao et al., 1981; Schrader, 1981; Schrader et
al., 1981; Tertian et al., 1981; Ginsburg et al., 1982;
Nakahata et al., 1982b; Sredni et al., 1983; Wedling et
al., 1983, 1985), which induce the anaphylactic release of
histamine when cross-linked by IgE and homologous antigen
(Ginsburg et al., 1978; Sredni et al., 1983) or anti-IgE
(Ginsburg et al., 1982). The number of IgE receptors per
cell has been estimated to be 2 to 3 x 10^, similar to the
number on serosal and mucosal mast cells (Razin et al..


Table IV-1. Frequency of Mast Cell Precursors in Adult Bone Marrow from Homozygous and Heterozygous Mice
Number of Colonies per Million Bone Marrow Cells3
Strain
Experiment 1
Experiment 2
Experiment 3
Experiment 4
All Experiments
BALB/cAN
450+/-150(2)
378+/-25.4(4)
371+/-20.9(3)
391+/-51.6(9)
BIO.BR/SgSn
352+/-34.7(3)
308+/-8.5(3)
290+/-10(2)
320+/-18.7(8)
CBA/J
435+/-18.7(3)
398+/-13.1(3)
408+/-16.5(3)
420+/-8.2(3)
415+/-14.1(12)
(BALB/c x BIO.BR)F1
301+/-13.4(4)
304+/-28.1(4)
377+/-28.7(3)
382+/-6.2(3)
336+/-19.3(14)
(BALB/c x CBA)F1
338+/-22.8(4)
350+/-36(4)
403+/-20.5(3)
402+/-19.3(3)
369+/-25.3(14)
a: Mast cell cultures in agar were prepared as described in Materials and Methods. Numbers represent
statistical means +/- one standard deviation (number of cultures). Data for all experiments were
calculated as weighted means and standard deviations (Bahn,1972).
162


CHAPTER III
CHARACTERIZATION OF MAST CELLS DERIVED FROM MIDGESTATION
EMBRYONIC TISSUES IN LIQUID CULTURE
Introduction
Mast cells have been the object of innumerable
scientific investigations since their initial description
by Ehrlich (1877). The study of mast cells experienced
renewed impetus in the nineteen-sixties based upon research
in two major areas. First, the development of in vitro
culture techniques confirmed the existence of mast cell
precursors in a variety of tissues and indicated that mast
cell differentiation was dependent on factor(s) elaborated
by stimulated lymphocytes. Secondly, the development of
improved cytological fixation and staining techniques
(Enerback, 1966a, 1966b) permitted the characterization of
a new class of "atypical" mast cells (mucosal mast cells)
which were were distinct from the connective tissue-
associated (serosal) mast cells studied in detail prior to
that time. Within a short period of time, a morphological,
biochemical, and functional relationship between the in
vitro-derived mast cells and mucosal mast cells was noted.
The distinction between the culture-derived and
serosal mast cells was facilitated by the development of a
series of monoclonal antibodies which discriminated between
the two subsets (Katz et al., 1983). The observation of
metachromatic granules in A-MuLV-transformed placental cell
105


100
suface glycoprotein (Omary et al., 1980), which has been
observed on cultured mast cells (Nabel et al., 1981) as
well as on mastocytomas (Scheid and Triglia, 1979). We
have also extended the scope of expression of Ly5 on mast
cell tumors to the methycholanthrene-induced P815 and to
two A-MuLV/pristane in vivo-induced mastocytomas.
Interestingly, the latter three mastocytomas and our Ly5-
positive cell lines express the epitope recognized by the
monoclonal antibody RA3-3A1, which was previously thought
to be B lineage specific (Coffman and Weissman, 1981a,
1981b). Mice homozygous for the mutant lpr gene, which
experience severe early onset autoimmune disease, exhibit
lymphoproliferation of a thymus-dependent Ly 5-positive,
cell population; the proliferating cells, however, lack
other B-lymphoid characteristics and appear to be of the T
lineage (Morse et al., 1982).
Three of the embryonic ceil lines also expressed early
thymocyte antigens. One of the cell lines, 11P0-1,
expressed the fetal thymocyte-specific epitope FT-1 (Kasai
et al., 1983) as well as the neutral giycolipid asialo-GMl,
while two other cell lines (9P1 and 10P6) expressed asialo-
GMl only. The latter differentiation antigen, which is
expressed on twelve to fifteen day fetal thymocytes (Habu
et al., 1980), has also been observed on natural killer
cells (Kasai et al., 1980; Young et al., 1980). However,
none of the embryonic cell lines exhibited natural killer


104
of interleukin 3-dependent FDC-P1 cells. The protein
kinase C translocation kinetics in FDC-P1 cells is
paralleled by the DNA synthesis dose response curve of
these cells, suggesting a relationship between enzyme
association with plasma membranes and cell proliferation.
The Abelson murine leukemia virus transforming gene product
is a transmembrane protein (Witte et al., 1979b) with known
tyrosine protein kinase activity (Witte et al., 1980) and a
normal cellular analogue (Witte et al., 1979a). Cells
transformed by the virus contain a number of cellular
proteins which are phoshorylated on tyrosine residues
(Cooper and Hunter, 1981); Sefton et al., 1981a, 1981b).
We therefore propose that the Abelson murine leukemia virus
transforming protein may act on the substrate of protein
kinase C or one of the other enzymes in the pathways that
are normally activated by extracellular messengers (like
interleukin 3) which generate transmembrane control of
cellular functions (Nishizuka, 1984; Marx, 1984; Michell,
1984). Further studies to determine which, if any, of
these mechanisms is in effect are in order to elucidate the
functional role of the A-MuLV transforming protein.


177
previously been observed on cultured mast cells (Nabel et
al., 1981; Wong et al., 1982) and mast cell tumors (Scheid
and Triglia, 1979); in addition, it was detected on two A-
MuLV-transformed placental mast cell lines and on the
murine mastocytoma P815 and two A-MuLV/pristane-induced
mastocytomas (Siegel et al., 1985; Chapter II). The
expression of Ly5 on the surface of mast cells may
represent a discreet stage in the maturation which is not
attained under the conditions of our liquid culture system
(Chapter III) .
The final aspect of our studies described in this
chapter dealt with the determination of cell surface
markers on mouse bone marrow-derived mast cell precursors.
Although the literature contains numerous references to
surface markers on liquid culture-derived mast cells
(reviewed by Katz et al., 1985a), we were only able to find
a single reference to expression of such markers on mast
cell precursors (Yung et al., 1983). Treatment of mouse
bone marrow cells with anti-la and rabbit complement
resulted in the loss of fifty percent of the interleukin 3-
responsive (proliferating) population; the same treatment
also depleted granulocyte-macrophage colony-forming units
(CFU-GM) by the same factor.
Three surface determinants detected on A-MuLV-
transformed embryonic mast-like cell lines were selected
for sorting mast cell precursors. The sorting methodology


40
however, that the cells were able to maintain their growth
in the presence of interleukin 3 at levels below those
detected in the assay. interestingly, both sets of factor-
independent mast cells were tumorigenic in syngeneic mice.
Although no retroviral particles were observed in one of
these cell lines (Ball et al., 1983), the observations are,
by the criteria of tumorigenicity and factor independence,
similar to those of a recent report of Abelson murine
leukemia virus-transformation of culture-derived mast cells
(Pierce et al., 1985). Two possible mechanisms could
reconcile the yet unexplained results. First, the
activation of a latent replication-defective viral genome
in the Long-term culture-derived mast cells of Ball and
colleagues could be the missing link. Under such
circumstances, no viral particles would be detected, but
the cells could become both factor independent and
tumorigenic by virtue of the viral transforming gene
product. Similarly, the activation of a cellular homologue
of a viral transforming gene, like c-abl (the normal
function of which is unknown), could be invoked to activate
the mechanisms necessary to generate the phenotype of the
factor-independent, tumorigenic mast cells.
Mast Cell Precursors
The development of in vitro techniques for the
differentiation and maturation of mast cells from
phenotypically immature progenitors permitted the analysis


52
receptors for IgE. Rat culture-derived mast cell granules
stained blue by the alcian blue-safranin technique and were
metachromatic when stained with toluidine blue. The
granule proteoglycan was identified as non-heparin,
although no report of its precise chemical composition has
been published to date. Such cells also contained
immunochemically detectable levels of rat mast cell
protease II, which was previously described as a marker of
mucosal, but not serosal mast cells. Rat mesenteric lymph
nodes (Denburg et al., 1980) and peripheral blood (Zucker-
Franklin et al., 1981) have also been shown to contain
precursors of culture-derived mast cells.
Reports of mast cells derived from human tissues are
clouded by difficulties in distinguishing between the
various types of basophilic cells when compared to in vivo
correlates, namely mucosal mast cells, connective tissue
mast cells, and basophils. Granulated cells with receptors
for IgE and low levels of histamine (50 to 450 nanograms
per million cells), thus resembling mouse culture-derived
mast cells, have been observed in cultures of human fetal
liver grown in unconditioned media (Razin et al., 1981b).
Adherent human blood mononuclear cells and pleural exudate
cells, which were propagated with L-cell conditioned media,
exhibited similar characteristics (Czarnetzki et al., 1983,
1984; Kruger et al., 1983). Cells with high affinity
receptors for IgE and slightly higher levels of histamine


156


56
to proliferate in the presence of mouse interleukin 3 (in
WEHI-3 conditioned media). Thus, in the human system, the
culture-derived mast cell-basophil dilemma is no longer
solely a matter of terminology and mistaken identity, but,
in fact, apppears to involve multiple growth promoters and,
most likely, multiple progenitor cells.
Relationship of In Vivo- and In Vitro-Derived Mast Cells
A small body of evidence supports the theory that
culture-derived mast cells are more than circumstantially
related to mucosal, and perhaps serosal, mast cells. A
number of characteristics, including morphology,
histochemical fixation and staining, dependence of
proliferation on T cell-derived factors, biogenic amine
content, protease content, presence of receptors for IgE,
and sensitivity to secretogogues, have been noted in this
review and cited by many of the authors as proof of the
relationship between culture-derived mast cells and the
mast cells of the mucosal surfaces. There is preliminary
evidence that culture-derived mast cells have natural
cytotoxic activity against tumors such as WEHI-3 and Meth A
which is enhanced by interleukin 3 (Ghiara et al., 1985).
Investigators have noted that in vivo-derived mast ceils
exhibited similar tumoricidal activity (Farram and Nelson,
1980) and that in vivo-derived cells with the


143
Neither cocultivation with adherent cells nor their
conditioned media resulted in the expression of the B220
antigen on long-term cultured mast cells (Table III-l),
while the expression of receptors for IgE, the epitope of
monoclonal antibody B23.1, and the presence of
metachromatic granules was unchanged with respect to
untreated mast cells. We therefore conclude that neither
the adherent cells used nor their cytokines are responsible
for the expression of Ly5 on the surface of mature mast
cells. Further studies are required to clarify the Ly5
expression enigma. It seems likely that Ly5 may only be
transiently expressed on mast cells during the course of
their differentiation. We propose that the expression of
the determinant is perhaps fixed when such cells are
transformed with Abelson murine leukemia virus and
protracted when immature mast cells are maintained in agar
culture. Alternatively, the B-220 variant of the Ly 5
antigen may be expressed on a more differentiated form of
mast cell which is not present in standard liquid cultures.


213
Sidman, C.L., and E.R. Unanue. Control of proliferation and
differentiation in B lymphocytes by anti-Ig antibodies
and a serum-derived cofactor. Proc. Natl. Acad. Sci.
USA 75: 2401-2405.
Siegel, M.L., R.J. Horwitz, T.D. Morris, R.M. DiVenere, and
E.J. Siden. 1985. Isolation and characterization of
Abelson murine leukemia virus transformed mast cell
lines from midgestation embryonic placenta. Eur. J.
Immunol. 15: 1136-1141.
Silverstone, A.E., N. Rosenberg, D. Baltimore, V.L. Sato,
M.P. Scheid, and E.A. Bovse. 1978. Correlating
terminal deoxynucleotidyl transferase and cell-surface
markers in the pathway of lymphocyte ontogeny. In B.
Clarkson, P. Marks, and J. Till (eds.). Differentiation
of Normal and Neoplastic Hematopoietic Cells. Cold
Spring Harbor Laboratory, New York.
Sklar, M.D., E.M. Shevach, I. Green, and M. Potter. 1975.
Transplantation and preliminary characterization of
lymphocyte surface markers of Abelson murine leukemia
virus-induced lymphomas. Nature 253: 550-552.
Soil, A.H., K.J. Lewin, and M.A. Beavin. 1981. Isolation
of histamine-containing cells from rat gastric mucosa:
Biochemical and morphologic differences from mast cells
Gastroenterology 80: 717-727.
Sonoda, T., Y. Kitamura, Y. Haku, H. Hara, and K.J. Mori.
1983. Mast cell precursors in various haematopoietic
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Biol. 98: 503-517.
Spivak, J.L., R.R.L. Smith, and J.N. Ihle. 1985.
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Invest. 76: 1613-1621.
Sredni, B., M.M. Friedman, C.A. Bland, and D.D. Metcalfe.
1983. Ultrastructural, biochemical, and functional
characteristics of histamine-containing cells cloned
from mouse bone marrow: Tentative identification as
mucosal mast cells. J. Immunol. 131: 915-922.
Stadler, B.M., K. Hirai, K. Tadokoro, and A.L. de Week.
1985. Distinction of the human basophil promoting
activity from human interleukin 3. Int. Archs. Allergy
Appl. Immunol. 77: 151-154.


76
cultures were seeded at 1x10^ per ml in 10P in bacterial-
grade petri dishes (Fisher Scientific Co.). Following four
days (92 to 100 hours) of incubation in humidified five
percent carbon dioxide, the conditioned media were
harvested by centrifugation and filtered through tissue
culture grade 0.2 micron nitrocellulose filters.
Conditioned media were concentrated by stirred-cell
ultrafiltration (Amicon, Danvers, MA), by dialysis against
polyethylene glycol 20,000 (Fisher Scientific), or by
ammonium sulfate (Fisher Scientific Co.) precipitation when
such procedures were desired.
Cell lysates were prepared from cells grown to 7 to
9x10^ per ml as follows: Cell cultures were centrifuged
(ten minutes at 200xg, 4) and the cells were washed twice
in PBS. The cells were resuspended in one milliliter 10P
and subjected to three rounds of alternate freezing (in dry
ice-ethanol) and thawing (at 37C). The lysates, which
contained no viable cells upon microscopic examination,
were cleared centrifuga 1ly at 2000xg for fifteen minutes
(4C) and at 12,000xg for fifteen minutes (4C), and were
filtered through 0.45 micron sterile, disposable filters
(Gelman Sciences, Ann Arbor, MI). Lysates were stored at
-20C prior to use.
Assay for Interleukin 3
Interleukin 3-like activity was analyzed by a
modification of the method of Razin and colleagues (1984a).


115
culture cluster (Costar, Broadway, MA). After six days in
culture (five percent carbon dioxide in air at 37C), the
supernatants, with nonadherent cells, were removed and 2.5
ml of fresh 50% W3CM were added. Two days later, the bone
marrow-derived adherent cell-conditioned media (bmadhCM)
were harvested by centrifugation (200xg, fifteen minutes)
and filtration through a PBS-washed, 0.2 micron disposable
filter (Gelman Sciences, Inc., Ann Arbor, MI). The
adherent cell monolayers were washed twice with cold PBS to
remove any residual nonadherent cells and conditioned media
and then overlayered with 2 ml of 0.3 percent agar (Difco,
Detroit, MI) in 50% W3CM.
P388D1 monocytic tumor cells were grown in RPMI 1640
with 10 percent v/v heat-inactivated fetal bovine serum,
2 mM glutamine and 0.05 mM 2-mercaptoethanol (10P) to a
density of 2x10^ to 1x10^ per ml. Cells were pelleted by
centrifugation (200xg for ten minutes at room temperature)
and resuspended at lxlO6 per ml in 10P made 20 micrograms
per ml with Escherichia coli serotype B5:055
lipopolysaccharide (LPS, Sigma Chemical Co.) (10P/LPS)
after the method of Lachman and colleagues (1979). Within
three hours, most of the cells, which were aliquoted at
1x10^, 0.5x10^ and 0.25x10^ per well in a six well cluster
(in 1 ml of 10P/LPS), had become adherent. P388/LPS-
conditioned media (P388/LPSCM) were harvested after two
additional days in culture in a manner identical to that


195
Doolittle, R.F., M. W. Hunkapiller, L.E. Hood, S.G. Devare,
K.C. Robbins, S.A. Aaronson, and H.M. Antonaides. 1983.
Simian sarcoma virus one gene, v-sis, is derived
from the gene (or genes) encoding a platelet derived
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Downward, J., Y. Yarden, E. Mayes, S. Scrase, N. Toddy, P.
Stockwell, A. Ullrich, J. Schlessinger, and M.D.
Waterfield. 1984. Close similarity of epidermal growth
factor receptor and v-erb B oncogene protein sequences.
Nature 307: 521-527.
Dunn, T.B., and M. Potter. 1957. A transplantable mast cell
neoplasm in the mouse. J. Natl. Cane. Inst. 30: 587-601
Dvorak, A.M., G. Nabel, K. Pyne, H. Cantor, H.F. Dvorak, and
S.J. Galli. 1982. Ultrastructure of the mouse basophil
Blood 59: 1279-1285.
Efrati, P., A. Klagman, and H. Spitz. 1957. Mast cell
leukemia? Malignant mastocytosis with leukemia-like
manifestations. Blood 12: 869-882.
Ehrlich, P. 1877. Bietrage zur Kenntnis der Anilinfarbungen
und ihrer Verwendung in der Mikroskopishen Technik.
Arch. f. Mikros. Anat. 13: 263-269.
Ehrlich, P. 1879. Bietrage zur Kenntis der granulierten
Bindegewebszellen und der eosinophilen Leukocythen.
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mucosa. 1. Effects of fixation. Acta Pathol.
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mucosa. 2. Dye-binding and metachromatic properties.
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Allergy 17: 222-232.


70
Rabbit anti-asiaio GM1 was received from W.W. Young
(University of Virginia Medical Center) as a delipidated
serum. Rabbit anti-rat immunoglobulins was purchased as a
lyophilized powder (IgG fraction of rabbit anti-rat IgG
(heavy and light chains), Miles Laboratories, Elkhardt, IN)
and reconstituted per manufacturer's specifications.
Rabbit anti-mouse immunoglobulins was purchased from
Gateway Biologicals (St. Louis, MO).
Normal rat serum was prepared from cardiac blood of an
unimmunized animal. Normal mouse serum was prepared from
pooled specimens of multiple unimmunized BALB/cAN mice.
Cell Surface Markers
Cell surface differentiation antigens were detected by
a modification of the method of Uchanska-Ziegler and
colleagues (1982). Formalin-treated, heat killed
Staphylococcus aureus (S.aureus) Cowan I (The Enzyme
Center, Inc.,Boston, MA, and later a generous gift of Dr.
Michael D.P. Boyle) were first coated with rabbit anti-rat
IgG (RAMIgG, Miles Laboratories, Elkhart, IN) and then with
rat monoclonal antibodies specific for mouse
differentiation markers. Either hybridoma cell culture
media or partially purified immunoglobulins were used as a
source of the latter antibodies.
Mouse cells (1x10^ or fewer) selected for analysis
were pelleted in round-bottom, PVC microtiter wells
(Dynatech, Alexandria, VA) and resuspended in a small


201
King, S.J., H.R.P. Miller, G.F.J. Newlands, and R.G. Woodbury.
1985. Depletion of mast cell protease by corticosteroids
Effect on intestinal anaphylaxis in the rat. Proc. Natl.
Acad. Sci. USA 82: 1214-1218.
King-Smith, E.A., and A. Morley. 1970. Computer simulation
of granulopoiesis. Normal and impaired granulopoiesis.
Blood 36: 254-262.
Kitamura, Y., S. Go, and K. Hatanaka. 1978. Decrease of
mast cells in W/Wv mice and their increase by bone
marrow transplantation. Blood 52: 447-452.
Kitamura, Y., K. Hatanaka, M. Murakami, and H. Shibata.
1979a. Presence of mast cell precursors in peripheral
blood of mice demonstrated by parabiosis. Blood 53:
1085-1088.
Kitamura, Y., M. Shimada, and S. Go. 1979c. Presence of
mast cell precursors in fetal liver of mice. Dev. Biol.
70: 510-514.
Kitamura, Y., M. Shimada, S. Go, H. Matsuda, K. Hatanaka, and
M. Seki. 1979d. Distribution of mast cell precursors
in hematopoietic and lymphopoietic tissues of mice. J.
Exp. Med. 150: 482-490.
Kitamura, Y., M. Shimada, K. Hatanaka, and Y. Mivano. 1977.
Development of mast cells from grafted bone marrow cells
in irradiated mice. Nature 268: 442-443.
Kitamura, Y., M. Yokoyama, H. Matsuda, and T. Ohno. 1981.
Spleen colony-forming cell as common precursor for tissue
mast cells and granulocytes. Nature 291: 159-160.
Kruger, G., W. Sterry, and B.M. Czarnetzki. 1983. Cultures
of mast cell-like (MCL) cells from human pleural exudate
cells. Blut 46: 143-148.
Lagunoff, D. 1972. Contributions of electron microscopy to
the study of mast cells. J. Invest. Dermatol. 58: 296-
311.
Lagunoff, D. 1985. The mast cell. In E.B. Weiss, M.S.
Segal, and M. Stein (eds.), Bronchial Asthma:
Mechanisms and Therapeutics. Little, Brown and Company,
Boston, MA.


198
Greenberger, J.S., P.B. Davisson, P.J. Gans, and W.C.
Moloney. 1979. In vitro induction of continuous acute
promyelocytic cell lines by Friend or Abelson murine
leukemia virus. Blood 53: 987-1001.
Guy-Grand, D., M. Dy, G. Luffau, and P. Vassalli. 1984.
Gut mucosal mast cells. Origin, traffic, and
differentiation. J. Exp. Med. 160: 12-28.
Habu, S., M. Kasai, Y. Nagai, N. Tamaoki, T. Tada, L.A.
Herzenberg, and K. Okumura. 1980. The glycolipid
asialo GM1 as a new differentiation antigen of fetal
thymocytes. J. Immunol. 125: 2284-2288.
Haig, D.M., T.A. McKee, E.E. Jarrett, R. Woodbury, and H.R.
Miller. 1982. Generation of mucosal mast cells is
stimulated in vitro by factors derived from T cells of
helminth-infected rats. Nature 300: 188-190.
Haig, D.M., C. McMenamin, C. Gunneberg, R. Woodbury, and
E.E.E. Jarrett. 1983. Stimulation of mucosal mast cell
growth in normal and nude rat bone marrow cultures.
Proc. Natl. Acad. Sci. USA 80: 4499-4503.
Hall, B.T. 1981. Mechanism of altered cell-mediated
immune responsiveness in mice infected with Trichinella
spiralis. Ph. D. dissertation, University of Florida.
Hara, H., and M. Ogawa. 1978. Murine hemopoietic colonies
in culture containing normoblasts, macrophages, and
megakaryocytes. A. J. Hematol. 4: 23-34.
Hasthorpe, S. 1980. A hemopoietic cell line dependent on
a factor in pokeweed mitogen-stimulated spleen cell
conditioned medium. J. Cell. Physiol. 105: 379-384.
Hatanaka, K., Y. Kitamura, and Y. Nishimune. 1979. Local
development of mast cells from bone marrow-derived
precursors in the skin of mice. Blood 53: 142-147.
Henderson, W.R., E.Y. Chi, E.C. Jong, and S.J. Klebanoff.
1981. Mast cell-mediated tumor cell cytotoxicity. Role
of the peroxidase system. J. Exp. Med. 153: 520-533.
Horton, M.A., and H.A.W. O'Brien. 1983. Characterization
of human mast cells in long term culture. Blood 62:
1251-1260.


129
transformed bone marrow pre-B cells which are present in
the early cultures. This was substantiated by the
morphology of the cells in the cultures. Figure III-6 shows
both RA3-3Al-positive agranular, lymphoid cells and RA3-
3Al-negative granular cells in the same day 28 culture.
Subsequent infections of mast cells with A-MuLV were
performed with long-term cultures which were deficient in
both adherent cells and RA3-3Al-positive cells. When
assayed as described above, neither A-MuLV- nor sham-
infected culture-derived mast cells had constituent
subpopulations expressing the lymphoid marker (data not
shown).
The presence of A-MuLV in cultures of cells treated
with the virus was assayed to confirm that the cells were
indeed productively infected with the same transforming
virus which provided us with the placental tumor cell lines
described in Chapter II. Biosynthetically -^^S-methionine-
labeled cells were lysed in detergent with proteinase
inhibitors and the cleared lysates were specifically
precipitated with goat anti-M-MuLV. The latter antiserum
precipitates all major M-MuLV proteins and will likewise
bind to the A-MuLV gag-abl transforming protein by nature
of its M-MuLV gag epitopes. The immunoprecipitated
proteins were separated electrophoretically on a 7.0
percent, SDS polyacrylamide gel. The autoradiograph of the
gel (Figure III-7) shows virus specific bands of
i


4
of pre-B ceils (Boss et al., 1979; Siden et al., 1979; Alt
et al., 1981, 1984). Using identical conditions for
transformation of mouse placenta and fetal liver, Waneck
and Rosenberg (1981) described colonies of cells expressing
various differentiated erythroid characteristics,including
cessation of growth. Unlike its Moloney leukemia virus
ancestor, A-MuLV transforms the NIH/3T3 fibroblast cell
line (Scher and Siegler, 1975).
Modifications of the original Rosenberg and Baltimore
in vitro transformation protocol have resulted in the
transformation of phenotypically disparate lineages.
Whitlock and colleagues (1983) and Serunian and Rosenberg
(1986) have reported the transformation of more
differentiated B lineage ceils from in vitro-derived
"normal" populations. More recently, permanent cell lines
expressing mast cell characteristics have been reported
following transformation of midgestation embryonic placenta
(Siegel et al., 1985) and third trimester fetal liver
targets (Pierce et al., 1985).
Recent reports from several laboratories indicate that
retroviruses may alter the growth factor requirement of
cells of several lineages. Rapp and colleagues (1985)
reported the development of factor-independent cell lines
following transformation of interleukin 2-dependent T
lineage and interleukin 3-dependent myeloid cell lines by
recombinant viruses bearing the v-myc oncogene. Abelson


39
to growth in interleukin 3-free liquid media. The
autonomous colonies secreted interleukin 3 into the culture
media, but also retained their receptors for the factor.
Furthermore, the autonomous cells generated more colonies
when plated at low density in the presence of exogenous
interleukin 3 in agar than in the absence of the growth
factor. It seems likely that the "autogenous cells were
not truly factor-independent, but rather were variants
which were able to proliferate, albeit at lower efficiency,
at the low levels of interleukin 3 provided by an autocrine
mechanism.
Similar factor-independent culture-derived mast cells
have been reported by a second group (Ball et al., 1983;
Conscience and Fisher, 1985). Several long-term bone
marrow-derived cultured mast cell lines were found, after
eleven months in culture, to contain variants which
proliferated at higher rates than similar cultures in the
presence of interleukin 3. The more proliferative cells
were able to continue cell growth in the absence of
exogenous interleukin 3, but the doubling time was
increased 160 percent when compared to cells of the same
line maintained in conditioned media. In contrast to the
autogenous cells of Schrader, the media conditioned by the
factor-independent mast cells described in the more recent
studies failed to support the growth of other factor-
dependent culture-derived mast cells. It is possible,


153
Washed and counted bone marrow cells were incubated
with PBS-dialyzed, filter-sterilized IgE anti-DNP2
hybridoma supernatants; again, 0.1 ml of antibody
preparation was used per 1x10^ cells (empirically
determined to be optimum) and 1x10^ to 3x10^ cells were
sorted at one time. The tubes containing the cells were
incubated for one hour at 37C, centrifuged (ten minutes at
200xg, 4C) and washed twice with cold BSS. The pelleted
cells were flicked, resuspended in heavily-modified TNP-
SRBC (in 100-fold excess) and centrifuged one minute at
375xg (4C). Following a twenty-minute incubation on ice,
the tubes were centrifuged at 375xg for an additional five
minutes, the supernatants removed and the resuspended
pellets carefully overlayered on 3 ml of Lympholyte M.
Remaining procedures were identical to those performed on
B23.1 and RA3-3Al-sorted cells, with the notable exception
that only hypotonic shock was found to be effective at
lysing TNP-SRBC.
Multiple sorting experiments were performed as
described for single sorting, with the following
exceptions: Bone marrow cells were incubated with one
antibody preparation, washed three times with cold PBS and
then resuspended in the second antibody preparation for an
additional thirty minute incubation. Following washes as
noted before, the bone marrow cells were mixed with either
200 fold excess of avidin-SRBC or 100 fold excess each of


68
ethanol, pH 0.5 after fixation for two minutes in Mota's
fixative (lead subacetate in acidic ethanol)(Yam et al.,
1971). All stained slides were coverslipped with Permount
(Fisher Scientific Co.) prior to observation by light
microscopy.
Antibodies
Lineage-specific determinants on the surfaces of
placental and control cell lines were probed with a panel
of monoclonal (mAb) and polyvalent antibodies whose
significant features are summarized in Table II-l. All
monoclonal antibody preparations, unless otherwise noted,
were used as filtered hybridoma supernatants from
stationary phase cultures grown (in our facility) in
Dulbecco's Modified Eagle's Minimal Essential Media (GIBCO)
with 10 percent heat-inactivated fetal bovine serum and
5x10~5 m 2-mercaptoethanol. Preliminary work with mAb
B23.1 was performed with partially purified antibodies
generously supplied by P. LeBlanc (University of Florida);
later studies were performed with filtered hybridoma
supernatants which had been grown in RPMI 1640 with 10
percent fetal bovine serum, generously supplied by G. Place
in the laboratory of S. Russell (University of Florida).
Monoclonal antibody FT-1 was generously supplied by M.
Kasai (National Institute of Health, Tokyo, Japan); the
nature of its production and processing have been
previously reported (Kasai et ai., 1983).


Table II-3. Continued
WEHI-3
MYELOMONOCYTIC
LEUKEMIA
RLd 11
THYMOMA +
B2-4-4
THYMOMA +
YAC-1
THYMOMA ND ND
18-81
Pre-B + ++
FLEI-4
Pre-B ++ ++
P815
MASTOCYTOMA ++ +
CB6ABMC4
MASTOCYTOMA
CONTROL CELL LINES
++
+ ND ND
ND ++ ++
++ ND +
++ +
++
+ ++
++ +
++ ++
++ +
ND ND ND
++
++ £
++
ND +
BALABMC20
MASTOCYTOMA --++++ ND +
* Cell surface determinants were analyzed as described in Materials and Methods.
Scoring: indicates less than 5 bacteria per mouse cell
+ indicates 5 to 50 bacteria per mouse cell
++ indicates more than 50 bacteria per mouse cell
ND indicates reactivity not analyzed


15
The study of mast cells, which has accelerated
dramatically in the past quarter century, has revealed that
Ehrlich's "Mastzellen" are heterogeneous both
evolutionarily (interspecies) and functionally
(intraspecies). Mast cell heterogeneity has been the topic
of numerous general reviews in recent years (Enerback,1981;
Bienenstock et al., 1982, 1983; Pearce, 1982, 1983;
Shanahan et al., 1984; Jarrett and Haig, 1984; Austen,
1984: Katz et al., 1985a; Lagunoff, 1985; Pearce et al.,
1985). From this sea of literature, the differences
between mast cells of various species are apparent. The
multipotent biogenic compound of mammalian mast cells,
histamine, has not been detectable in fishes and
amphibians, while serotonin is apparently unique to rodent
and dopamine to bovine mast cells. Heparin proteoglycan
from porcine, bovine, rat, and human sources is
heterogeneous in molecular weight and charge (Stevens and
Austen, 1981). The proteolytic enzymes of mast cells are
also phylogeneticallv disparate; rodent mast cells contain
an alpha-chymotrypsin-like activity, while dog, human, and
turtle mast cells have trypsin-like activity and bird and
fish mast cells have no esteroprotease activity (Woodbury
and Neurath, 1980; Lagunoff, 1985). Ultrastructurally, the
granules of human mast cells appear to be organized in
crystalline scrolls, while rat mast cell granules are


47
1981a; Ginsburg et al., 1982). Some investigators have
also noted the presence of receptors for IgG (Schrader,
1981; Tertian et al., 1981); the cells, however, did not
phagocytose opsonized or unopsonized targets (Schrader,
1981; Sredni et al., 1983). Liquid cultured mast cells in
conconavalin A-stimulated spleen conditioned media also
express the lymphocyte marker Ly 5 (Nabel et al., 1981;
Tertian et al., 1981). Contradictory observations of
histocompatability Class II (la) antigens, or the lack
thereof, on culture-derived mast cells were resolved by
Wong and colleagues (1982), who showed that such cells,
grown in the presence of immune interferon (interferon
gammma, found in the supernatants of concanavalin A-
stimulated splenocytes) expressed the marker while cells
grown in the absence of interferon (as in WEHI-3
conditioned media) were devoid of la. Culture-derived mast
cells were also shown to express Class 1 histocompatability
antigens and receptors for peanut agglutinin (Schrader,
1981; Schrader et al., 1981; Tertian et al., 1981).
Russell and colleagues developed a panel of rat
monoclonal antibodies against murine mononuclear phagocytes
which could discriminate between culture-derived mast cells
and connective tissue mast cells (Leblanc et al., 1982).
The same group observed that culture-derived mast cells
expressed the phenotype B1.1-/B23.1+/ B54.2T, while in
contrast peritoneal mast cells were B1.1T/ B23.1-/B54.2T


171
linear in two experiments. The number of mast cell
colonies observed after reconstitution of both fractions at
the correct ratio was very close to the anticipated value
in one experiment and slightly greater than (140 percent)
the anticipated value in the second experiment, perhaps
indicating synergism between the partitioned cell types in
the formation of colonies. Further experiments to test the
cooperative hypothesis would be required before the latter
could be concluded.
Bone marrow sorted with IgE anti-TNP and TNP-SRBC
exhibited partition characteristics similar to that sorted
with RA3-3A1. Eighty-five to ninety-nine percent of the
mast cell precursors were recovered following sorting in
two independent experiments using two different methods to
lyse the SRBC. Cells seeded versus colonies enumerated
were linear in both experiments, and the number of colonies
counted in reconstitution experiments was similar to those
anticipated if only cells from the marker-negative
(supernatant) population were seeded, indicating that the
mast cell precursors found in the IgE receptor-negative
fraction were independent of IgE receptor-positive cells in
their ability to form mast cell colonies.


217
Wysocki, L.J., and V.L. Sato. 1978. "Panning" for
lymphocytes: A method for cell selection. Proc. Natl.
Acad. Sci. USA 75: 2844-2848.
Yam, L.T., C.Y. Li, and W.H. Crosby. 1971. Cytochemical
identification of monocytes and granulocytes. Amer. J.
Clin. Path. 55: 283-290.
Yoffe, J.R., D.J. Taylor, and D.E. Woolley. 1984. Mast
cell products stimulate collagenase and prostaglandin E
production by cultures of adherent rheumatoid synovial
cells. Biochem. Biophys. Res. Comm. 122: 270-276.
Yoffe, J.R., D.J. Taylor, and D.E. Woolley. 1985. Mast
cell products and heparin stimulate the production of
mononuclear-cell factor by cultured human
monocyte/macrophages. Biochem. J. 230: 83-88.
Yokota, T., F. Lee, D. Rennick, C. Hall, N. Arai, T. Mossman,
G. Nabel, H. Cantor, and K. Arai. 1984. Isolation and
characterization of a mouse cDNA clone that expresses
mast cell growth factor activity in monkey cells. Proc.
Natl. Acad. Sci. USA 81: 1070-1074.
Young, W.W., Jr., S.-I. Hakomori, J.M. Durdik, and C.S.
Henney. 1980. Identification of ganglio-N-
tetrasylceramide as a new cell surface marker for murine
natural killer (NK) cells. J. Immunol. 124: 199-201.
Yung, Y.P., R. Eger, G.Tertian, and M.A.S. Moore. 1981.
Long-term in vitro culture of murine mast cells.
II. Purification of a mast cell growth factor and its
dissociation from TCGF. J. Immunol. 127: 794-799.
Yung, Y.-P., and M.A.S. Moore. 1982. Long term in vitro
culture of murine mast cells. III. Discrimination of
mast cell growth factor and granulocyte-CSF. J.
Immunol. 129: 1256-1261.
Yung, Y.-P., and M.A.S. Moore. 1985. Mast-cell growth
factor: Its role in mast-cell differentiation,
proliferation, and maturation. Contemp. Top. Mol.
Immunol. 10: 147-179.
Yung, Y.-P., S.-Y. Wang, and M.A.S. Moore. 1983.
Characterization of mast cell precursors by physical
means: Dissociation from T cells and T cell precursors.
J. Immunol. 130: 2843-2848.
Yurt, R.W., R.W. Leid, Jr., K.F. Austen, and J.E. Silbert.
1977. Native heparin from rat peritoneal mast cells.
J. Biol. Chem. 252: 518-521.


TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS ii
LIST OF TABLES vi
LIST OF FIGURES vii
ABSTRACT viii
CHAPTERS
I INTRODUCTION AND REVIEW OF THE LITERATURE I
Introduction: Hematopoietic Cell Differentiation
and Tumor Models 1
Mast Cell Function, Origin, Ontogeny, and
Heterogeneity 6
Connective Tissue Mast Cells 18
Mucosal Mast Cells 19
Basophils 25
Mastocytomas 26
Culture-Derived Mast Cells 28
Other In Vitro-Derived Metachromatic Cells 53
Relationship of In Vivo- and In Vitro-Derived
Mast Cells 56
Epilogue 59
IIABELSON MURINE LEUKEMIA VIRUS-INFECTED CELLS
FROM MIDGESTATION PLACENTA EXHIBIT MAST CELL
AND LYMPHOID CHARACTERISTICS 61
Introduction 61
Materials and Methods 63
Results 77
Discussion 95
III CHARACTERIZATION OF MAST CELLS DERIVED FROM
MIDGESTATION EMBRYONIC TISSUES IN LIQUID
CULTURE 105
Introduction 105
Materials and Methods 106
Results 117
Discussion 135
IV


139
precursors were more immature) in contrast to bone marrow-
derived mast cells cultures (Figures III-l and III-3) for
the first week of in vitro growth but subsequently reached
similar proportions by week four. Expression of the B23.1
epitope, however, demonstrated similar population dynamics
for both adult and embryonic tissue-derived cells following
the first week in culture.
Reports of the progression of markers and cell types
in mast cell cultures have previously lacked quantitative
data of the kind presented here. In a series of studies
spanning two decades, Ginsburg (Ginsburg, 1963; Ginsburg
and Sachs, 1963; Ginsburg and Lagunoff, 1967; Davidson et
al., 1983) presented observations of sequential changes in
population morphology, size, granule content, nucleus-to-
cytoplasm ratio, and mitotic activity of cultures of mouse
thymocytes on feeder layers. The four stages of In vitro
mast cell development began with small colonies of
primitive mast cells and progressed to large lymphocytes
(or mastoblasts), young mast cells with mitotic figures,
and amitotic mature mast cells. Ishizaka et al. (1976)
reported that although less than 0.05 percent of similarly
prepared rat thymocytes initially possessed receptors for
IgE, most of the cells at the end of one week in culture
were blasts which had variable numbers of granules but were
still incapable of binding IgE. IgE receptors were not
detected in the Ishizaka cultures before day fourteen.


102
with B lineage characteristics (Rosenberg and Baltimore,
1976; Siden et al., 1979; Alt et al., 1981), and to induce
terminally differentiated, erythroid colonies from early
placenta and fetal liver (Waneck and Rosenberg, 1981).
Although A-MuLV induces mastocytomas in vivo (Mendoza and
Metzger, 1976; Risser et al., 1978), and can infect and
immortalize cultured mast cells (Pierce et al., 1985;
Chapter III of this work), the proliferation of continuous,
exogenous growth factor-independent mast cells from
midgestation embryonic tissue in the absence of interleukin
3 is unprecedented. Pierce and colleagues (1985) recently
reported the generation of interleukin 3-independent mast
like cell lines from day eighteen fetal liver cells
infected with A-MuLV and subsequently selected in media
containing the growth factor. Those authors speculated
that the omission of beta-mercaptoethanol from their tissue
culture media was probably responsible for the
proliferation of transformed mast cells. Since our cell
lines were produced in mercaptoethanol-containing media, we
suggest rather that the generation of interleukin 3-
independent mast cell lines may be due to unique conditions
of transformation which incoporate the B cell mitogen
dextran sulfate or the specific targets used.
Abeison murine leukemia virus has been shown to
transform, ini vitro, macrophages (Greenberger et al.,
1979), erythroid cells (Waneck and Rosenberg, 1981), and


187
adherent cells and their factors on culture-derived mast
cells. Long-term culture-derived mast cells did not
express the B lineage variant of Ly 5 before or after
coculture with adherent cells of several sources. We have
therefore concluded that neither Abelson murine leukemia
virus nor adherent cell monolayers were responsible for the
expression of a B lymphoid determinant on the mast-like A-
MuLV-transformed cell lines.
In the final chapter of this dissertation, we
enumerated and characterized mast cell precursors in
embryonic tissues and in adult bone marrow. Mast cells
derived from semisolid agar cultures expressed the B
lymphoid Ly 5 variant previously noted on the surfaces of
A-MuLV-transformed mast-like cells. Based on these and
previous observations, we have proposed that the expression
of such surface markers on agar culture-derived mast cells
represents a discreet stage of mast cell differentiation
which is not observed in liquid culture.
The ability to ascribe surface determinants to
cultured mast cell precursors may provide us with a better
understanding of the differentiation of all mast cells. We
have sorted primary bone marrow cells with monoclonal
antibodies which recognize surface determinants previously
noted on culture-derived mast cells and on A-MuLV-
transformed mast-like cells. Cells lacking the B
lymphoid variant of Ly 5 and receptors for IgE were


3
1980) in a prednisolone-treated BALB/cCR mouse (Abelson and
Rabstein, 1970). A-MuLV is capable of rapid transformation
of bone marrow-derived thymus-independent lymphoid cells in
vivo (Sklar et al., 1975; Premkumar et al., 1975) and in
vitro (Rosenberg et al., 1975; Baltimore et al., 1979;
Rosenberg and Baltimore, 1976a, 1980).
Although most of the in vivo transformants reportedly
have been of the B cell phenotype (Premkumar et al., 1975),
plasmacytomas have also been reported (Potter et al.,
1973). T cell lymphomas (Cook, 1982), myelomonocytic
leukemias (Raschke et al., 1978), and mast cell tumors
(Mendoza and Metzger, 1976; Risser et al., 1978; Pierce et
al., 1985) have also resulted from in vivo infections,
indicating that Abelson virus may affect the growth and
differentiation of multiple hematopoietic lineages.
Abelson murine Leukemia virus is capable of
transforming both hematopoietic and nonhematopoietic cells
in vitro. Rosenberg and Baltimore (Rosenberg et al.,
1975; Rosenberg and Baltimore, 1976a, 1976b) have developed
an jji vitro culture system for the transformation and
clonal proliferation of lymphoid cells from murine
hematopoietic tissues. Under well-defined conditions,
permanent cell lines with pre-B lymphoid characteristics
(Siden et al., 1979) have been generated. These cell lines
are believed to exhibit the earliest known differentiation
markers and immunoglobulin gene organizational structures


121
/
Figure III-2. Metachromatic Granules of Long-Term, Culture-
Derived Embryonic Mast Cells.
Mast cells were propagated, cytocentrifuge smears were prepared,
and slides were fixed and stained with 0.1 percent acidic
toluidine blue as described in Materials and Methods.


38
Bazill and colleagues ("multi-hematopoietic cell growth
factor", MCGF; Bazill et al., 1983) and Iscove
("multilineage hematopoietic growth factor", multi-HGF;
Iscove, 1985).
Interleukin 3, derived from a variety of sources, has
been purified to homogeneity (Yung and Moore, 1982; Ihle et
al., 1982b; Bazill et al., 1983; Clark-Lewis et al., 1984)
and, more recently, the genes for IL 3 have been molecularly
cloned and expressed (Fung et al., 1984; Yokota et al.,
1984; Rennick et al., 1985). Extensive reviews of the
literature describing interleukin 3 (per se, and the
related activities called by various other names) have been
published recently and should be consulted for additional
information (Clark-Lewis et al., 1985; Ihle, 1985; Iscove,
1985; Schrader et al., 1985; Whetton et al., 1985; Yung and
Moore, 1985).
Interleukin 3-Independent Mast Cells
Several reports of interleukin 3-independent mast cells
have appeared in the literature in recent years.
Schrader's group (Schrader and Crapper, 1983; Schrader et
al., 1983a) observed the emergence of factor-independent
variants from factor-dependent cells. In one experiment,
factor-dependent cells were plated in agar in the absence
of exogenous interleukin 3. From these cultures, several
colonies of autonomous culture-derived mast cells (P cells)
arose which, after several weeks, were subsequently adapted


98
subsets of mast cells which have been contrasted on the
basis of morphology, size, thymus-dependent proliferation,
fixation and staining requirements, proteases, proteoglycan
composition, and histamine content and release (reviewed by
Jarrett and Haig, 1984; Shanahan et al., 1984; Katz et al.,
1985a). The original mast cells studied by Ehrlich and his
proteges have been termed connective tissue or serosal mast
cells, while those isolated initially from intestinal
mucosa were termed atypical or mucosal mast cells for their
aberrant fixation and staining properties (Enerback, 1966a,
1966b). The relationship of the two subsets is still
unclear.
A third subset of mast cells was observed with the
development of techniques to propagate hematopoietic cell
in vitro. The culture-derived mast cells are
phenotypically similar to mucosal mast cells by a number of
criteria including morphology, granule number, size, and
staining requirements, histamine content, and proteoglycan
composition. The development of in vitro mast cell
precursor culture (Schrader, 1981; Nakahata et al., 1982b;
Crapper and Schrader, 1983) has permitted the cultivation
of mast cells from a number of adult tissues as well as
from day thirteen fetal mouse liver.
Some of the cell lines resulting from the
transformation of embryonic placental cells bv Abelson
murine leukemia virus are morphologically and


135
metachromatic granules remained unchanged as well in mast
cells derived from adult bone marrow or embryonic (day
eleven) placenta.
Discussion
The observation of mast-like cells in lines derived by
the transformation of midgestation embryonic placenta by
Abelson murine leukemia virus (Chapter II) prompted a number
of questions which were addressed in this chapter: Are
there mast cells in the placental and nonplacental
embryonic tissues at days ten and eleven of gestation? Are
there mast cell precursors in these tissues? Is A-MuLV
responsible for the expression of lymphocyte
characteristics on mast cells? The results of our
experiments have provided new and significant insight to
our understanding of mast cell differentiation.
Analysis of dissociated, fresh bone marrow, placenta
and nonembryonic placental embryonic tissues indicated few
or no cells with toluidine blue-stained metachromatic
granules indicative of mast cells. Mast cells, therefore,
comprise at most a minor, and perhaps insignificant,
portion of the cells in the tissues examined.
We have also documented, for the first time, the
presence of mast cell precursors in both embryonic placenta
and nonplacental embryonic tissues. Mast cell precursors


179
efforts to minimize nonspecific depletion of cell
populations, will be required to better define the surface
phenotype of precursors to cultured mast cells.
The midgestation murine conceptus is the site of a
number of interesting and perhaps time-related phenomena
which may be associated with the presence of culture-
derived mast cell precursors. The pluripotent
hematopoietic stem cell, which gives rise to all of the
hematopoietic lineages and is quantitated in the in vivo
CFU-S assay (Till and McCulloch, 1961), has been isolated
during embryogenisis from the blood islands of the yolk sac
between days eight and ten and from the fetal liver
throughout the remainder of gestation (Moore and Metcalf,
1970). Mast cells have been shown to arise from CFU-S
(Kitamura et al., 1981) as well as multipotent in vitro
colony forming cells (Schrader et al., 1981; Sonoda et al.,
1983; Pharr et al., 1984). Furthermore, CFU-S and their in
vitro correlates are responsive to the proliferative
effects of interleukin 3 (Goldwasser et al., 1983; Garland
and Crompton, 1983; Spivak et al., 1985; Rennick et al.,
1985), the mast cell growth factor prevalent in WEHI-3
conditioned media. The presence of mast cell precursors
in embryonic tissues as early as eight days of gestation
may thus be an indication of multipotent hematopoietic stem
cells or their interleukin 3-responsive progeny which
differentiate into mast cells in the continued presence of


165
Table IV-2. Expression of Surface and Cytochemical
Markers on Colony-Derived Mast Cells3.
Percent cells expressing marker^
Colony
Source
RA3-3A1
B23.1
IgE
Metachromatic
granules
dlO embryo
67
84
56
100
dlO placenta
55
65
48
89
dll embryo
36
83
51
100
dll placenta
47
78
53
96
dl2 placenta
37
89
55
99
bone marrow
43
94
52
94
bone marrow
47
91
54
97
3 cells were cultured in 0.3% agar, 50% W3CM, for three to
four weeks before analysis,
k cells were analyzed for cell surface markers and
toluidine blue metachromasia as indicated in Materials
and Methods.


69
Table II-l.
Designation
(isotype)
14.8
(IgG2b)
RA3-2C2
(IgM)
RA3-3A1
(IgM)
M6
(IgM)
anti-Asialo
(polyclonal)
T24/31.7
(igG)
5H1
(IgM)
B23.1
(IgM)
Lineage-Specific Antibodies Used in Surface
Marker Analysis
Specificity (Literature Citation)
200 Kd Ly5 antigen on lymphoid cells from spleen,
lymph node, and bone marrow (Kincade et al., 1981b).
Ly5 antigen on lymphoid cells from spleen, bone
marrow, lymph node, and plasma cells but not on
thymocytes or CFU-S (Coffman and Weissman, 1981a).
220 Kd Ly5 variant (B220) on B lineage cells on
spleen, lymph node, and bone marrow, but not on
thymocytes (Coffman and Weissman, 1981b).
Probably recognizes Dolichus biflorus agglutinin
receptor on early fetal thymocytes and on some
thymic leukemia cells (Kasai et al., 1983).
GM1 Neutral glycolipid asialo GM1 on early fetal
thymocytes, some fetal liver cells, and few adult
bone marrow, spleen, and lymph node cells as well
as some thymic leukemia cells and natural killer
cells. Not on adult and embryonic, Thy-1 positive
thymocytes (Habu et al., 1980; Kasai et al., 1980;
Young et al., 1980).
Thy 1 glycoprotein on thymocytes and T cells from
spleen but not on bone marrow prothymocytes
(Dennert et al., 1980).
Abelson transforming antigen on bone marrow targets
of A-MuLV, on most thymocytes, some bone marrow and
spleen cells, fetal liver erythrocytes and pre-B
cells, and bone marrow pre-B cells, but not on lymph
node cells, CFU-S or stem cells committed to myeloid
lineages (Shinefeld et al., 1980).
Antigen on resident and elicited macrophages, adherent
cultured bone marrow cells and macrophage-like cell
lines as well as on culture-derived mast cells but not
on resident peritoneal mast cells (Katz et al., 1983;
Leblanc et al., 1982).


syngeneic wild type controls. Since athymic mice do not
exhibit the profound mucosal mastocytosis found in
appropriate controls (Olson and Levy, 1976), the last
observations could be interpreted to indicate that athymic
mice lack the inductive conditions required for the
proliferation of seemingly normal numbers of mast cell
precursors.
Mast cell precursors were enumerated by Nakahata and
colleagues (1982), using a modification of the semisolid
methylcellulose media culture system previously used to
identify erythroid and myeloid precursors. In the latter
and subsequent report using this system (Pharr et al.,
1984), which used pokeweed mitogen activated spleen
conditioned media for a source of interleukin 3, the
investigators found between twenty and 140 mast cell
precursors per million BDF1 mouse spleen cells and 200 mast
cell precursors per million bone marrow cells. Suda and
colleagues (1985), using the Nakahata culture system,
demonstrated that W/Wv mice, which are severly deficient in
mast cells of both serosal and mucosal subsets, had the
same number of peripheral blood mast cell precursors as
wild type mice (approximately thirty per million nucleated
cells), thus indicating that the mast cell defect was in a
homing or developmental step, rather than at the stem cell
or migratory level.
A third method for the enumeration of culture-derived


147
tissues following a modification of the method used to
propagate culture-derived mast cells in liquid suspension
culture. Disaggregated cells were prepared from tissues as
described in Chapter III. Washed, enumerated cell
suspensions from various tissues were resuspended at twice
the concentration desired for the most concentrated
innoculum in 50% W3CM. The cells were then aliquoted into
12 well tissue culture clusters (Costar, Broadway, MA) in
volumes necessary to provide the total number of cells
desired per culture, and the total volume of each well was
brought to 0.5 ml with 50%W3CM. BACTO agar (DIFCO
Laboratories, Detroit, MI) was prepared in sterile water
(Travenol Laboratories Inc., Deerfield, IL) at 3.0% w/v,
autoclaved for twenty minutes and cooled to 42 to 45
degrees in a water bath. The agar was added to prewarmed
(37C) 50% W3CM at one part agar to four parts medium and
mixed thoroughly by pipeting. One-half ml of the 0.6% agar
was added to each well of cell suspension and the contents
of each well were mixed well by pipeting. The cultures
were then allowed to gel at room temperature for ten to
twenty minutes before being transferred to a five percent
carbon dioxide incubator.
Cultures were fed by diluting freshly prepared, 42 to
45C autoclaved agar in prewarmed 50% W3CM at one volume
of agar per nine volumes of media. One ml of the 0.3% agar
was overlayered onto each well and allowed to gel at room


78
with basophilic granules which stained metachromatically
with acidic toluidine blue (H.R. Katz, personal
communication). Since the cells fit the working definition
of mast cells by these criteria, we began our
characterizations by assaying for intracellular histamine.
All but two of the placental cell lines analyzed contained
histamine as detected by a sensitive, isotopic-enzymatic
microassay (Table II-2). The quantity of histamine detected
in the cells (5 to greater than 500 nanograms per million
cells) was similar to that found in cultured mast cells
derived from mouse spleen (450 to 500 nanograms per million
cells; Razin et al., 1981a), bone marrow (80 to 150
nanograms per million cells; Razin et al., 1982a), and
fetal liver (200 to 1400 nanograms per million cells; Nabel
et al., 1981), and mucosal mast cells (160 to 2000
nanograms per million cells; Befus et al., 1982b;
Bienenstock et al., 1982), but one order of magnitude less
than that found in serosal mast cells (15 micrograms per
million ceils; Bienenstock et al., 1982). Histamine was
not detected in lymphoid or myelomonocytic cell controls,
nor was it detected in the mastocytoma P815, which
reportedly has variants which are devoid of mast cell
granules (Mori et al., 1979). Histamine biosynthesis was
confirmed by chromatographic identification (Galli et al.,
1976) of [^H]-histamine in extracts of f^H]-histidine-
labeled cells (data not shown).


107
WEHI-3 Conditioned Medium (W3CM)
W3CM was prepared following the method of Razin and
colleagues (1984a). WEHI-3 myelomonocytic leukemia cells
from log phase cultures were seeded at 1x10^ per ml into an
enriched medium (EM) consisting of RPMI 1640 (GIBCO, Grand
Island, NY) supplemented with 10% v/v native fetal bovine
serum (Sterile Systems, Logan, UT), 2mM L-glutamine, 0.1 mM
nonessential amino acids, 100 Units per ml penicillin, 100
micrograms per ml streptomycin (GIBCO), and 0.05 mM 2-
mercaptoethanol (Fisher Scientific Co., Medford, MA).
Cultures were maintained in one of two ways: 200 ml
cultures in 17x150 mm dishes (Falcon Plastics, Oxnard, CA)
were incubated at 37C in a humid, five percent carbon
dioxide incubator (Forma Scientific, Marietta, OH);
alternatively, 1000 ml cultures were seeded into pregassed
EM in polycarbonate roller bottles (Corning Glass Works,
Corning, NY) which were placed a horizontal roller
apparatus (New Brunswick Scientific, New Brunswick, NJ) in
a controlled environment room (37C). Following four days
(92-100 hours) of incubation, the conditioned media were
harvested by centrifugation (15 minutes at 2000xg, 4C) and
filtered through tissue culture grade 0.2 micron
nitrocellulose membranes (Nalgene, Rochester, NY). Media
harvested from small cultures were pooled into 1000 ml lots
prior to filtration. W3CM was aliquoted in smaller volumes
(100, 250, 500 ml) in sterile bottles and stored for up to


and my mother, Frances Heifer Siegel, who showed me the
value of patience and determination. My brother, Victor,
is also acknowledged for encouraging me to read and learn.
I owe much of my recent success to the support of my
fellow graduate students. In particular, I express my
thanks to Randy Horwitz, who has helped me stay young,
sharpened my cynical wit, shared my most profane moments,
and provided me with friendship which has endured almost
six years in Gainesville.
Finally, I wish to recognize the faculty and staff
(past and present) of the Department of Immunology and
Medical Microbiology who have made my experience more
fulfilling. In particular, I wish to thank Ken Berns, who
encouraged me to return to graduate studies after a seven
year hiatus. I am also indebted to Catherine and Richard
Crandall, George Gifford, and Michael Boyle, for a
seemingly endless supply of reagents and advice. Last, but
not least, I thank Muriel Reddish, Patrice Boyd, Ellen
Boukari and their superb staffs, without whom the work
would probably have taken six additional years.
iii


Table IV-4. Sorting of Bone Marrow Cells by Surface Determinants
Number of Colonies per 10^ Cells Analyzed0
Experiment3
Rosetting
Agents^
Unfractionated
Control1^
Marker
Positivee
Marker
Negative^
Reconstructed
Population
1A
B23.1-Biotin/
Avidin-SRBC
212+/
-13(3)
185+/-23(3)
ND§
178+/
-2(2)
IB
RA3-3Al-Biotin/
Avidin-SRBC
212
10(3)
ND
190+/
-22(3)
185
5(2)
1C
IgE/TNP-SRBC
240
14(3)
ND
205
5(2)
148
8(2)
2A
IgE/TNP-SRBC
232
26(3)
2 2(3)
230
22(3)
175h
2B
B23.1-Biotin/
Avidin-SRBC
220
20(2)
222 20(2)
ND
215
15(2)
2C
RA3-3A1-Biotin/
Avidin-SRBC
ND
0(1)
215
55(2)
232
8(2)
a: Experiments 1A, IB, 1C were performed with 3 to 6 x 10^ cells prior to sorting and
with ammonium chloride-mediated SRBC lysis. Experiments 2A, 2B, 2C were performed with
0.7 to 2.8 x 10? cells prior to sorting and with hypotonic shock-mediated SRBC lysis,
b: Sorting was performed with the antibody/SRBC combination as indicated in Materials and
Methods.
c: Numbers represent mean number of colonies (standardized per 10^ cells per well)
+/- 1 standard deviation (number of wells analyzed),
d: Unfractionate controls were kept on ice without rosetting agents for the duration of the
sorting experiment and plated in agar at the same time as the sorted cells,
e: The "marker positive" fraction consisted of sorted cells from the separation medium
pellet which were subsequently depleted of sheep red blood cells,
f: The "marker negative" fraction consisted of sorted cells from the separation medium
supernatant which were subsequently depleted of sheep red blood cells,
g: ND: experiment not done due to insufficient cells in the indicated fractions,
h: Only one well plated due to insufficient cells.
169


151
number ratio was used in the depletion experiments.
Following a thirty minute incubation at 4C, the cells were
washed three times by centrifugation in cold BSS and
resuspended in avidin-modified SRBC at a ratio of 100 SRBC
per bone marrow cell. The cells were pelleted for one
minute at 200xg (4C) and then incubated on ice for twenty
minutes. The cells were then centrifuged for an additional
five minutes, the supernatant was removed and the pellet
resuspended by flicking. The cells were overlayered on 3
ml of isotonic Ficoll-Hypaque (Lympholyte M, Accurate
Chemical and Scientific Corp., Hicksville, NY) and
centrifuged for twenty minutes at 800xg (room temperature).
The separation medium and its contained cells were
carefully transferred to a second tube, the pelleted
rosettes and free SRBC were flicked, and both were washed
twice in cold PBS prior to lysis with 0.17M ammonium
chloride, 0.01M HEPES, pH 7.35 (Hall, 1981).
Alternatively, the density-separated populations were
depleted of red blood cells by hypotonic shock (Mishell and
Shiigi, 1980) after two washes in room temperature BSS.
The red blood cell-depleted, sorted bone marrow cells were
washed two times with cold PBS or room temperature BSS (as
appropriate) and resuspended in EM for counting. Counted
cells were resuspended in 50% W3CM for plating in 0.3
percent agar as previously described.


37
Yung et al., 1981; Yung and Moore, 1982). Mast cell-
promoting activities have also been demonstrated in
conditioned media from concanavalin A-stimulated T cell
hybridoma cells (Clark-Lewis and Schrader, 1981), cloned T
cell lines (Nabel et al., 1981), lectin-stimulated T
leukemias (Yung et al., 1981; Yung and Moore, 1982; Metcalf
and Kelso, 1985), and B lymphoma cells (Clark-Lewis et al.,
1982) .
The biologically active factor in lymphocyte and WEHI-
3 conditioned media has been given a variety of names. It
was first termed "multi-CSF" by Burgess and colleagues
(1980), due to its ability to support the differentiation
of multiple hematopoietic lineages fn vitro. Schrader and
colleagues (Schrader and Nossal, 1980; Schrader, 1981;
Schrader et al., 1981; Clark-Lewis and Schrader, 1981)
reported that "P cell stimulating factor" (PSF), which
supported the growth of persisting, mast-like cells in
vitro, was present in concanavalin A spleen conditioned
media. Ihle and colleagues (1981, 1982) proposed the name
"interleukin 3", or IL 3, for the factor which induced the
enzyme 20-alpha-hvdroxysteroid dehydrogenase in nude mouse
spleen cells as well as effecting a number of
differentiative and supportive activities in multiple cell
lineages. Although Ihle's terminology has achieved the
greatest usage in recent literature, alternative
nomenclature for the same activity has been proposed by