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In vitro production of rabbit macrophage tumor cytotoxin

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Title:
In vitro production of rabbit macrophage tumor cytotoxin its role in macrophage mediated tumor cell killing
Creator:
Fisch, Harvey, 1946-
Publication Date:
Language:
English
Physical Description:
xi, 112 leaves : ill. ; 29 cm.

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Subjects / Keywords:
Actinomycin ( jstor )
Cattle ( jstor )
Cells ( jstor )
Cultured cells ( jstor )
Cytotoxicity ( jstor )
Cytotoxins ( jstor )
Irrigation ( jstor )
Macrophages ( jstor )
Rabbits ( jstor )
Tumors ( jstor )
Cytotoxins ( mesh )
Macrophages ( mesh )
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bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida.
Bibliography:
Includes bibliographical references (leaves 103-111).
General Note:
Photocopy of typescript.
General Note:
Vita.
Statement of Responsibility:
by Harvey Fisch.

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University of Florida
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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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08039004 ( OCLC )
ocm08039004
00342537 ( ALEPH )

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Full Text














IN VITRO PRODUCTION OF RABBIT MACROPHAGE TUMOR CYTOTOXIN:
ITS ROLE IN MACROPHAGE MEDIATED TUMOR CELL KILLING













BY

HARVEY FISCH


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



UNIVERSITY OF FLORIDA


1981




IN VITRO PRODUCTION OF RABBIT MACROPHAGE TUMOR CYTOTOXIN
ITS ROLE IN MACROPHAGE MEDIATED TUMOR CELL KILLING
BY
HARVEY FISCH
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1981


To my parents, Irving and Martha Fisch, for their years of
love and concern and for setting the proper example.


ACKNOWLEDGEMENTS
First, I wish to thank my advisor, Dr. George E. Gifford, for
his guidance and patience throughout these studies, and for instilling
within me the elements of the scientific method. I also thank the
members of my committee for their helpful comments and suggestions.
The good nod to my fellow graduate students for their helpful
comments and encouragement as well. As a former colleague of mine once
said, "You guys are the only ones who know what it's really like."
Last, but not least, thanks to Mike Duke and Joe Brown for
technical assistance and light hearted humor.
ii i


TABLE OF CONTENTS
PAGE
ACKNOWLEDGEMENTS iii
LIST OF TABLES vi
LIST OF FIGURES vi i
ABBREVIATIONS USED ix
ABSTRACT x
INTRODUCTION 1
Immune System Components: In Vitro Cytotoxicity for
Tumors 1
The Mononuclear Phagocyte System: A Macrophage
Network 2
Significance of the Mononuclear Phagocyte Systan in
Tumor Regression 5
The Process of Macrophage Activation 6
The Elicitation of Serum Tumor Necrotic and Cytotoxic
Activities 9
Tumor Necrosis and Tumor Regression 13
Activated Macrophages Produce Soluble Cytotoxic
Activity in Vitro 15
Purpose of These Studies 15
MATERIALS AND METHODS 17
Animals 17
Cell Lines 17
Culture Systems 17
Effector Cel 1 s (Macrophages) 18
Production of Tumor Necrosis Serum 21
Production of Rabbit Macrophage Cytotoxins 21
iv


Cytotoxicity Assays 22
Column Chromatography 28
Effect of Trypsin Inhibitors, Phenylrnethyl Sulfonyl
Fluoride, Chloroquine and o-Phenanthrolene on
Rabbit Macrophage Cytotoxin Activities 28
Effects of Prostaglandin E2 on Rabbit Macrophage
Cytotoxin Production 29
Effects of Actinomycin D on Rabbit Pulmonary Lavage
Cell Cytotoxin Production 29
Effect of Tunicamycin on Molecular Weight of Rabbit
Pulmonary Lavage Cell Cytotoxin 29
Freeze-Thawing of Activated Rabbit Pulmonary Lavage
Cells 29
Susceptibility of Tumor Necrosis Serum Cytotoxin
Resistant L-929 Cells to Rabbit Macrophage
Cytotoxins and Pulmonary Lavage Cells 30
Time Lapse Cinematography 30
RESULTS 32
Percent Macrophages in Rabbit Effector Cell Populations 32
Increasing the Macrophage Content of Bone Marrow
Cell Cultures 32
In Vitro Cytotoxin Production by Rabbit Macrophages ... 35
Physicochemical and Biological Properties of Rabbit
Macrophage Cytotoxins and Tumor Necrosis Serum
Cytotoxin 63
Cocultivation of Mouse Peritoneal Exudate Cells or
Rabbit Pulmonary Lavage Cells with Actinomycin D
Pretreated L-929 Targets 75
DISCUSSION 93
BIBLIOGRAPHY 103
BIOGRAPHICAL SKETCH 112
v


LIST OF TABLES
TABLE PAGE
I.RABBIT CELL POPULATIONS UTILIZED AS MACROPHAGE
SOURCES 33
II.PERCENT RABBIT BONE MARROW CELLS WITH MACROPHAGE
PROPERTIES BEFORE AND AFTER TPA TREATMENT .... 34
III. ITEMS TESTED WITH LIMULUS AMEBOCYTE LYSATE REAGENT. 33
IV.RABBIT BONE MARROW CYTOTOXIN PRODUCTION: OPTIMUM
PLATING DENSITY 43
V. EFFECT OF TPA ON TUMOR NECROSIS SERUM (TNS) TITER . 45
VI.CYTOTOXIN PRODUCTION BY RABBIT MACROPHAGES FROM
BLOOD MONONUCLEAR, TPA-PRETREATED BONE
MARROW AND PULMONARY LAVAGE CELLS:
COMPARISON OF TITERS PRODUCED PER
1 x 106 CELLS 46
VII.DATA FROM TABLE VI CONSIDERING PERCENT MACROPHAGE
CONTENT OF EACH CELL TYPE 48
VIII.EFFECTS OF CHLOROQUINE AND o-PHENANTHROLENE ON
CYTOTOXIN ACTIVITY 72
IX.CELL SUSCEPTIBILITY PROFILES OF RABBIT CYTOTOXINS . 74
X.SUMMARY: COMPARISON OF PHYSICOCHEMICAL AND
BIOLOGICAL PROPERTIES OF RABBIT TUMOR NECROSIS
SERUM CYTOTOXIN (TNSCT) WITH RABBIT
MACROPHAGE CYTOTOXINS 76
XI.51Cr RELEASE BY L-929 CELLS COCULTURED WITH
MOUSE PERITONEAL EXUDATE CELLS 77
XII.EFFECTS OF TNSCT, PLCCT, BMCCT, AND PLC ON TNSCT -
RESISTANT AND CONVENTIONAL L-929 CELLS 92
vi


LIST OF FIGURES
FIGURE PAGE
1. Comparison of automated and manual photocell
in the photometric cytotoxicity assay. ... 25
2. Rabbit macrophage cytotoxin production with
and without added LPS 37
3. Rabbit macrophage cytotoxin production in the
absence and presence of varying levels of
exogenous LPS 41
4. Effects of bovine and fetal bovine serum on
pulmonary lavage cell cytotoxin
production 50
5. Comparison of effects of 1% and 10% fetal bovine
serum on cytotoxin production by rabbit
pulmonary lavage cell cultures containing
various levels of LPS 52
6. Time kinetics of rabbit pulmonary lavage cell
cytotoxin production 54
7. Time kinetics of cytotoxin production by
TPA-pretreated and washed rabbit bone
marrow cells 57
8. Effects of actinomycin D on cytotoxin
production by rabbit pulmonary lavage
cells 60
9. Effects of prostaglandin E2 and prostaglandin
antiserum on cytotoxin production by rabbit
pulmonary lavage cells 62
10. Gel filtration of rabbit macrophage cytotoxins
on Sephacryl S-200 65
11. Ion exchange chromatography of rabbit macrophage
cytotoxins on DEAE-Sephadex 68
12. Thermal stability of rabbit TNS cytotoxin
and macrophage cytotoxins 71
13. C3H/HeN mouse peritoneal exudate cells on
actinomycin D pretreated L-929 cells:
ninety-six well tray
vii
79


14.
Effector cell cytotoxicity for actinomycin D
pretreated L-929 targets: Determination
of S5Q endpoint 82
C3H/HeN mouse peritoneal exudate cells on
actinomycin D pretreated L-929 cells:
Twenty-four well tray 84
16. Rabbit pulmonary lavage cells on actinomycin D
pretreated L-929 cells: Twenty-four
well tray 86
17. Linearity between number of plaques counted
vs. number of mouse peritoneal exudate
cel Is plated 88
vi i i


ABBREVIATIONS USED
BCG
BLM
BLMCT
BMC
BMCCT
BPTI
CT
FMEM
HEPES
LPS
MAF
MEF
MEM
MPS
PEC
PGE
PLC
PLCCT
PMSF
RES
SBTI
TNF
TNS
TNSCT
TPA
Mycobacterium bovis, strain BCG
blood mononuclear cells
blood monocyte cytotoxin
bone marrow cel 1s
bone marrow cell cytotoxin
bovine pancreatic trypsin inhibitor
cytotoxin(s)
fortified Eagle's minimum essential medium
N-2-hydroxyethy1 piperazine-N1-2-ethane
sulfonic acid
1ipopolysaccharide
macrophage activating factor
mouse embryo fibroblasts
Eagle's minimum essential medium
mononuclear phagocyte system
peritoneal exudate cells
prostaglandin E
pulmonary lavage cells
pulmonary lavage cell cytotoxin
phenylmethyl sul fonyl fl uoride
reticuloendothelial system
soybean trypsin inhibitor
tumor necrosis factor
tumor necrosis serum
tumor necrosis serum cytotoxin
12-o-tetradecanoylphorbol 13-acetate
IX


Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
IN VITRO PRODUCTION OF RABBIT MACROPHAGE TUMOR CYTOTOXIN:
ITS ROLE IN MACROPHAGE MEDIATED TUMOR CELL KILLING
By
Harvey Fisch
December 1981
Chairman: George E. Gifford
Major Department: Immunology and Medical Microbiology
Rabbit macrophages derived from pulmonary lavage cells (PLC),
phorbol ester pretreated and washed bone marrow cells (BMC), or
blood mononuclear cells (BLM), cultured in the presence of LPS,
secreted tumor cell cytotoxins (CT) that were similar to each other
and tumor necrosis serum cytotoxin (TNSCT).
All CT's had molecular weights of approximately 48,000 D by
gel filtration and eluted from DEAE-Sephadex between 0.28 and 0.32 M
salt. All were stable to 56C for 60 minutes, but labile to 70C
for 20 minutes. Actinomycin D (AcD) enhanced sensitivity of L-929
cells to CT. BI6C3 melanana cells and mouse embryo fibroblasts
were resistant to CT.
By 3 hours in culture, all effector cells secreted detectable
levels of CT. Titers increased for PLC and BLM between 3 and 5
hours. After that time, BLM titers ranained constant through 24
hours. PLC titers rose again from 18 to 30 hours. BMC cultures
x


demonstrated increasing titers from 3 through 14 hours. After that,
no increase in titer was demonstrated. No additional CT production
could be demonstrated after 30 hours in BMC or PLC cultures, despite
a change to fresh medium with LPS.
On an adjusted basis of 1 x 10^ macrophages/ml PLC produced
4 to 25 times more CT than BMC or BLM after 28 hours culture.
CT production by LPS-activated PLC could occur in serum-free
medium. Fetal bovine serum (10%) enhanced production up to 4-fold.
Bovine serum (>4%), or prostaglandin E2 (10~M) inhibited CT
production. AcD (1 pg/ml) added with LPS inhibited CT production
(>95%) by PLC. Delaying addition of AcD after LPS demonstrated
messenger RNA production for CT was completed by 2 to 6 hours.
AcD pretreatment of L-929 cells (2 yg/ml 2 to 3 hours)
followed by washing enhanced their sensitivity to PLC killing. From
this, a plaque assay was generated demonstrating a linear
relationship between number of PLC plated and plaques formed.
Therefore, 1 PLC was capable of forming a plaque. Time lapse
cinematography demonstrated that contact between PLC and targets was
unnecessary for target cell killing.
BI6C3 cells and L-929 cells selected for resistance to TNSCT
were resistant to PLC mediated cytotoxicity.
The conclusion drawn is that secreted CT may play a major role
in macrophage mediated tumor cytotoxicity.
XI


INTRODUCTION
The problems encountered in biological disciplines are often
frustrating and at times seemingly insurmountable. This is the case in
the broad field of tumor immunology, an area filled with complexity and
apparent contradiction.
Immune System Components: In Vitro Cytotoxicity for Tumors
The interactions of the various humoral and cellular components of
the immune systan with tumors as well as with each other in vivo and in
vitro have been well documented. This has been described as a "network"
(Jerne, 1974, 1977) as well as a surveillance system seeking out tumors
as they arise within the host (Thomas, 1959; Burnet, 1967, 1970). Some
investigators seriously question the validity of these sweeping concepts
in the face of spontaneously arising tumors (Hewitt et al., 1976).
Evidence also exists illustrating that some tumors grow as a result of an
immune response (Prehn, 1976).
Despite these difficulties, there has been much effort put forth to
investigate and understand just what the components of the immune system
can do when confronted with tumors both in the test tube and in the tumor
bearing host. More specifically, sensitized cytotoxic T lymphocytes
(Brunner and Cerottini, 1971), natural killer lymphocytes (Herberman et
al., 1975), and armed and activated macrophages (Alexander, 1974)
represent cell types that can directly kill tumor cells subsequent to
contact with them. Some of the reported humoral activities cytotoxic for
1


2
tumor cells include specific antibody and complement (Hellstrom et al.,
1968), lymphotoxin, a product of lymphocytes (Granger and Kolb, 1968),
tumor necrosis factor, and cytotoxic factor (Carswell et al. 1975),
presumed macrophage products (Matthews, 1978; Mannel et al. 1979, 1980a,
1980b). Examples of humoral and cellular elements working together to
destroy tumor cells include specific antibody with nonimmune lymphocytes
(Pollack et al., 1972) and specific antibody with macrophages (Whitcomb,
1979).
The Mononuclear Phagocyte System: A Macrophage Network
The mononuclear phagocytes or macrophages represent a large,
morphologically heterogeneous group of cells found widely distributed
throughout the body (Vernon-Roberts, 1972). Metchnikoff (1905)
recognized the presence of mononuclear cells in the blood, lymph, as well
as throughout the various organs, that could engulf small particles such
as bacteria by projecting ameboid processes. He coined the term
"phagocytosis" (from the Greek word, "phagein," to eat) and designated
the large free and fixed phagocytic cells as "macrophages." He also
demonstrated the significance of these phagocytic cells concerning host
defense by reconfirming their presence in inflamed tissue as well as
noting their phagocytic capacity for bacteria.
Utilizing vital dyes which are preferentially taken up by
macrophages and to a lesser extent by other types of cells, Aschoff
(1924) demonstrated the distribution throughout the body of a functional
system of cells which included macrophages. He referred to this overall


3
network of cells as the "reticuloendothelial system (RES)." It was
believed that these cells were responsible for the formation of the
extrasinusoidal solid pulp of the lymph nodes and spleen (reticulum). In
addition, they were found lining the sinusoids of lymph nodes and other
organs (endothelium). Additional investigations extendea the functional
RES to include the microglia of the central nervous system (del Rio
Hortega and de Asua, 1924) and blood monocytes (Marshall, 1956).
Later studies reviewed by van Furth et al. (1975) revealed that the
original concept of the RES as a unified system is inappropriate in that
it really consists of at least two distinct systems. One of these, the
"RES proper" is composed of cells that are nonphagocytic and make up the
framework of connective tissue. Distinct from these cells are the
macrophages, which often exist either fixed or free, in close proximity
to connective tissue. As a result, the tern "mononuclear phagocyte
system" (MPS) has been proposed to denote a functional network made up
exclusively of macrophages (van Furth et al., 1975).
As previously mentioned, macrophages display considerable
morphological heterogeneity (Vernon-Roberts, 1972). This no doubt
created considerable confusion among early investigators regarding what
was and was not to be considered a macrophage. Perhaps the most
enlightening studies concerning this problem, as well as revealing the
source of macrophages, have dealt with their ontogeny. Macrophages
originate from a common source in the bone marrow, enter the circulation
and finally pass into the tissues, changing their morphological and many
of their functional character!-sties as they proceed (van Furth et al.,


4
1975; van Oud Alblas and van Furth, 1979). Therefore, the varied
descendants of the bone marrow precursor merely represent different
stages or various end cells of a progressive differentiation process.
Aside from their mononuclear appearance, intense phagocytic
capability and common origin, macrophages share an array of other
morphological, chemical and functional characteristics. Included among
these properties are the ability to adhere tightly to glass, motility,
possession of abundant lysosomes containing various enzymes, production
of various immune modulators, complement components, superoxide free
radical and hydrogen peroxide and possession of Fc and complement
receptors (Eisen, 1980). In additional to morphological features,
heterogeneity among macrophages can be extended to these properties as
well since different macrophages vary qualitatively and quantitatively
with respect to these characteristics (van Oud Alblas and van Furth,
1979; Keller, 1980). This overall heterogeneity reflects the existence
of clearly separable subpopulations (Keller, 1980) with either distinct
genetic differences or changes that come about with pluripotent
macrophage maturation or activation.
Many of these properties contribute to the ability of macrophages
to either act alone or in conjunction with other cells or products of the
"immune system" (T and B lymphocytes, antibody and complement) to help
maintain a state of homeostasis within the host. More specifically, this
involves elimination of exogenously introduced toxins, invading
microorganisms, or other foreign antigens, as well as the destruction and
eventual elimination of host cells that are effete or have undergone
certain alterations. This latter case includes the changes seen in cells
associated with neoplasia.


Significance of the Mononuclear Phagocyte
System in Tumor Regression
5
Nearly a century ago, Coley (1891) reported on the palliative (and
occasional curing) effects of using bacterial culture filtrates to treat
patients with neoplastic disease. The rationale for this treatment of
neoplasia was based on the observation (made long before that) that
cancer patients with concurrent infectious disease often displayed
regressing neoplasia or tumors that advanced less rapidly than those of
cancer patients without concurrent infection. More recent reviews
(Pearl, 1929; Jacobsen, 1934; Nauts et al. 1953) have discussed related
phenomena in detail.
Later investigations revealed that the MPS could expand and
increase its activity during the course of a neoplastic disease (Old et
al., 1960) or following the administration of such agents as
Mycobacteriurn bovis, strain BCG (Old et al., 1960; Leake and Myrvik,
1968), zymosan (Old et al., 1960), or Corynebacteriurn parvum (Hal pern et
al., 1974). Similar studies also showed that such agents could protect
challenged animals against transplanted tumors or increase the survival
time of these animals (Old et al., 1959; 1960).
Further evidence that contributed credibility to the macrophage as
a critical component of the j_n vivo effector response to tumors was later
provided. Macrophages could be made nonspecifical ly cytotoxic to tumor
cells by culturing them in the presence of endotoxin (Alexander and
Evans, 1971), or supernatant fluids from antigen sensitized lymphocyte
cultures (Piessens et al., 1975; Leonard et al., 1978). Alternatively,
macrophages could be rendered specifically cytotoxic or "armed" to a


6
particular tumor by cocultivating them with spleen lymphocytes from an
animal previously inoculated with the tumor (Alexander, 1974). These
macrophages could then be rendered nonspecifical ly cytotoxic (activated)
to different tumors by continued cocultivation with the original
immunizing tumors (Alexander, 1974).
More convincing evidence indicating that macrophages play a
significant role in the overall scheme of tumor defense mechanisms was
revealed by their actual presence within tumors (Evans, 1972; Alexander,
1974; Eccles and Alexander, 1974). Furthermore, tumor regression could
be correlated with macrophage infiltration (Russell et al., 1976).
The Process of Macrophage Activation
The term "activated" as it pertains to the macrophage was
introduced by MacKaness (1970) and it is a term which has received wide
usage by many investigators who study the macrophage. Depending upon
one's interests, the activated macrophage possesses many character!'sties
which distinguish it from a macrophage which is not activated. Allison
and Davies (1975) have described biochemical and biological changes in
macrophages undergoing activation. These include increases in the
following: glucose oxidation, lysosomal enzyme levels, synthesis of
membrane components, pinocytosis, phagocytosis, adhesion, spreading and
motility. In addition, activated macrophages, as opposed to resting
macrophages, can inhibit the proliferation of and have demonstrated
cytotoxicity for tumor cells (Allison and Davies, 1975; Hibbs, 1976;
Keller, 1980).
The cytotoxicity that activated macrophages demonstrate for
transformed cells is most interesting. Aside from being nonspecific


7
as seen by the ability of activated macrophages to kill different types
of tumors from allogeneic, syngeneic or xenogeneic sources (Keller,
1980), most nontra ns formed cells are not killed by activated macrophages
(Hibbs, 1974a; Keller, 1980). Therefore, the activated macrophage
appears to be capable of distinguishing some common feature(s) of the
tumor state from that of normal cells. Currently, the exact distinction
between tumor and nontra ns formed cells made by the activated macrophage
is unknown. However, some evidence suggests the difference lies within
the plasma membrane (Hibbs, 1974a).
The process of macrophage activation is not a simple, absolute,
one-step event, but involves a cascade of transient, intermediary
reactions (Hibbs et al., 1980; Meltzer et al., 1980) dependent upon the
presence of signals in the local environment of the macrophage (Hibbs et
al., 1977). Some of these signals are stimulatory while others are
inhibitory to macrophage activation (Hibbs et al., 1977; Chapman and
Hibbs, 1977). Ruco and Meltzer (1978) have extended this model by
describing it as a series of "priming" signals followed by activation
signal(s).
As mentioned previously, some of the signals that can affect the
state of the macrophage and modulate activation have been identified.
They include endotoxin (Alexander and Evans, 1971), macrophage activating
factor, a T lymphocyte product (Leonard et al., 1978) and type I
interferon (Schultz et al., 1977; Jett et al., 1980). As work in this
field progresses, it is becoming more and more evident that many of these
activation signals are related to one another as part of the macrophage
activation cascade, particularly as priming or activation signals.


8
This is the case concerning macrophage activating factor (MAF) and the
active component of endotoxin, 1 ipopolysaccharide (LPS). It has been
established using highly purified materials that MAF alone cannot
activate macrophages, but merely sensitizes or "primes" them to the
activating capabilities of LPS (Weinberg and Hibbs, 1979; Weinberg and
Hibbs, 1980; Hibbs et al., 1980; Weinberg, 1981; Pace and Russell, 1981).
Earlier studies concerning these two signals in this scheme were
complicated by the presence of small amounts of LPS contaminating the
lymphokine preparations as well as other materi al s (Hibbs et al., 1980).
Aside from clarifying the roles of MAF and LPS in the macrophage
activation scheme, many of these studies demonstrate the importance of
determining the presence of contaminating LPS when working with
macrophages (Levin et al., 1970; Fumarola, 1981).
Despite the apparent clarity of this simple model utilizing MAF (a
priming signal) and LPS (a triggering signal), there are many points
regarding macrophage activation and its associated signals that are still
not clear. First, it is apparent that LPS is not the only triggering
signal. This has been demonstrated in several studies which either
exclude LPS from cultures or utilize macrophages derived from mice that
respond very poorly to LPS (Hibbs, 1976; Schultz and Chirigos, 1980;
Boraschi and Tagliabue, 1981). Secondly, type I interferon has been
described as a priming signal distinct from MAF (Boraschi and Tagliabue,
1981) as well as serving as the endogenous macrophage generated mediator
for LPS induced triggering of macrophage activation (Schultz and Chirigos,
1979). Further studies are needed to determine the identity of other


9
triggering signals as well as the precise role of interferon in the
scheme of macrophage activation.
The Elicitation of Serum Tumor Necrotic
and Cytotoxic Activities
Many of the foregoing mechanisms concerning macrophage activation
are now believed to represent accurate in vitro correlates of host
mediated responses to such agents as BCG, zymosan, Corynebacterium parvum
and LPS. Although the early investigators were not aware of the details
behind the host's response to these agents, it was quite apparent that
they had value in inducing favorable clinical responses in tumor bearing
animals. Ribi et al. ( 1975) gave tumor bearing guinea pigs both BCG and
LPS. The results of this double regimen were rather impressive. Test
animals that had received both BCG and LPS displayed a survival rate of
about 90% compared to a survival rate of about 66% for those receiving
BCG alone.
Carswell et al. (1975) found that they could cause a dramatic
necrosis of transplanted subcutaneous tumors in otherwise unmanipulated
mice that were injected with sera from other mice, rabbits, or rats that
had previously been inoculated intravenously with BCG, Corynebacteriurn
species or zymosan followed by an intravenous injection of LPS 2 weeks
later. They called the agent(s) in the sera responsible for this
phenomenon, tumor necrosis factor (TNF). It was also demonstrated by the
same group (Carswell et al., 1975; Helson et al., 1975; Green et al.,
1976) that crude tumor necrosis serum (TNS) or material that was
partially purified from TNS could kill many but not all types of tumor
cells in vivo and in vitro while being nontoxic for mouse embryo


10
fibroblasts in vitro. Others have reported similar findings for TNS
derived from the mouse (Mannel et al., 1980a; Mannel et al., 1980c) and
rabbit (Matthews and Watkins, 1978; Ostrove and Gifford, 1979). In
addition, some of these as well as other studies demonstrate that neither
tumor necrosis or in vitro cytotoxicity are due to LPS (Carswell et al .,
1975; Oettgen et al., 1980). LPS levels in the serum transferred from
donor animals are substantially below the levels required to cause tumor
necrosis. Furthermore, LPS is not toxic for tumor cells in vitro and has
a profile distinctly different from that of TNF regarding other
biological activities (Oettgen et al., 1980).
Biochemical and Physical Properties of Tumor
Necrosis Factor and Cytotoxins in Tumor
Necrosis Serum
Green et al. (1976) partially purified TNF from mouse TNS and
determined it to have a molecular weight of 125,000 to 150,000 D by gel
filtration. Further studies in the mouse (Mannel et al., 1980a; Mannel
et al., 1980c; Oettgen et al., 1980) demonstrated the molecular weight of
the cytotoxic activity (CT) and TNF respectively (by gel filtration) to
be about 60,000 D, after exposure to high salt concentrations (0.2 M or
greater). This may indicate ionic aggregation of polymeric subunits or
aggregation with some other molecular species found in the serum. Other
properties of mouse TNF and tumor necrosis serum cytotoxin (TNSCT)
include stability to 56 C exposure for 30 minutes, an isoelectric point
of pH 4.8, pronase sensitivity, and the presence of sialic acid and
galactosamine residues (Green et al., 1976; Mannel et al., 1980c; Oettgen
et al., 1980).


11
Studies in the rabbit employing gel filtration have determined the
molecular weight of TNSCT to be 55,000 D (Ruff and Gifford, 1980) and
39,000 D (Matthews et al., 1980). The two groups reported approximately
the same molecular weight (67,000-68,000 D) utilizing polyacrylamide gel
electrophoretic techniques. The consistently higher molecular weight as
determined by electrophoresis over that of gel filtration could possibly
be explained by alteration of configuration of the cytotoxic molecule by
denaturing conditions (Ruff and Gifford, 1980) or interaction of the
molecule with the electrophoretic matrix resulting in retardation of
migration (Matthews et al., 1980). The reasons for the difference between
the two groups concerning the molecular weight as determined by gel
filtration are unclear. Additional characteristics of rabbit TNSCT
include relative stability to 70C for 20 minutes (Matthews et al.,
1980), an isoelectric point of pH 5.1, and high sensitivity to pronase
and relative insensitivity to trypsin (Ruff and Gifford, 1980; Matthews
et al., 1980). These and other studies (Ruff and Gifford, 1981) suggest
that the rabbit CT molecule does not contain large amounts of exposed
carbohydrate contains little or no carbohydrate, as based upon inability
to bind cytotoxic activity to various plant lectins.
Are Tumor Necrosis Serum-Perived
Necrotic and Cytotoxic Activities
Mediated by the Same Effectors?
It is tempting to speculate from results of many of the
aforementioned studies that the TNS agents responsible for tumor necrosis
(TNF) and in vitro cytotoxicity are one and the same. However, the
complexity of TNS is underscored by the demonstration that a variety of
biological products such as interferon (Sauter and Gifford, 1966), B cell
differentiating factor (Hoffman et al., 1976), bone marrow colony


12
stimulating factor (Hoffman et al., 1977) and lysosomal enzymes (Sauter
and Gifford, 1966; Green et al., 1976; Old, 1976) are released into the
serum under conditions used for induction of tumor necrosis. It is
therefore possible that many mediators may contribute to tumor necrosis
and cytotoxicity and the rnediator(s) responsible for one of these
phenomena may not be responsible for the other.
Initial studies employing crude TNS (Carswell et al., 1975) implied
that a single entity, TNF, could be responsible for both in vivo tumor
necrosis and in vitro cytotoxicity. As a result, many of the studies that
followed exclusively utilized either the in vivo assay for necrosis
Green et al., 1976) or an in vitro assay for cytotoxicity (Matthews et al.,
1980; Mannel et al., 1980d) to study purified effector material. It was
felt that either assay probably reflected the activity of the same
effector. Along these lines, Oettgen (1980) and Kull and Cuatrecasas
(1981), working in the mouse system, were able to partially copurify both
cytotoxic and necrotic activities from TNS. Additional studies
demonstrated that partially purified CT derived frail rabbit TNS could
elicit tumor necrosis (Ruff and Gifford, 1980). The data of Kull and
Cuatrecasas (1981) are particularly interesting because they demonstrated
material with cytotoxic activities having molecular weights of 50,000,
160,000 and 225,000 D, but only the 160,000 D containing fractions also
displayed necrotic activity. In addition, they found that high molecular
weight species persisted after salt fractionation, and gel filtration of
the high molecular weight form in high salt led to loss of activity without
an increase in activity of low molecular weight species (50,000 0). This


13
is contrary to the hypothesis concerning high salt deaggregation of high
molecular weight forms (Mannel et al., 1980a; 1980c).
The results of the aforementioned studies do not definitively
demonstrate whether or not TNS necrotic and cytotoxic activities can be
attributed to a single effector molecule. Further investigation is
needed to clarify this point.
Tumor Necrosis and Tumor Regression
Early studies utilizing animals with transplanted subcutaneous
tumors demonstrated a "hemorrhagic necrosis" of their tumors after being
administered bacterial culture filtrates of such organisms as Escherichia
coli (Gratia and Linz, 1931). Later, Shear and Turner (1943) purified a
polysaccharide containing lipid material from Serratia marcescens (now
known to be LPS) which alone could cause a rapid and dramatic hemorrhagic
necrosis of tumors. Although the mechanism behind this phenomenon
remains unclear at this time, there is evidence suggesting an LPS induced
vasoconstriction and intravascular coagulation within the tumor (Algire
et al., 1952) as well as a need for lymphoreticular cells that are
sensitive to LPS (Mannel et al., 1979). As already mentioned, a similar
phenomenon can be seen in tumor bearing animals administered TNS
(Carswell et al.,1975) or partially purified TNF (Ruff and Gifford,
1980). It is not yet clear (but is circumstantially possible) that TNF
plays a role in the tumor necrosis induced by LPS alone. As rapid (often
less than a day) and dramatic as this phenomenon is, a ring of viable
tumor cells usually survives at the periphery of the neoplasm to grow and
kill the host (Shear, 1943). This demonstrates that tumor necrosis


14
in itself is usually insufficient to lead to total recovery from
neoplastic disease.
Tumor regression, on the other hand, involves all those events
which contribute and ultimately lead to total recovery of the host from
naturally occurring or experimentally induced tumors. It is possible
that tumor necrosis may contribute to or allow subsequent tumor
regression to take place more readily by destroying those parts of the
tumor (the center) which effector mechanisms (such as activated
macrophages) may have difficulty reaching.
As previously discussed, much evidence has been provided concerning
the significance of the macrophage in overall tumor regression (Evans,
1972; Alexander, 1974; Eccles and Alexander, 1974; Russell et al., 1976).
It is possible that in many cases, the macrophage may be directly
involved in tumor regression that has been attributed to other types of
effector cells (natural killer cells, cytotoxic T lymphocytes). As an
example, studies by Berendt et al., (1978a; 1978b) have indicated that
LPS induced tumor regression requires concomitant T cell immunity and
that only those tumors that are immunogenic enough to evoke T cell
immunity will regress. At first inspection it is difficult to envision a
role for macrophages in such a scheme. However, the macrophage
activation scheme involves a series of complex events dependent upon
various signals such as MAF, a T lymphocyte product (Leonard et al.,
1978; Weinberg and Hibbs, 1979). It is quite possible that macrophages
may be "primed" by MAF produced by immunized T cells and subsequently
activated by the administered LPS, thereby allowing them to be
responsible at least in part for LPS induced tumor regression.


15
Activated Macrophages Produce Soluble Cytotoxic
Activity in Vitro
The involvement of the macrophage in the production of tumor
necrotic and cytotoxic serum activities is suggested by the coincident
hyperplasia of the MPS accompanying the administration of many TNS
eliciting agents (Carswell et al., 1975; Old, 1976). In addition,
evidence demonstrating that macrophages can kill tumor cells in vitro
(Alexander, 1974; Hibbs, 1976) prompted studies that sought to better
understand the relationship of TNF and TNSCT to activated macrophages.
Studies in the mouse have demonstrated that LPS sensitive cells are
needed to induce TNF in serum (Mannel et al., 1980d) and that biochemical
and physical properties of serum-derived TNSCT are similar to those of CT
derived from cultures of activated macrophages and macrophage cell lines
(Mannel et al., 1980a, 1980b, 1980d). In addition, these same studies
demonstrated that a rabbit antiserum directed against serum-derived TNSCT
inhibited all soluble cytotoxicity generated in the macrophage cultures.
Working in the rabbit, Matthews (1978) has demonstrated that blood
monocytes produce soluble tumor CT in vitro. This CT has striking
similarity to rabbit TNSCT in terms of selective toxicity for various
tumor cells and biochemical characteristics.
Purpose of These Studies
The aims of these studies are as follows:
1. To study the production of rabbit macrophage CT j_n vitro
utilizing macrophages from different sources (pulmonary alveolar, bone
marrow, and blood mononuclear cells).


16
2. To compare physicochanical and biological properties of these
CT with one another as well as with those of TNSCT.
3. To determine the significance these CT play in macrophage
mediated tumor cytotoxicity.


MATERIALS AND METHODS
Animals
New Zealand white female rabbits, ranging in age from 3 to 5 months
(mean weights of 3 to 5 kilograms) were employed. Mice (CgH/HeN and
C57B1/6, female) used ranged in age from 6 to 12 weeks. All animals
were obtained from commercial sources and housed and fed conventionally.
Cell Lines
L-929 cells (a C3H mouse cell line) were grown in Eagle's minimum
essential medium (MEM) supplemented with 10% bovine serum and 250 units of
penicillin/ml and 125 yg of streptomycin/ml. BI6C3 melanoma cells (a
C57B1/6 mouse cell line) were grown in fortified MEM supplemented with
10% fetal bovine serum. Fortified MEM contained 2x concentrations of
essential and nonessential amino acids and vitamins. Glutamine and sodium
pyruvate were also added. Primary mouse embryo fioroblasts (MEF) were
prepared by mincing near term embryos followed by trypsinization for 60
minutes (0.1% trypsin, 0.04% ethylene diamine tetraacetic acid), washing
in Gey's balanced salt solution and culturing in glass bottles containing
FMEM with 10% fetal bovine serum.
Culture Systems
Media
These have been described above (see Cell Lines). For use in
cultures where macrophages were employed, N-2-hydroxyethyl piperazine-N'-
2-ethane sulfonic acid (HEPES buffer, Cal biochan, LaJolla, CA) was added
at .03M.
17


18
Sera
Two lots of bovine sera were used: Gibco (Grand Island, NY), lot
number R290222 and Sterile Systens, Inc. (Logan, UT), lot number 400168.
The latter was certified to contain less than 1 ng lipopolysaccharide/rnl .
Unless otherwise stated, this was the lot used. One lot of fetal bovine
serum was used for all experiments in this study (Kansas City Biological,
Lenexa. KS).
Lipopolysaccharide-Free Culture System
Using a Limulus amebocyte lysate reagent kit (Pyrogent, Mallinckrodt
Inc., St. Louis, MO) to test various media components for contaminating
1ipopolysaccharide (LPS), a culture medium was established which contained
less than 0.025 ng LPS/ml (see Table III for the various materials
screened). This "LPS-free" culture system was used for most of the
studies to be discussed.
Effector Cells (Macrophages)
Rabbit Macrophage Sources
Rabbit macrophages were derived from three sources, pulmonary lavage
cells (PLC), bone marrow cells (BMC), and blood mononuclear cells (BLM).
For harvesting PLC and BMC, rabbits were anesthetized with a combination
general anesthetic consisting of ketamine hydrochloride (Bristol
Laboratories, Syracuse, NY) and xylazine hydrochloride (Cutter
Laboratories, Shawnee, KS) followed by intravenous air embolism sacrifice.
After hair removal and cleansing of the skin with 70% ethanol, the trachea
and/or femurs were exposed. For PLC harvest, the trachea was transected
and the lungs flushed out with six aliquots of 50 ml cold sterile pyrogen-
free saline using a sterile disposable syringe. Cells were immediately
removed from the cold saline by centrifugation and resuspended


19
in cold MEM supplemented with 10% fetal bovine serum and .03M HEPES
buffer. Cells were generally washed twice by centrifugation in medium.
About 20 to 40 x 10 PLC were obtained per rabbit. For BMC, femurs were
removed and trimmed free of as much soft tissue as possible. After
the bones had been rinsed well with sterile, pyrogen-free saline, the ends
were removed with sterilized bone cutters and the contents of the marrow
cavity flushed out with FMEM supplemented with 10% fetal bovine serum and
.03M HEPES buffer, using a 16 gauge needle attached to a 20 ml sterile
disposable syringe. Up to 2 x 10^ bone marrow cells were obtained per
rabbit.
For harvesting BLM, rabbits were anesthetized and bled from the
marginal ear vein into sterile tubes containing pyrogen-free heparin.
After collection, the blood was underlayered with half its volume of
Histopaque-1077 (Sigma Chemical Co., St. Louis, M0) and centrifuged at 400
g for 40 minutes at room temperature. The leukocytes at the interface
were collected, washed 3 times with cold pyrogen-free saline and suspended
in MEM, supplemented as for PLC. Generally, 50 to 100 x 10 BLM were
obtained from one rabbit. Where necessary to minimize effector cell
clumping and to insure a majority of single effector cells (see Plaque
Assay), PLC were pushed through a 19, 23 and finally a 25 gauge hypodermic
needle, using a syringe.
Leake and Myrvik (1968) have described a technique to significantly
increase the yield of PLC from rabbit lungs. When this technique was
modified by intravenous injection of 0.25 ml Freund's complete adjuvant
(Gibco, Grand Island, NY) instead of Mycobacterium bovis, strain BCG
(Leake and Myrvik, 1968), PLC yields were significantly increased up to
about 200 x 10 per rabbit, if cells were harvested 2 to 3 weeks after
the injection. These rabbits will be referred to as "primed" throughout


20
these studies. Those rabbits which were previously unmanipulated prior to
sacrifice will be referred to as "normal."
Mouse Macrophage Source
Mouse macrophages were derived from peritoneal exudate cells (PEC).
CgH/HeN and C57B1/6 mice were injected intraperitoneal ly with 2.5 to
3.0 ml of fluid thioglycollate medium (Bacto, Difco, Detroit, MI). Three
days later, the mice were sacrificed by cervical dislocation and peri
toneal exudate cells (PEC) harvested by washing out the peritoneal
cavities with 8 to 10 ml of cold medium. The cells were washed twice by
centrifugation and resuspended in fresh medium. As with rabbit PLC, where
necessary to minimize cell clumping and insure a majority of single cells,
PEC were pushed through 19, 23 and finally 25 gauge hypodermic needles,
using a syringe.
Activation of Macrophages in Culture
LPS (Escherichia col i 055:B5, Westphal, Difco, Detroit, MI) was used
to induce macrophage activation in all cultures. Unless otherwise stated,
culture conditions were as follows: for mouse PEC or rabbit PLC and BLM,
LPS was added at 10 yg/ml in MEM, supplemented with 10% fetal bovine serum
plus .03M HEPES buffer; for rabbit BMC, cells were cultured in FMEM
supplemented with 10% fetal bovine serum plus .03M HEPES buffer. Further
more, concerning BMC, the phorbol ester, 12-o-tetradecanoylphorbol
13-acetate (TPA) was added at 0.1 or 1 yg/ml and the cells generally
cultured overnight, after which time they were washed free of TPA (4 to 6
times) and refed with fresh medium containing LPS (see Increasing the
Macrophage Content of Bone Marrow Cultures, under Results). Unless other
wise stated, all effector cells were cultured at 1 x 10^ cells/rnl.


21
Viability of all effector cell populations was detennined by trypan
blue dye exclusion. All effector cells used had 95% viability or greater
by this criterion and were always cultured at a density based on the
percentage of viable cells.
Identification of Macrophages in Effector
Cell Populations
Identification of macrophages was based on morphological criteria
(Giemsa's blood stain), phagocytic capability of latex beads (Dow Chemical
Co., Indianapolis, IN) and peroxidase content (Leukocyte Peroxidase,
Histozyme Kit No. 390A, Sigma Chemical Co., St. Louis, MO).
Production of Tumor Necrosis Serum
The basic technique has been described by Carswell et al. (1975).
Rabbits were injected intravenously with viable BCG (Tice strain, 3 x
108 organisms). Two to three weeks later, LPS was injected
intravenously and the rabbits bled 2 to 3 hours later. The blood was
allowed to clot and the serum was withdrawn, further clarified by
centrifugation, and aliquoted. Aliquots were frozen at -20C until used.
Once thawed, tumor necrosis serum (TNS) could maintain cytotoxicity titer
(see assays) when kept at 4C for several months.
Production of Rabbit Macrophage Cytotoxins
Rabbit PLC, BLM or TPA-pretreated and washed BMC were cultured in
appropriate medium containing LPS. (Occasionally BMC were cultured in 175
cm8 plastic tissue culture flasks at 2 to 5 x 108 cells/ml to increase
titer yields. Otherwise, PLC and BLM were generally cultured at 1 x
108 cells/ml in 25 cm8 plastic tissue culture flasks or 24 well
plastic tissue culture plates.) After an appropriate time at 37C,
supernatants were harvested and cells removed by centrifugation.


22
Supernatants were titered and used within 48 hours if stored at 4C (no
appreciable loss of titer occurred if stored at this temperature for this
time) or frozen at -80C. Supernatants frozen at -80C maintained titers
for several months. Cytotoxins (CT) generated by rabbit macrophages are
designated as follows: pulmonary lavage cells (PLCCT), TPA-pretreated and
washed bone marrow cells (BMCCT), and blood mononuclear cells (BLMCT).
Cytotoxicity Assays
Photometric Assay
A basic photometric technique has been previously described (Ruff
and Gifford, 1980). L-929 cells were plated in 96-well tissue culture
trays at 50,000 cell s/0.1 ml to establish a dense monolayer. Dilutions of
CT in 0.1 ml were added along with actinomycin D (Calbiochem, LaJolla, CA)
to give a final concentration of 1 ug/ml and the trays incubated at 37C
for 18 hours in a humidified 5% COg atmosphere. After the culture
period, the plates were stained for 10 minutes with crystal violet (0.2%
in 2% ethanol), washed with water and allowed to dry.
Destruction of monolayers was evaluated by placing trays on an X-ray
viewing screen and measuring light transmission through monolayers with a
photocell. The reciprocal of the dilution of CT required to kill 50% of
the targets was taken as the 50% endpoint (S5Q). Two kinds of
photocells were used in these studies. A manually operated photocell unit
was constructed using a photoconductive cell (cadmium sulfide, No. 05HL,
Cl airex Electronics, Mt. Vernon, NY). The cell was cemented to a small
piece of copper tubing that had an outside diameter approximating the
inside diameter of culture wells. The photocell was attached to a
multipurpose meter and read as a variable resistance at a fixed voltage as
a function of light transmitted through the monolayers. For convenience


23
an automated photocell (Titertek Multiskan, Flow Laboratories, Inc.,
Inglewood, CA) was also used for some of the experiments. Figure 1
compares the two photocells reading the same sample wells. They are
fairly similar, although it appears that the automated photocell is
slightly more sensitive and can detect very small numbers of target cells
retained at lower CT dilutions that the manual photocell cannot.
Nevertheless, since an entire experiment was always read with the same
photocell, and the enhanced sensitivity demonstrated by the automated
photocell was not much greater than the manual one (about 15% greater, see
Figure 1), this difference in photocell sensitivity is insignificant.
This photometric technique was slightly modified to measure effector
cell killing of targets. The only difference from the aforementioned
procedure used to titer CT involved adding actincmycin D at 2 ug/ml for 2
to 3 hours, followed by removal of this drug from the medium by decanting,
washing the monolayer with medium and replacing with fresh medium
containing effector cells. It was previously determined that 2 washings
were sufficient to remove virtually all extracellular actincmycin D
(washings at this point could not inhibit H^-uridine incorporation into
L-929 cells).
Plaque Assay
L-929 cells were grown to monolayers in plastic 24 well flat bottom
tissue culture trays, plating 250,000 cells in 1 ml medium. After the
monolayers were pretreated with actincmycin D (2 ug/ml) for 2 to 3 hours,
they were washed twice with medium. Small numbers of well mixed effector
cells (mouse PEC or rabbit PLC) in 2-fold increments were added carefully
to washed and drained monolayers of targets in a final volume of 0.2 ml
medium. Control target monolayers received only 0.2 ml medium. No agar


Figure 1. Comparison of automated and manual photocell in
the photometric cytotoxicity assay.
Each panel (upper and lower) represents titration of a
single sample by each of the photocells. Each point represents
percent target cell survival of a single culture well.
S = nuntier of cells surviving in cultures, S0 = number
of cells plated.


100
80
60
40
20
100
80
60
40
20
Automated Photocell
O Mon u o I Photocel I
25
<3
9
O
o
o
o
2
9
o
o
o
40 160 640 2560 0,240
[CYTOTOXIN DILUTION] '*


26
or methyl cellulose overlays were used. LPS was added at a final
concentration of 10 ug/ml to PEC and PLC cultures. Plates were cultured
undisturbed at 37C for 18 to 20 hours. Supernatant contents were then
decanted and the plates were stained for 10 minutes with crystal violet,
washed with water and allowed to dry. In all cases, at least 95% of the
effector cells plated were single cells and the number plated was based on
number found to be viable (always >95%).
Cr51 Release Assay
L-929 cells were suspended into two 1 ml aliquots of 2 x 10^
cells. One aliquot was treated with actinomycin D for 1 hour. At that
time, 100 pCi sodium^ chromate (^Cr; ICN Chemical, Waltham, MA)
was added to each aliquot and the cells gently mixed by shaking. After 1
hour at 37C the target cells were washed 5 times by repeated centrifuga
tion and resuspension in fresh medium (MEM with 10% fetal bovine serum,
gentamicin and HEPES buffer) to remove unincorporated ^Cr and from
one of the aliquots, actinomycin D. The concentration of the suspension
was adjusted with medium to 3 x 10^ cel 1 s/0.1 ml. BI6C3 cells were
labelled in a similar way only in the absence of actinomycin D.
These labelled targets were used to determine cytotoxicity of mouse
PEC (L-929) or CT generated by rabbit macrophage cultures (L-929,
BI6C3).
Mouse PEC were seeded into 96 well flat bottom plastic tissue
culture plates in MEM with 15% fetal bovine serum, gentamicin, and HEPES
buffer at 3 x 10^ cells in 0.1 ml volumes. Plates were centrifuged at
200 g for 30 seconds to sediment the cells to well bottoms. After 2 hours
at 37C in a humidified air atmosphere containing 5% 0039 the nonad
herent cells were removed by decanting the supernatants and washing


27
the adherent PEC monolayers twice gently with warn medium. The great
majority of these adherent PEC, greater than 90%, had macrophage
morphology as revealed by staining with Gieinsa's blood stain and could
phagocytize latex particles. After the second wash, 0.1 ml MEM with 10%
fetal bovine serum, HEPES buffer and gentamicin was added to each well.
In an additional 0.1 ml of similar medium, ^^Cr-labelled targets were
added. LPS was then added. Plates were centrifuged at 200 g for 30
seconds to sediment target cells upon effector monolayers. Plates were
incubated at 37C, in a humidified air atmosphere containing 5% C0£. At
appropriate times for harvesting the supernatants, 0.05 ml of medium was
added to each well to insure an even distribution of released ^-*-Cr
throughout the cultures. Cr^ labelled targets (L-929 or BI6C3
cells) were added to microtiter wells along with TNSCT, PLCCT, or BMCCT.
Again, at harvest times, 0.05 ml of medium was added to wells to evenly
distribute released Cr^.
For harvest, aliquots of 0.1 ml were ratioved and counted in a gamma
counter (Gamma 300 Radiation Counter, Beckman Instruments, Inc.,
Fullerton, CA). For each time period, the percent specific ^Cr
released was determined by use of the following formula: percent release
= [(E-S -5 (T-S)] x 100, where E = counts released from labelled targets in
test cultures containing effectors and targets, S = spontaneous counts
released from labelled targets cultured alone and T = total releasable
counts from labelled targets, as determined by maximum lysis of targets
with 1% dodecyl sodium sulfate. Appropriate volume correction factors
were employed to calculate counts released in each case.


28
Column Chromatography
Gel Filtration
All gel filtration chromatography was performed using a single,
calibrated coluim of Sephacryl, S-200 (Pharmacia Fine Chemicals,
Piscataway, NJ) of gel bed dimensions 83 x 2.5 cm. Flow rate was 21 ml/hr
using phosphate buffered saline. Samples were concentrated by pressure
dialysis in an Anicon Diaflo ultrafiltration unit (Anicon Corp.,
Lexington, MA) prior to loading 1 ml samples onto the column. Fractions
(2 ml) were collected and assayed for cytotoxicity on L-929 cells.
Ion Exchange Chromatography
Fractions with cytotoxicity for L-929 cells from the Sephacryl S-200
column were pooled and concentrated by pressure dialysis and loaded onto a
column of DEAE-Sephadex (Pharmacia Fine Chemicals, Piscataway, NJ)
previously equilibrated with phosphate buffered saline (pH 7.2, 0.15 M).
The column was washed with 2 column volumes of starting buffer to remove
unbound proteins. A linear salt gradient (0.15 to 0.60 M) was applied to
the column and fractions (4 ml) collected and assayed for cytotoxicity.
Flow rate was 20 ml/hr.
All chromatography was performed at 4C.
Effect of Trypsin Inhibitors, Phenylmethyl Sulfonyl-
fluoride, Chloroquine and o-Phenanthrolene on
Rabbit Macrophage Cytotoxin Activities
Various drugs were tested in the actinomycin D cytotoxicity assay to
determine their effects on CT activity. The following were tested:
soybean trypsin inhibitor (SBTI, Worthington Biochemical Corp., Freehold,
NJ), bovine pancreatic trypsin inhibitor (BPTI, Sigma Chemical Co., St.
Louis, M0), at 100 ug/ml each; phenylmethyl sulfonylfluoride (PMSF) at
10~^M; chloroquine at 10^M, and o-phenanthrolene at 10"^M
(last 3 drugs from Sigma Chemical Co., St. Louis, M0).


29
Effects of Prostaglandin Eg on Rabbit Macrophage
Cytotoxin Production
Prostaglandin E2 (PGE2 Sigma Chanical Co., St. Louis, MO) was
dissolved in ethanol and diluted in serum-free medium immediately before
addition to duplicate PLC cultures in serum-free medium. PGE2 was added
at 10'^M, 6 hours after initiation of the cultures with LPS (10
ug/ml). Controls received no PGE2* PGE2 antiserum (0.1 ml of lyoph-
ilized stock reconstituted with 2.5 ml buffer, Sigma Chemical Co., St.
Louis, MO) was added to another set of cultures at the same time as LPS.
Controls for this received no PGE2 antiserum. Tnese latter experiments
were performed in the conventional MEM containing fetal bovine serum.
Effects of Actinomycin D on Rabbit Pulmonary
Lavage Cell Cytotoxin Production
Actinomycin D was added at 1 yg/ml at the same time and at various
times after addition of LPS to PLC cultures. Cultures were incubated for
30 hours, supernatants harvested and assayed.
Effect of Tunicamycin on Molecular Weight of Rabbit
Pulmonary Lavage Cell Cytotoxin
Tunicamycin (Sigma Chemical Co., St. Louis, MO) was added to test
PLC cultures at 0.5 and 5 ug/ml both 2 hours before and at the same time
as LPS addition. Cultures were incubated for 30 hours at 37C at which
time, supernatants were harvested and titrated for cytotoxic activities.
Cytotoxin was subjected to gel filtration chromatography on Sephacryl
S-200 (previously described).
Freeze-Thawing of Activated Rabbit Pulmonary Lavage Cells
Pulmonary lavage cells were cultured in the presence of LPS (10
ug/ml) and at various times, replicate cultures were terminated. Fran
some, supernatants were harvested and titered as previously described.


30
Other cultures were subjected to five freeze-thaw cycles in a dry
ice-ethanol bath. After freeze-thawing, the culture contents were
centrifuged and both supernatants and pellets titrated for cytotoxicity.
Susceptibility of Tumor Necrosis Serum Cytotoxin
Resistant L-929 Cells to Rabbit Macrophage
Cytotoxins and Pulmonary Lavage Cells
Matthews (1978) has demonstrated that it is possible to select for
TNSCT-resistant L-929 cells by growing L-929 cells in the presence of
small amounts of TNSCT. Using a lot of TNS which had an S^q titer
(See Cytotoxicity Assays, Photometric Assay) in the actinomycin D
cytotoxicity photometric assay of approximately 8000 units, L-929
resistant cells were selected for by growing cells in the presence of a
1:500 dilution of this CT. After about 2 weeks, during which time dead
cells were decanted and viable cells were refed twice a week with fresh
medium containing TNS, resistant cells were trypsinized, washed and plated
into microtiter wells (10,000/well). Conventional L-929 cells were plated
into another set of wells. After allowing cells to adhere overnight in
conventional medium, TNSCT, PLCCT, BMCCT, or PLC (100 or 1000 cells) were
added to test wells (control wells received only medium). In those wells
containing PLC, LPS (10 yg/ml) was added. After 48 hours, plates were
stained and read as previously described.
Time Lapse Cinematography
L-929 cells were plated into 25 cm^ tissue culture flasks and
allowed to adhere and form a monolayer overnight. The cells were
treated with actinomycin D (2 yg/ml) for 2 to 3 hours, then washed
extensively. Rabbit PLC were added in small numbers (10 x 10^ to 50 x
10^) to the washed monolayers in 3 ml medium at which time, LPS was
added (10 yg/ml). Plates were allowed to equilibrate for an hour in a
lucite incubator chamber mounted on an inverted phase contrast microscope


31
connected to a tine lapse motion picture camera. A temperature of 37 to
38C was maintained within the lucite chamber by a heated air curtain
with a thermostat sensing device. An appropriate field was located
(containing as few macrophages as possible). The field of view was
photographed through a green filter. Photography was generally commenced
4 to 6 hours after addition of effector cells and LPS. Exposure rate for
the first 2 to 3 hours of filming was 1 frame/15 seconds. Generally after
that time, exposure rate was increased to 1 frame/8 seconds. Several
films were made at magnifications of 150 and 200 X. Films were viewed at
24 frames/second.


RESULTS
Percent Macrophages in Rabbit Effector Cell Populations
Table I illustrates the percentage of cells that are macrophages
from three sources, in several rabbits, as judged by morphological and
phagocytic criteria. Pulmonary lavage cells represented a population
made up almost exclusively of macrophages (90-95%). Blood mononuclear
cells were 12-20% macrophages while bone marrow cells contained no more
than 0.1-0.5% macrophages.
Increasing the Macrophage Content of Bone
Marrow Cell Cultures
It is obvious from Table I that bone marrow cells provided only
negligible amounts of macrophages. Lotem and Sachs (1979) have demon
strated that the phorbol ester, TPA, can cause both mouse and human
myeloid leukemic cells to differentiate into cells with varying degrees
of several macrophage characteristies (adherence to surfaces, Fc and C3
receptors, phagocytic capability, lysozyme activity, and macrophage
morpho logy).
Treatment of rabbit bone marrow cell cultures with TPA (1 yg/ml )
resulted in adherence of many cells to the culture dish by 30 minutes.
By 5 hours, over 90% of the cells were adherent (Table II). In addition,
about one-third of the adherent cells had macrophage morphology and
phagocytized latex beads. Twenty percent stained positive for
peroxidase. (Later studies indicated that 0.1 yg/ml TPA was adequate to
cause similar differentiation changes in rabbit bone marrow cells.)
32


33
TABLE I
RABBIT CELL POPULATIONS UTILIZED AS MACROPHAGE SOURCES
Source
Perce nt
Macrophages3
Pulmonary Lavage Cells
90 95
Blood Mononuclear Cells
12 20
Bone Marrow Cel Is
<1
aMacrophage morphology (Giemsa's blood stain) and phagocytosis of latex
beads.


34
TABLE II
PERCENT RABBIT BONE MARROW CELLS WITH MACROPHAGE PROPERTIES BEFORE AND
AFTER TPA TREATMENT
Macrophage Property
Before
Percent of Cells
After TPAa
Adherence
<1
ro
cr
Morphology
<1
32c
Phagocytosis (latex beads)
NT*1
35c
Peroxidase positive
NT
20c
a5-28 hours after adding TPA at 1 yg/ml .
^Percent of total bone marrow cells added to culture.
cPercent of total adherent bone marrow cells.
^Not tested.


35
Considering the variable degrees of each property present, this
process can be envisioned as a spectrum of variable differentiation in
the direction of the mature macrophage state.
In Vitro Cytotoxin Production by Rabbit Macrophages
Requirement for Activating Agent
Matthews (1978) has reported on the apparently consistent, spontane
ous production of CT by rabbit BLM cultures in the absence of known added
activating agent (e.g., LPS). These studies did not determine the possible
presence of contaminating LPS in the culture medium. This is often a prob
lem in studying macrophages and can lead to serious misinterpretation of
experimental results (Fumarola, 1981). Figure 2 demonstrates the results
from experiments employing rabbit PLC and BLM cultures, where no attention
was given to the possibility of contaminating LPS. Depending upon the time
cell supernatants were harvested, the cytotoxin titers in the cultures to
which LPS was added (1 pg/ml) were consistently greater than titers gener
ated in cultures to which no LPS was added (3 to 20 times greater for BLM,
10 to 50 times greater for PLC, replicate cultures varied from mean values
by 50%).
Using the Limulus amebocyte lysate test (Levin et al., 1970), it was
determined that the culture system used for the experiments demonstrated in
Figure 2 contained LPS (>0.025 ng/ml ). Table III illustrates the various
materials tested. The recycled glassware used to store prepared media
tested positive for LPS. This was probably contaminated with the LPS-
positive bovine serum lot (Gibco) also shown in Table III. Other than the
glassware and Gibco bovine serum, only the trypsin stock concentrate
(Microbiological Associates) and thioglycollate broth base powder (Difco)
tested positive in the Limulus assay.
If no LPS was added to culture systans testing negative for LPS
(<0.025 ng/ml) by the Limulus assay, rabbit PLC generally produced no CT


Figure 2. Rabbit macrophage cytotoxin production with and
without added LPS.
Cytotoxin production by rabbit macrophage cultures in the
presence (1 pg/ml) or absence of added LPS. Culture
supernatants were harvested for assay at times indicated. Blood
monocytes were cultured at 4 x 106/ml pulmonary lavage cells
at 1 x lO^/rnl.


37
PULMONARY LAVAGE CELLS
LPS ADDED NO LPS ADDED
HOURS


38
TABLE III
ITEMS TESTED WITH LIMULUS AMEBOCYTE LYSATE REAGENT
Test
Item Results3
Bovine calf serum
Gibco; lot No. R290222 +
Sterile Systems, Inc., lot No. 400168 0
Fetal bovine serum
K.C. Biol., lot No. 309447 0
Trypsin
Stock concentrate, Microbiol. Assoc. +
Crystalline, Sigma; bovine, porcine pancreas 0
Eagle's minimum essential medium, base concentrate 0
Glutamine, stock concentrate 0
Gentamicin, stock concentrate 0
HEPES buffer, stock powder 0
Deionized water from ion exchange tap 0
Thioglycollate broth, base powder, Difco +
Reusable autoclaved laboratory glassware^ +
aLimulus lysate test results interpreted as follows: ( + ) = contains at
least 0.025 ng/ml LPS; (0) = contains less than 0.025 ng/ml LPS.
^Sterilized glassware rinsed with pyrogen free water; washings used for
Limulus assay.


39
(Figure 3). However, one out of the ten rabbits sacrificed for these
studies (only four rabbits are shown in Figure 3) had PLC that could
produce detectable levels of CT in LPS-free cultures. Pasturella species
was cultured from lung washings from this rabbit as well as from its PLC
cultures. The presence of a Gram negative infection in the lungs probably
resulted in in vivo activation of PLC. Despite this problem, the great
majority (90%) of the rabbits utilized provided PLC that would not produce
CT unless LPS was added to the culture (Figure 3). As little as 0.1 to 1
ng/ml could induce CT production. As a precaution, PLC ranoved from all
rabbits to be utilized in other experiments (other than to merely generate
CT for physicochemical studies) were tested in LPS-free culture systems for
their ability to produce CT in the absence of added LPS. This insured that
CT was not produced unless LPS was added to the culture systan, allowing
control over PLC activation.
As previously mentioned, many agents such as BCG (Old et al., 1960),
Corynebacteriurn parvum (Hal pern et al., 1974), etc., can cause a general
ized macrophage hyperplasia in vivo. Leake and Myrvik (1968) have demon
strated this locally in the lungs of rabbits that were injected intraven
ously with BCG in mineral oil. They reported up to a 10-fold increase in
the number of PLC obtained from rabbits injected intravenously over 1 week
before sacrifice. They also reported qualitative changes in the PLC (an
increase in cell lysozyme content as well as ultrastructural changes).
Figure 3 shows that PLC from rabbits injected intravenously with complete
Freund's adjuvant 2 to 3 weeks previously, produced no CT in the absence of
added LPS and variable amounts of CT per 1 x 10^ cells cultured in the
presence of different levels of LPS. In addition, the normal rabbit shown
(not administered Freund's adjuvant) produced significantly greater levels
of CT at lower doses of LPS. Other experiments employing single dose
levels of LPS in cultures of PLC demonstrated variable and overlapping


Figure 3. Rabbit macrophage cytotoxin production in the
absence and presence of varying levels of exogenous LPS.
Pulmonary lavage cells frail normal rabbits, or rabbits
primed with Freund's complete adjuvant 2 to 3 weeks previously,
were cultured in the absence or presence of varying levels of
LPS. Supernatants were harvested after 24 hours. Culture
systems contained <0.025 ng/rnl LPS prior to LPS addition.


41
O Normal Rabbit
Primed Rabbit a
A Primed Rabbit b
B Primed Rabbit c
LPS CONC., ng/ml


42
results for CT production by LPS-activated PLC from normal and primed
rabbits (data not shown). From this, it appears that both primed and
normal rabbit PLC required the presence of LPS to produce CT in vitro. In
addition, CT production by each cell type occurred to a similarly variable
extent. Primed rabbits yielded 4 to 10 times more PLC (up to 200 x 10^
cells) than normal rabbits, 80 to 90% of which had macrophage morphology
(data not shown). In addition, LPS added to PLC cultures from primed or
normal rabbits in amounts exceeding 10 yg/ml produced no more CT than at 10
ug/ml LPS (data not shown).
As with PLC, TPA-pretreated and washed BMC would not produce CT in
LPS-free cultures unless LPS was added. In addition, no CT was produced
from bone marrow cells that were not pretreated with TPA although exposed
to LPS (bone marrow contains <1% macrophages, see Table I). Optimum CT
production resulted when BMC were pretreated with TPA (0.1 and 1 yg/ml
yielded similar results) for a minimum of 7 hours, then washed extensively
(4 to 6 times) prior to activation with LPS (data not shown). This demon
strates the prerequisites for production of significant levels of CT by
BMC. These are differentiation of sufficient bone marrow precursor cells
to macrophages and subsequent activation of these macrophages by LPS.
Taking advantage of the strong and efficient adherence of BMC
induced by TPA, BMC were plated at varying densities to determine the most
efficient plating density for CT production (Table IV). Cultures producing
the most CT had cells plated at approximately 0.5 x 10^ and 1.5 x
lO^/cm^. This range of plating densities yielded consistently high CT
titers for PLC as well (data not shown).
It was important that the TPA-pretreated BMC were washed extensively
prior to adding LPS. Initial experiments with BMC were performed by adding
LPS to TPA treated BMC without washing out the TPA. Virtually no CT was


43
TABLE IV
RABBIT BONE MARROW CYTOTOXIN PRODUCTION: OPTIMUM PLATING DENSITY3
Number Cel 1 s
x lO^/cm^
s50 Titerb
SgoTiter5
per 10 x 10^ cel Is
8.80
2290
104
5.92
2500
169
2.96
2500
338
1.48
2089
564
0.54
933
691
aCells were cultured in MEM with 10% fetal bovine serum and 1 yg/ml TPA
for 12 hours. TPA was removed by washing cultures extensively and LPS
(1 ug/ml) was added.
^30 hour titers.


44
produced by these cultures. In order to determine if TPA could have an
inhibitory effect on the CT, TPA was added to a TNSCT assay (Table V). A
linear relationship was found between the logarithm of the amount of TPA
added to the assay and the decrease in the titer of TNSCT. Pretreating the
target cells (L-929) used in the assay with 100 ng TPA for 18 hours and
then washing out the TPA prior to adding TNSCT to the cultures resulted in
less inhibition of TNSCT titer (21% instead of 36% where only 2.4 ng TPA
was left in the assay with TNSCT). These results demonstrate that TPA
exerts an inhibitory effect on CT activity in general.
Comparison of Cytotoxin Production Capabilities
by Rabbit Macrophages Derived from the
Different Sources
Rabbit PLC, BMC and BLM were cultured under conditions that optim
ized CT production for each of the cell types. BMC cultured in a fortified
medium (see Materials and Methods) generated about 4 times more CT than if
cultured in the standard medium used for PLC and BLM. This was done to
determine which of the cell types had the greatest potential for generating
highest CT titers. Based upon 1 x 10^ cells cultured per ml of medium,
PLC were most efficient under the tested conditions (Table VI, 20 to 140
times more CT than BMC or BLM produced). Also as previously seen regarding
sensitivity to LPS, PLC demonstrated much variability (about 7-fold) when
comparing different rabbits with each other. BMC demonstrated much less
variability (about 2-fold).
The overall cell populations used varied greatly in their macrophage
content (Tables I and II). In addition, the BMC demonstrated a broad
range of cells with macrophage characteristics depending upon which prop
erty was being utilized to call a cell a macrophage. It was therefore
necessary to normalize the different cell populations for their macro
phage content to compare the ability of macrophages from these


45
TABLE V
EFFECT OF TPA ON TUMOR
NECROSIS SERUM (TNS)
TITER
Experiment
TPA(ng)
Percent Inhibition
TNSCT Activity
TNS titrated in presence of TPAa
2.4
36
9.75
48
78
70
TNS titrated on TPA-pretreated and
100
21
washed targets'5
aTPA was added to the standard actinanycin D serum cytotoxin photometric
assay (see Materials and Methods).
^L-929 cells were preincubated with TPA for 18 hours, then washed. Serum
cytotoxin photometric assay was then performed.


46
TABLE VI
CYTOTOXIN PRODUCTION BY RABBIT MACROPHAGES FROM BLOOD MONONUCLEAR, TPA-
PRETREATED BONE MARROW AND PULMONARY LAVAGE CELLS: COMPARISON OF
TITERS PRODUCED PER 1 x 106 CELLS
Cel 1 Type3
Rabbi t
S50Titer
per 1 x 106 Cel Is
Pulmonary lavage*3
1
14,000
2
4,562 526
3
2,200 100
TPA-pretreated bone marrowc
4
70 4d
5
77 6d
6
119 0d
Blood monocytes*3
1
79
aAl1 cells derived from normal rabbits and cultured at 1 x 10^/ml in
medium containing 10% fetal bovine serum, LPS at 10 yg/ml and 3 x 10^M
HEPES buffer. Plating densities, 0.5-1.5 x 10/cm .
^Cultured in Eagle's minimum essential medium.
cCultured in Eagle's minimum essential medium fortified with 2 X vita
mins and essential amino acids, nonessential amino acids and sodium
pyruvate.
^Corrected for loss of bone marrow cells that do not adhere following
TPA-pretreatment (<10%).


47
different sources to generate CT. Considering the potential ranges of
percent macrophages in each population as well as the extremes of CT
titer produced by each of the cell types, the PLC were still most
efficient at generating CT (see Table VII) on a basis of 1 x 10^
macrophages/ml culture medium (4 to 250 times greater than BMC and 4 to
40 times greater than BLM).
Effects of Bovine and Fetal Bovine Serum on
Rabbit Macrophage Cytotoxin Production
The cytotoxic capabilities of macrophages can be enhanced or
inhibited by various serum components (Hibbs et al., 1977; Chapman and
Hibbs, 1977). Mechanisms which could help explain this phenomenon may
involve production or function of macrophage CT. PLC were cultured in
the absence or presence of varying levels of bovine or fetal bovine serum
(Figure 4). Under the conditions tested, it can be seen that PLC could
generate CT in serum-free medium (PLC from two different rabbits generated
330 to 400 S5Q CT units under the culture conditions described for
Figure 4, panel a). Bovine serum was inhibitory to CT production at levels
generally greater than 4%. Fetal bovine serum enhanced CT production by
PLC up to 3-fold (at 10%). Figure 5 further illustrates that 10% fetal
bovine serum was more effective than lower levels (1%) at potentiating
LPS-induced PLCCT production. This was the case at 3 levels of LPS
concentration. It can also be concluded that to the extent to which they
were tested, LPS at 10 pg/ml with 10% fetal bovine serum were optimal
together for potentiating the activation of PLC by LPS.
Time Kinetics of Macrophage
Cytotoxin Production
Figure 6 demonstrates in vitro production of CT by LPS-activated
PLC with respect to time. Generally, significant levels of CT appeared


48
TABLE VII
DATA FROM TABLE VI CONSIDERING PERCENT MACROPHAGE
CONTENT OF EACH CELL TYPE
Perce nt
Theoretical Range of
Macrophages
Suiter per
Cell Type
Possible Range
1 x 10 Macrophages
Pulmonary lavage
90 95
14,737 15,556
2,316 2,444
TPA-pretreated bone marrow
20 100a
70 350
119 595
Blood monocytes
12 20
395 658
Considering all macrophage properties of TPA-pretreated and washed
adherent bone marrow cells (e.g. peroxidase staining positive cells
represent 20% of total adherent cell population. See Table I).


Figure 4. Effects of bovine and fetal bovine serum on
pulmonary lavage cell cytotoxin production.
Rabbit pulmonary lavage cells were cultured for 24 hours
in the absence or presence of various levels of bovine or fetal
bovine serum. Experiments employing cells from 2 rabbits are
shown: a. LPS present at 1 pg/ml ; b. LPS present at 10 pg/ml.
Control cultures contained no serum.


50


Figure 5. Comparison of effects of 1% and 10% fetal
bovine serum on cytotoxin production by rabbit pulmonary lavage
cell cultures containing various levels of LPS.
Supernatants were harvested from triplicate cultures that
were incubated for 30 hours.


L P S fJLq/ m I
CYTOTOXIN S
c_n
i
50 TITER x ICf3
OJ £>
i i
c_n
ro
Fetal Bovine Serum:


Figure 6. Time kinetics of rabbit pulmonary lavage cell
cytotoxin production.
Pulmonary lavage cells from a normal rabbit or rabbits
primed with Freund's complete adjuvant 2 to 3 weeks previously
were cultured in the presence of LPS. Supernatants were
harvested at times indicated.


54
PRIMED RABBIT I
o PRIMED RABBIT #2
a NORMAL RABBIT
HOURS


55
by 3 hours and titers increased significantly between 3 and 5 hours.
Levels remained fairly constant at times tested between 5 to 7 and 18
hours and then increased significantly again after about 18 hours in
culture. Variation of replicate cultures was mean 20% for the Freund's
adjuvant primed rabbits and mean 50% for the normal rabbit. Figure 6
also demonstrates the variation among rabbits (normal and primed) in the
CT titers generated by their PLC at any single given time. Other experi
ments involving culturing PLC from several normal and primed rabbits under
identical conditions for the same time resulted in overlapping and simi
larly variable CT titers for both groups (data not shown).
Time kinetics of CT production by TPA-pretreated and washed, LPS-
activated BMC are given in Figure 7. Detectable levels of CT (20 to 26
S50 units) appeared by 3 hours and increased through 14 hours. Levels
measured at 14 and 30 hours were similar.
Time kinetics of CT production by LPS-activated BLM are included in
Figure 2. Detectable levels of CT were present by 3 hours, increased
between 3 and 7 hours and were similar at 7 and 24 hours. These observa
tions are consistent with the data of Matthews (1978). From these observa
tions, it is apparent that time kinetics of CT production by PLC, BMC and
BLM are somewhat different after an initial production time during the
first 3 hours. After 30 hours culture, essentially no CT was produced by
PLC and BMC that were refed with fresh LPS-containing medium (data not
shown).
Effects of Actinomycin D on Cytotoxin
Production by Rabbit Pulmonary Lavage Cells
Cameron (1980) has described an actinomycin D-induced inhibition of
cytotoxicity of human macrophages for tumor cells. The data presented


Figure 7. Time kinetics of cytotoxin production by
TPA-pretreated and washed rabuit bone marrow cells.
Rabbit bone marrow cells were plated into 24-well tissue
culture dishes and treated with TPA (0.1 u9/ml) overnight.
After removing TPA from cultures (washed 5 times), fresh medium
containing LPS (10 yg/ml) was added. Supernatants were
harvested at various times.


120
100
80
60
40
20
57
Rabbi t A
ARabbit B
5 ¡0 15 20 25 30
HOURS


58
in Figure 8 show that actinomycin D (1 ug/nl ) consistently inhibited
PLCCT production if added to cultures at the same time as LPS.
(Inhibition of CT titers of test cultures was greater than 95% below
those of control cultures to which no actinomycin D was added.) Delaying
addition of actinanycin D resulted in a decrease of inhibition of CT
titers. This varied considerably among rabbits, especially when compar
ing primed to normal rabbits. Fran the data of Figure 8, it is apparent
that in no case was there active CT messenger RNA or active CT in PLC
prior to the addition of LPS to the cultures. In addition, active CT
messenger RNA was fully synthesized by 2 to 6 hours after LPS activation
of primed PLC and about 3 hours after LPS-activation of normal PLC.
Effects of Prostaglandin E (PGE) and Prostaglandin
Antiserum on Cytotoxin Production by Rabbit
Pulmonary Lavage Cells
It has been demonstrated that PGE can be produced by macrophages in
culture and can shut off macrophage cytotoxicity for tumor cells
apparently after a period of macrophage activation (Taffet and Russell,
1981). Figure 9 demonstrates that PGE (10 M) added to PLC cultures
6 hours after LPS (lOug/ml) resulted in inhibition of CT production down
to about 30% of control culture levels. In addition, adding PGE anti
serum to cultures at the same time as LPS increased CT production by 50%
over the control levels. These data are consistent with the conclusions
of Taffet and Russell (1981) concerning regulation of macrophage cytotox
icity by endogenously produced PGE.
Disruption of Rabbit Pulmonary Lavage Cells at Various
Times after LPS Activation: Failure to Demonstrate
Significant Active Intracellular Cytotoxin Pool
Unanue and Kiely (1977) have demonstrated that a macrophage
protein, mitogenic for thymocytes, accumulates within the macrophage in


Figure 8. Effects of actinomycin D on cytotoxin
production by rabbit pulmonary lavage cells.
Actinomycin D (lug/ml) was added to cultures of rabbit
pulmonary lavage cells at the same time and various times after
addition of LPS. Culture supernatants were harvested for ass^y
after 30 hours. Panel a represents 2 primed rabbits, panel b, 2
normal rabbits.


200
150
100
50
150
100
50
60
Primed Rabbit I
APnmed Rabbit 2
Rabbit I
A Rabbit 2
JRS
I 2 3
ACTINOMYCIN D
4 5 6
ADDED AFTER LPS


Figure 9. Effects of prostaglandin E2 and prostaglandin
antiserum on cytotoxin production by rabbit pulmonary lavage
cel Is.
Rabbit pulmonary lavage cells were cultured in the
presence of LPS at 10yg/ml. To 1 set of cultures in serum-free
FMEM, prostaglandin E2 was added at 10" M. To another
set of cultures in standard MEM supplemented with 10% fetal
bovine serum, prostaglandin antiserum was added (see Materials
and Methods). Cultures were incubated for 28 hours and
supernatants harvested.


% CONTROL CYTOTOXIN S50 TITER
62


63
the form of an intracellular pool. Only a small portion of this pool may
be secreted by the macrophage depending upon its state of activation. By
repeated freeze-thawing of thioglycollate-stimulated mouse PEC, they
could demonstrate higher levels of mitogenic protein within the cell than
were secreted under certain conditions.
Rabbit PLC were activated by LPS and at various times after LPS
addition (30 minutes, 1, 3, 7 and 24 hours), culture supernatants and
freeze-thawed lysates were analyzed for CT. As previously, supernatant
CT became measurable at 3 hours but at no time, except one (1 hour after
addition of LPS) did freeze-thawed lysate contain measurable CT (data not
shown). At 1 hour after LPS addition to cultures, only about 2 S5Q
units of CT appeared in freeze-thawed lysates while none appeared in the
culture supernatant. Appropriate controls indicated total stability of
PLCCT to repeated freeze-thawing in this culture system. In addition,
assaying the freeze-thawed PLC (none viable by inability to exclude
trypan blue dye) on L-929 cells in the presence of actinomycin D revealed
no cytotoxicity. These studies indicate that no significant levels of
active freeze-thaw-releasable CT were retained by the activated
macrophage.
Physicochemical and Biological Properties of Rabbit
Macrophage Cytotoxins and Tumor Necrosis
Serum Cytotoxin
Gel Filtration Chromatography
All 4 of the cytotoxins were eluted through the same Sephacryl
S-200 column. All eluted as single peaks. The elution volumes for all
corresponded to a molecular weight of about 48,000 0 (Figure 10).


Figure 10. Gel filtration of rabbit macrophage cytotoxins
on Sephacryl S-200.
Macrophage culture supernatants were concentrated by
pressure dialysis prior to gel filtration. The column was
calibrated with standards of known molecular weight,
ribonuclease (not shown), chymotrypsin (C), ovalbumin (0V),
bovine serum albumin (BSA).


CYTOTOXIN S Sn TITERS
65
TNS
OPLC
ABMC
A BLM
Cytotoxic
Activities
ELUTION VOLUME (ml)


66
Ion Exchange Chromatography
The 4 cytotoxins were eluted through freshly poured DEAE-Sephadex
columns with gel beds of approximately similar size. All eluted with the
same linear salt gradient between 0.28 and 0.32 M NaCl (Figure 11).
Effect of Tunicamycin on the Molecular Weight
of Cytotoxin Generated by Rabbit Pulmonary
Lavage Cells
Previous studies failed to demonstrate significant levels of
exposed carbohydrate on rabbit TNSCT (Matthews et al., 1980; Ruff and
Gifford, 1981). These studies drew their conclusions from the inability
to bind TNSCT to a variety of sugars and plant lectins.
Tunicamycin has been shown to inhibit N-glycosylation of
glycoproteins by blocking formation of N-acetylglucosainine-1 ipid
intermediates (Hickman et al., 1977). This has been demonstrated for
several types of glycoprotein including interferon (Fujisawa et al.,
1978) and immunoglobulin (Hickman et al., 1977). Rabbit PLC were
cultured with LPS both in the presence and absence of tunicamycin.
Tunicamycin at 1 yg/ml had no effect on production of CT, but at 5 u9/ml ,
CT titers were decreased by 30% when compared to control cultures which
contained no tunicamycin. As high as 5 yg/ml tunicamycin had no effect
on the molecular weight of CT produced as determined by gel filtration
chromatography on Sephacryl S-200 (data not shown). This demonstrates
that CT has no covalently linked carbohydrate or has a covalently linked
carbohydrate of very small molecular weight or could have a carbohydrate
portion of substantial size that is covalently linked to the rest of the
molecule by a tunicamycin resistant mechanism.


Figure 11. Ion exchange chromatography of rabbit
macrophage cytotoxins on DEAE-Sephadex.
Rabbit TNS or pooled cytotoxin containing fractions from
the Sephacryl S-200 column were concentrated by pressure
dialysis and loaded in starting buffer. Unbound proteins were
washed off the column, followed by elution with a linear salt
gradient from 150 to 600 mM phosphate buffered saline.


CYTOTOXIN S50 TITER
68
TNS
PLC CYTOTOXIC
FRACTION NUMBER
No Cl CONC. (mM)


69
Temperature Stability of Rabbit
Macrophage Cytotoxins
Each of the cytotoxins were subjected to 56C and 70C in separate
experiments (Figure 12). All four of the cytotoxins were stable to 56C
for 60 minutes. In addition, the titers of all were greater than the
controls, which were maintained at 4C during the course of the
experiment. This may suggest a heat labile inhibitor of CT present. At
70C, all cytotoxins demonstrated decay kinetics that were fairly similar
and by 60 minutes, all lost about 80% of activity compared to controls.
Effect of Serine Protease Inhibitors and
o-Phenanthrolene on Cytotoxin Activities
There is evidence in the murine system that serine proteases may
play a role in macrophage mediated tumor cytolysis (Adams, 1980; Adams et
al., 1980). Rabbit PLCCT was not inactivated by phenylmethyl sulfonyl
fluoride (10^M), an inhibitor of serine proteases (data not shown).
In addition, neither bovine pancreatic trypsin inhibitor nor soybean
trypsin inhibitor inactivated PLCCT or BMCCT activity (data now shown).
None of these enzyme inhibitors inactivated TNSCT either, consistent with
the findings previously reported (Ruff and Gifford, 1981). On the other
hand as this latter group has reported for TNSCT, o-phenanthrolene, a
metal chelator had significant inhibitory effects on the macrophage CT
(Table VIII).
Effect of Chloroquine on Rabbit
Macrophage Cytotoxin Activities
Chloroquine is a drug which can apparently stabilize cell lysozomes
(Lie and Schofield, 1973). Previous work with TNSCT (Ruff and Gifford,
1981) has demonstrated an inhibitory effect of this drug on cytotoxin
activity in the actinomycin D assay. All 4 cytotoxins were tested in the
standard actinomycin D assay with chloroquine (10~4M) and all were
inhibited (Table VIII).


Figure 12. Thermal stability of rabbit TNS cytotoxin and macrophage
cytotoxins.
Rabbit macrophage culture supernatants and TNS diluted with culture
medium were exposed to 56C for 1 hour or 70C for 20, 40 or 60 minutes.
Control TNS and culture supernatants were maintained at 4C for 1 hour.


160
^
CYTOTOX I IMS:
TNS
BMC
PLC
BLM


TABLE VIII
72
EFFECTS OF CHLOROQUINE AND o-PHENANTHROLENE ON CYTOTOXIN ACTIVITY
Drug
PLCa
Percent of
BMCb
Control S5QI
BLMC
iter
TNSd
Chloroquine, 10"^M
55
38
37
31
o-Phenanthrolene, 10^M
46
48
35
50
Cytotoxins from:
aPLC, pulmonary lavage cells
bBMC, TPA-pretreated bone marrow cells
CBLM, blood monocytes
dTNS, tumor necrosis serum


73
Effects of High Arginine Levels on Rabbit
Macrophage Cytotoxin Activities
Many reports indicate that arginase plays some role in macrophage
mediated tumor cytotoxicity (Currie and Basham, 1975, 1978; Currie, 1978;
Chen and Broome, 1980). These same reports indicate inhibition of
macrophage merdiated tumor cytotoxicity by adding excess arginine to the
medium. Arginine added to our cytotoxin assay at 2 x 10"^M (5 to 10
times the level employed by these latter investigators) did not inhibit
cytotoxicity of any of the CT (data not shown). Fran this, we conclude
that none of our cytotoxins is arginase.
Relative Titers of Rabbit Macrophage Cytotoxins
in the Presence and Absence of Actinomycin D
in the Cytotoxicity Assay
0strove and Gifford (1979) have reported an enhanced sensitivity of
L-929 cells to TNSCT in the presence of actinomycin D. Similar results
were demonstrated with PLCCT and BMCCT. Target cells demonstrated up to
250 times more sensitivity to CT in the presence of actinomycin D than if
actinomycin D was not employed (data not shown).
Target Cell Susceptibility Profiles of Rabbit
Macrophage Cytotoxins and Tumor Necrosis
Serum Cytotoxin
Table IX compares PLCCT and BMCCT with TNSCT target cell
susceptibility. Both macrophage derived cytotoxins are not toxic for
mouse embryo fibroblasts or BI6C3 melanoma, two types of TNSCT-
resistant cell, while L-929 cells, a TNSCT-sensitive cell, is also
sensitive to macrophage CT. Chromium^ release studies demonstrated
L-92 9 sensitivity to PLCCT and BMCCT, while BI6C3 cells were resistant.
L929 cells released up to 18% of their label by 8 hours (taking into
consideration spontaneous release of label by target cells) while BI6C3
cells released none of their label by 21 hours (data not shown).


74
TABLE IX
CELL SUSCEPTIBILITY PROFILES OF RABBIT CYTOTOXINS3
Target Cel 1
Cytotoxin
Source
S50Titerc
L 92 9
TNSb
>128
PLCb
>128
BMCb
>128
Mouse embryo fibroblasts
TNS
<2
PLC
<2
BMC
<2
B16C3
TNS
<2
PLC
<2
BMC
<2
aPhotometric assay without added actinomycin D was used to titer the
same aliquot of each cytotoxin on the cells indicated.
bTNS, tumor necrosis serum; PLC, pulmonary lavage cells; BMC, bone
marrow cells (TPA pretreated).
cForty-eight hour titers.


75
Table X summarizes biological and physicochemical characteristics
of rabbit macrophage cytotoxins and TNSCT.
Cocultivation of Mouse Peritoneal Exudate Cells or
Rabbit Pulmonary Lavage Cells with Actinomycin D
Pretreated L-929 Targets
Target cells can be rendered increasingly sensitive to TNSCT (a
putative macrophage product) by actinomycin D (Ostrove and Gifford, 1979;
Ruff and Gifford, 1981). In addition, Kunkel and Welsh (1981) have
reported that pretreatment of L-929 cells with actinomycin D followed by
washing rendered them increasingly sensitive to lysis by natural killer
cells. Considering these observations, it was of interest to determine
what effect actinomycin D pretreatment of L-929 cells followed by washing
would have on macrophage mediated killing of L-929 targets. Table XI
illustrates increased sensitivity of L-929 cells to mouse PEC cytotox
icity using a ^Cr release assay. By 6 hours, nearly 40% of the
^Cr was released by actinomycin D pretreated targets cocultured with
LPS activated PEC, while by 12 hours, those targets not pretreated with
actinomycin D released less than 10% of their label in the presence of
effector cells. In this experiment, the effector:target cell ratio was
between 1:1 and 10:1 (see Materials and Methods).
Demonstration of Cytotoxicity of Actinomycin D
Pretreated L-929 Cells with Effector:Target Ratios <1
Actinomycin D pretreatment and washing of monolayers was followed
by addition of small numbers of mouse PEC or rabbit PLC activated by the
addition of LPS. Significant levels of killing could be detected by 18
hours using the photometric assay (see Figure 13). If actinomycin D was
omitted, no killing was detected at 18 hours. It was possible to
determine the number of effector cells required to kill 50% of the


TABLE X
SUMMARY: COMPARISON OF PHYSICOCHEMICAL AND BIOLOGICAL PROPERTIES OF RABBIT TUMOR NECROSIS
SERUM CYTOTOXIN (TNSCT) WITH RABBIT MACROPHAGE CYTOTOXINS
Property
TNSCT
Cytotoxin (CT)
PLCa BMCb
BLMC
Molecular weight, gel filtration
48 K
48 K
48 K
48K
Elution from DEAE Sephadex
0.30-0.32M
0.28-0.3 OM
0.30-0.31M
0.32M
Glycoprotein
No^e
No^ ?
ND9
ND
Temperature stability: 56C, 60 min.
All stable
70C (after 20 minutes)
All unstable
Trypsin inhibitors (soybean and bovine pancreatic)
NIh
NI
NI
ND
Phenylmethyl sul fonylfl uoride
NIe
NI
ND
ND
o-Phenanthrolene
All inhibited
Chloroquine
All inhibited
Arginine, 2 x 10"^M
No Effect
No Effect
No Effect
ND
Cytotoxicity Assay with actinornycin D
All activities enhanced
Target cell cytotoxicity: L-929
Susceptible
to al 1
B16C3
Resistant to all
Mouse embryo fibroblasts
Resistant tc
) all
aPulmonary lavage cells eRuff and Gifford, 1981
bTPA-pretreated bone marrow cells ffunicamycin did not decrease molecular
cBlood monocytes weight of CT produced by PLC
^Matthews et al., 1980 9Not determined
bNot inhibited


77
TABLE XI
5lCr RELEASE BY L-929 CELLS COCULTURED WITH MOUSE PERITONEAL
EXUDATE CELLS
Time (hours)
Percent
Actinomycin D
Pretreatment
^Cr Released3^
No Actinomycin D
Pretreatment
3
1.3 0
0
6
38.3 1.8
1.9 0
12c
46.8 1.3
9.4 0
aPercent specific
^Cr released into supernatants = E S
T S
(100)
where E = experimental release, S = spontaneous release and T = total
releasable counts.
S = amount of ^Cr released by target cells cultured alone.
T = total releasable counts by lysis of 3 x 10^ ^Cr labelled
L-929 cells with 1% dodecyl sodium sulfate.
^Samples run in triplicate for determination of experimental and
spontaneous counts released. Samples run in quadruplicate for total
releasable counts.
cMaximum spontaneous counts released = 22% of total releasable counts
by 12 hours.


Figure 13. C^H/HeN mouse peritoneal exude cells on actinomycin D
pretreated L-929 cells: ninety-six well tray.
L-929 monolayers were pretreated with actincmycin D for 2 to 3 hours
and then washed. Mouse peritoneal exudate cells were plated into the wells
in 2-fold increments (numbers below the well columns indicate number of
peritoneal exudate cells plated in 0.2 ml). LPS was added at 10 pg/ml.
Eighteen hours later the plate was stained. Two sets of peritoneal exudate
cells were run in triplicate.


o-l
T
t
j '
-
0 10 20 40 80 160 320 640 1280 2560
C3H peritoneal exudate cells on actinomycin d
PRETREATED L 929 CELLS
i i
i 11
/i ! i
>
i
i
VO


80
targets. Figure 14 demonstrates this point. Approximately 17 5 rabbit
PLC and 300 mouse PEC could kill 50% of the actinomycin D pretreated
monolayers. If we take into account the fact that 95% of the rabbit PLC
and 70% of the mouse (C3H/HeN) PEC were macrophages, we can then
determine the average number of targets killed by a single macrophage.
Knowing that approximately 50,000 L-929 cells composed a monolayer, it is
determined that a single rabbit macrophage killed an average of 25,000f
(175x0.95) or 150 targets and a single mouse macrophage killed an average
of 25,0004(300x0.70) or 119 targets. These are only average determin
ations, since, as will be shown, not every macrophage necessarily killed
targets, nor were the number of targets killed by a single macrophage the
same.
Controls for these experiments previously determined that the
manual photocell employed for these studies could at no time detect the
small number of effector cells plated. In addition, removal of adherent
cell populations from mouse PEC virtually eliminated their ability to
generate killing. We therefore concluded that the macrophage was the
effector cell in the PEC population.
Demonstration of Single Effector Cell
Cytotoxicity: Plaque Assay
Repeating the same experiment on a larger monolayer surface (24
well plates) resulted in a greater dispersal of the effector cells.
Here, killing was detectable by the formation of plaques (see Figures 15
and 16). If the number of effector cells plated was compared with the
number of plaques that resulted, a linear relationship between the two
was obtained (Figure 17). This dictates that a single effector cell was
capable of forming a single plaque, though not every effector cell


Figure 14. Effector cell cytotoxicity for actincmycin D
pretreated L-929 targets: Determination of Sgg endpoint.
L-929 monolayers were pretreated with actinomycin D
(2ug/ml ) for 2 to 3 hours. After washing monolayers, effector
cells (a, mouse peritoneal exudate cells; b, rabbit pulmonary
lavage cells) were added in 2-fold increments. Control target
monolayers received no effector cells. LPS was added at 10
pg/ml. Target monolayers were stained after 18 hours.


% TARGET CYTOLYSIS
82
NO RABBIT PULMONARY LAVAGE CELLS PLATED


Figure 15. C^H/HeN mouse peritoneal exudate cells on actinomycin D
pretreated L-929 cells: Twenty-four well tray.
Procedure was the same as for peritoneal exudate cells on L-929
targets, 96 well tray (Figure 13). Numbers below the well columns depict
number of peritoneal exudate cells plated in 0.2 ml. Each number of
peritoneal exudate cells run in quadruplicate.


CELLS
40 80 160
ON ACTINOMYCIN D PRETREATED L 929
00


Figure 16. Rabbit pulmonary lavage cells on actincmycin L) pretreated
L-929 cells: Twenty-four well tray.
Procedure as for mouse peritoneal exudate cells (Figures 13 and 15).


* ft
RABBIT PULMONARY LAVAGE CELLS ON ACTINOMYCIN D PRETREATED L 929 CELLS
03
cn


Figure 17. Linearity between number of plaques counted
vs. number of mouse peritoneal exudate cells plated.
Each point represents the mean of 4 replicates 1
standard deviation. Panel a, represents peritoneal exudate
cells from C57B1/6 mouse; panel b, represents peritoneal exudate
cells from C3H/HeN mouse. Botn lines represent the best fit
by linear regression and nave correlation coefficients of
>0.990.


100
80
60
40
20
100
80
60
40
20
88
NUMBER OF PERITONEAL EXUDATE CELLS PLATED


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IN VITRO PRODUCTION OF RABBIT MACROPHAGE TUMOR CYTOTOXIN
ITS ROLE IN MACROPHAGE MEDIATED TUMOR CELL KILLING
BY
HARVEY FISCH
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1981

To my parents, Irving and Martha Fisch, for their years of
love and concern and for setting the proper example.

ACKNOWLEDGEMENTS
First, I wish to thank my advisor, Dr. George E. Gifford, for
his guidance and patience throughout these studies, and for instilling
within me the elements of the scientific method. I also thank the
members of my committee for their helpful comments and suggestions.
The good nod to my fellow graduate students for their helpful
comments and encouragement as well. As a former colleague of mine once
said, "You guys are the only ones who know what it's really like."
Last, but not least, thanks to Mike Duke and Joe Brown for
technical assistance and light hearted humor.
ii i

TABLE OF CONTENTS
PAGE
ACKNOWLEDGEMENTS iii
LIST OF TABLES vi
LIST OF FIGURES vi i
ABBREVIATIONS USED ix
ABSTRACT x
INTRODUCTION 1
Immune System Components: In Vitro Cytotoxicity for
Tumors 1
The Mononuclear Phagocyte System: A Macrophage
Network 2
Significance of the Mononuclear Phagocyte Systan in
Tumor Regression 5
The Process of Macrophage Activation 6
The Elicitation of Serum Tumor Necrotic and Cytotoxic
Activities 9
Tumor Necrosis and Tumor Regression 13
Activated Macrophages Produce Soluble Cytotoxic
Activity in Vitro 15
Purpose of These Studies 15
MATERIALS AND METHODS 17
Animals 17
Cell Lines 17
Culture Systems 17
Effector Cel 1 s (Macrophages) 18
Production of Tumor Necrosis Serum 21
Production of Rabbit Macrophage Cytotoxins 21
iv

Cytotoxicity Assays 22
Column Chromatography 28
Effect of Trypsin Inhibitors, Phenylrnethyl Sulfonyl
Fluoride, Chloroquine and o-Phenanthrolene on
Rabbit Macrophage Cytotoxin Activities 28
Effects of Prostaglandin E2 on Rabbit Macrophage
Cytotoxin Production 29
Effects of Actinomycin D on Rabbit Pulmonary Lavage
Cell Cytotoxin Production 29
Effect of Tunicamycin on Molecular Weight of Rabbit
Pulmonary Lavage Cell Cytotoxin 29
Freeze-Thawing of Activated Rabbit Pulmonary Lavage
Cells 29
Susceptibility of Tumor Necrosis Serum Cytotoxin
Resistant L-929 Cells to Rabbit Macrophage
Cytotoxins and Pulmonary Lavage Cells 30
Time Lapse Cinematography 30
RESULTS 32
Percent Macrophages in Rabbit Effector Cell Populations . 32
Increasing the Macrophage Content of Bone Marrow
Cell Cultures 32
In Vitro Cytotoxin Production by Rabbit Macrophages ... 35
Physicochemical and Biological Properties of Rabbit
Macrophage Cytotoxins and Tumor Necrosis Serum
Cytotoxin 63
Cocultivation of Mouse Peritoneal Exudate Cells or
Rabbit Pulmonary Lavage Cells with Actinomycin D
Pretreated L-929 Targets 75
DISCUSSION 93
BIBLIOGRAPHY 103
BIOGRAPHICAL SKETCH 112
v

LIST OF TABLES
TABLE PAGE
I.RABBIT CELL POPULATIONS UTILIZED AS MACROPHAGE
SOURCES 33
II.PERCENT RABBIT BONE MARROW CELLS WITH MACROPHAGE
PROPERTIES BEFORE AND AFTER TPA TREATMENT .... 34
III. ITEMS TESTED WITH LIMULUS AMEBOCYTE LYSATE REAGENT. . 33
IV.RABBIT BONE MARROW CYTOTOXIN PRODUCTION: OPTIMUM
PLATING DENSITY 43
V. EFFECT OF TPA ON TUMOR NECROSIS SERUM (TNS) TITER . . 45
VI.CYTOTOXIN PRODUCTION BY RABBIT MACROPHAGES FROM
BLOOD MONONUCLEAR, TPA-PRETREATED BONE
MARROW AND PULMONARY LAVAGE CELLS:
COMPARISON OF TITERS PRODUCED PER
1 x 106 CELLS 46
VII.DATA FROM TABLE VI CONSIDERING PERCENT MACROPHAGE
CONTENT OF EACH CELL TYPE 48
VIII.EFFECTS OF CHLOROQUINE AND o-PHENANTHROLENE ON
CYTOTOXIN ACTIVITY 72
IX.CELL SUSCEPTIBILITY PROFILES OF RABBIT CYTOTOXINS . . 74
X.SUMMARY: COMPARISON OF PHYSICOCHEMICAL AND
BIOLOGICAL PROPERTIES OF RABBIT TUMOR NECROSIS
SERUM CYTOTOXIN (TNSCT) WITH RABBIT
MACROPHAGE CYTOTOXINS 76
XI.51Cr RELEASE BY L-929 CELLS COCULTURED WITH
MOUSE PERITONEAL EXUDATE CELLS 77
XII.EFFECTS OF TNSCT, PLCCT, BMCCT, AND PLC ON TNSCT -
RESISTANT AND CONVENTIONAL L-929 CELLS 92
vi

LIST OF FIGURES
FIGURE PAGE
1. Comparison of automated and manual photocell
in the photometric cytotoxicity assay. ... 25
2. Rabbit macrophage cytotoxin production with
and without added LPS 37
3. Rabbit macrophage cytotoxin production in the
absence and presence of varying levels of
exogenous LPS 41
4. Effects of bovine and fetal bovine serum on
pulmonary lavage cell cytotoxin
production 50
5. Comparison of effects of 1% and 10% fetal bovine
serum on cytotoxin production by rabbit
pulmonary lavage cell cultures containing
various levels of LPS 52
6. Time kinetics of rabbit pulmonary lavage cell
cytotoxin production 54
7. Time kinetics of cytotoxin production by
TPA-pretreated and washed rabbit bone
marrow cells 57
8. Effects of actinomycin D on cytotoxin
production by rabbit pulmonary lavage
cells 60
9. Effects of prostaglandin E2 and prostaglandin
antiserum on cytotoxin production by rabbit
pulmonary lavage cells 62
10. Gel filtration of rabbit macrophage cytotoxins
on Sephacryl S-200 65
11. Ion exchange chromatography of rabbit macrophage
cytotoxins on DEAE-Sephadex 68
12. Thermal stability of rabbit TNS cytotoxin
and macrophage cytotoxins 71
13. CjH/HeN mouse peritoneal exudate cells on
actinomycin D pretreated L-929 cells:
ninety-six well tray
vii
79

14.
Effector cell cytotoxicity for actinomycin D
pretreated L-929 targets: Determination
of S5Q endpoint 82
C3H/HeN mouse peritoneal exudate cells on
actinomycin D pretreated L-929 cells:
Twenty-four well tray 84
16. Rabbit pulmonary lavage cells on actinomycin D
pretreated L-929 cells: Twenty-four
well tray 86
17. Linearity between number of plaques counted
vs. number of mouse peritoneal exudate
cel Is plated 88
vi i i

ABBREVIATIONS USED
BCG
BLM
BLMCT
BMC
BMCCT
BPTI
CT
FMEM
HEPES
LPS
MAF
MEF
MEM
MPS
PEC
PGE
PLC
PLCCT
PMSF
RES
SBTI
TNF
TNS
TNSCT
TPA
Mycobacterium bovis, strain BCG
blood mononuclear cells
blood monocyte cytotoxin
bone marrow cel 1s
bone marrow cell cytotoxin
bovine pancreatic trypsin inhibitor
cytotoxin(s)
fortified Eagle's minimum essential medium
N-2-hydroxyethy1 piperazine-N1-2-ethane
sulfonic acid
1ipopolysaccharide
macrophage activating factor
mouse embryo fibroblasts
Eagle's minimum essential medium
mononuclear phagocyte system
peritoneal exudate cells
prostaglandin E
pulmonary lavage cells
pulmonary lavage cell cytotoxin
phenylmethyl sul fonyl fl uoride
reticuloendothelial system
soybean trypsin inhibitor
tumor necrosis factor
tumor necrosis serum
tumor necrosis serum cytotoxin
12-o-tetradecanoylphorbol 13-acetate
IX

Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
IN VITRO PRODUCTION OF RABBIT MACROPHAGE TUMOR CYTOTOXIN:
ITS ROLE IN MACROPHAGE MEDIATED TUMOR CELL KILLING
By
Harvey Fisch
December 1981
Chairman: George E. Gifford
Major Department: Immunology and Medical Microbiology
Rabbit macrophages derived from pulmonary lavage cells (PLC),
phorbol ester pretreated and washed bone marrow cells (BMC), or
blood mononuclear cells (BLM), cultured in the presence of LPS,
secreted tumor cell cytotoxins (CT) that were similar to each other
and tumor necrosis serum cytotoxin (TNSCT).
All CT's had molecular weights of approximately 48,000 D by
gel filtration and eluted from DEAE-Sephadex between 0.28 and 0.32 M
salt. All were stable to 56°C for 60 minutes, but labile to 70°C
for 20 minutes. Actinomycin D (AcD) enhanced sensitivity of L-929
cells to CT. BI6C3 me!anana cells and mouse embryo fibroblasts
were resistant to CT.
By 3 hours in culture, all effector cells secreted detectable
levels of CT. Titers increased for PLC and BLM between 3 and 5
hours. After that time, BLM titers ranained constant through 24
hours. PLC titers rose again from 18 to 30 hours. BMC cultures
x

demonstrated increasing titers from 3 through 14 hours. After that,
no increase in titer was demonstrated. No additional CT production
could be demonstrated after 30 hours in BMC or PLC cultures, despite
a change to fresh medium with LPS.
On an adjusted basis of 1 x 10^ macrophages/ml , PLC produced
4 to 25 times more CT than BMC or BLM after 28 hours culture.
CT production by LPS-activated PLC could occur in serum-free
medium. Fetal bovine serum (10%) enhanced production up to 4-fold.
Bovine serum (>4%), or prostaglandin E2 (10~^M) inhibited CT
production. AcD (1 yg/ml) added with LPS inhibited CT production
(>95%) by PLC. Delaying addition of AcD after LPS demonstrated
messenger RNA production for CT was completed by 2 to 6 hours.
AcD pretreatment of L-929 cells (2 yg/ml , 2 to 3 hours)
followed by washing enhanced their sensitivity to PLC killing. From
this, a plaque assay was generated demonstrating a linear
relationship between number of PLC plated and plaques formed.
Therefore, 1 PLC was capable of forming a plaque. Time lapse
cinematography demonstrated that contact between PLC and targets was
unnecessary for target cell killing.
BI6C3 cells and L-929 cells selected for resistance to TNSCT
were resistant to PLC mediated cytotoxicity.
The conclusion drawn is that secreted CT may play a major role
in macrophage mediated tumor cytotoxicity.
xi

INTRODUCTION
The problems encountered in biological disciplines are often
frustrating and at times seemingly insurmountable. This is the case in
the broad field of tumor immunology, an area filled with complexity and
apparent contradiction.
Immune System Components: In Vitro Cytotoxicity for Tumors
The interactions of the various humoral and cellular components of
the immune systan with tumors as well as with each other in vivo and in
vitro have been well documented. This has been described as a "network"
(Jerne, 1974, 1977) as well as a surveillance system seeking out tumors
as they arise within the host (Thomas, 1959; Burnet, 1967, 1970). Some
investigators seriously question the validity of these sweeping concepts
in the face of spontaneously arising tumors (Hewitt et al., 1976).
Evidence also exists illustrating that some tumors grow as a result of an
immune response (Prehn, 1976).
Despite these difficulties, there has been much effort put forth to
investigate and understand just what the components of the immune system
can do when confronted with tumors both in the test tube and in the tumor
bearing host. More specifically, sensitized cytotoxic T lymphocytes
(Brunner and Cerottini, 1971), natural killer lymphocytes (Herberman et
al., 1975), and armed and activated macrophages (Alexander, 1974)
represent cell types that can directly kill tumor cells subsequent to
contact with them. Some of the reported humoral activities cytotoxic for
1

2
tumor cells include specific antibody and complement (Hellstrom et al.,
1968), lymphotoxin, a product of lymphocytes (Granger and Kolb, 1968),
tumor necrosis factor, and cytotoxic factor (Carswell et al. 1975),
presumed macrophage products (Matthews, 1978; Mannel et al. 1979, 1980a,
1980b). Examples of humoral and cellular elements working together to
destroy tumor cells include specific antibody with nonimmune lymphocytes
(Pollack et al., 1972) and specific antibody with macrophages (Whitcomb,
1979).
The Mononuclear Phagocyte System: A Macrophage Network
The mononuclear phagocytes or macrophages represent a large,
morphologically heterogeneous group of cells found widely distributed
throughout the body (Vernon-Roberts, 1972). Metchnikoff (1905)
recognized the presence of mononuclear cells in the blood, lymph, as well
as throughout the various organs, that could engulf small particles such
as bacteria by projecting ameboid processes. He coined the term
"phagocytosis" (from the Greek word, "phagein," to eat) and designated
the large free and fixed phagocytic cells as "macrophages." He also
demonstrated the significance of these phagocytic cells concerning host
defense by reconfirming their presence in inflamed tissue as well as
noting their phagocytic capacity for bacteria.
Utilizing vital dyes which are preferentially taken up by
macrophages and to a lesser extent by other types of cells, Aschoff
(1924) demonstrated the distribution throughout the body of a functional
system of cells which included macrophages. He referred to this overall

3
network of cells as the "reticuloendothelial system (RES)." It was
believed that these cells were responsible for the formation of the
extrasinusoidal solid pulp of the lymph nodes and spleen (reticulum). In
addition, they were found lining the sinusoids of lymph nodes and other
organs (endothelium). Additional investigations extendea the functional
RES to include the microglia of the central nervous system (del Rio
Hortega and de Asua, 1924) and blood monocytes (Marshall, 1956).
Later studies reviewed by van Furth et al. (1975) revealed that the
original concept of the RES as a unified system is inappropriate in that
it really consists of at least two distinct systems. One of these, the
"RES proper" is composed of cells that are nonphagocytic and make up the
framework of connective tissue. Distinct from these cells are the
macrophages, which often exist either fixed or free, in close proximity
to connective tissue. As a result, the tern "mononuclear phagocyte
system" (MPS) has been proposed to denote a functional network made up
exclusively of macrophages (van Furth et al., 1975).
As previously mentioned, macrophages display considerable
morphological heterogeneity (Vernon-Roberts, 1972). This no doubt
created considerable confusion among early investigators regarding what
was and was not to be considered a macrophage. Perhaps the most
enlightening studies concerning this problem, as well as revealing the
source of macrophages, have dealt with their ontogeny. Macrophages
originate from a common source in the bone marrow, enter the circulation
and finally pass into the tissues, changing their morphological and many
of their functional character!-sties as they proceed (van Furth et al.,

4
1975; van Oud Alblas and van Furth, 1979). Therefore, the varied
descendants of the bone marrow precursor merely represent different
stages or various end cells of a progressive differentiation process.
Aside from their mononuclear appearance, intense phagocytic
capability and common origin, macrophages share an array of other
morphological, chemical and functional characteristics. Included among
these properties are the ability to adhere tightly to glass, motility,
possession of abundant lysosomes containing various enzymes, production
of various immune modulators, complement components, superoxide free
radical and hydrogen peroxide and possession of Fc and complement
receptors (Eisen, 1980). In additional to morphological features,
heterogeneity among macrophages can be extended to these properties as
well since different macrophages vary qualitatively and quantitatively
with respect to these characteristics (van Oud Alblas and van Furth,
1979; Keller, 1980). This overall heterogeneity reflects the existence
of clearly separable subpopulations (Keller, 1980) with either distinct
genetic differences or changes that come about with pluripotent
macrophage maturation or activation.
Many of these properties contribute to the ability of macrophages
to either act alone or in conjunction with other cells or products of the
"immune system" (T and B lymphocytes, antibody and complement) to help
maintain a state of homeostasis within the host. More specifically, this
involves elimination of exogenously introduced toxins, invading
microorganisms, or other foreign antigens, as well as the destruction and
eventual elimination of host cells that are effete or have undergone
certain alterations. This latter case includes the changes seen in cells
associated with neoplasia.

Significance of the Mononuclear Phagocyte
System in Tumor Regression
5
Nearly a century ago, Coley (1891) reported on the palliative (and
occasional curing) effects of using bacterial culture filtrates to treat
patients with neoplastic disease. The rationale for this treatment of
neoplasia was based on the observation (made long before that) that
cancer patients with concurrent infectious disease often displayed
regressing neoplasia or tumors that advanced less rapidly than those of
cancer patients without concurrent infection. More recent reviews
(Pearl, 1929; Jacobsen, 1934; Nauts et al. , 1953) have discussed related
phenomena in detail.
Later investigations revealed that the MPS could expand and
increase its activity during the course of a neoplastic disease (Old et
al., 1960) or following the administration of such agents as
Mycobacteriurn bovis, strain BCG (Old et al., 1960; Leake and Myrvik,
1968), zymosan (Old et al., 1960), or Corynebacteriurn parvum (Hal pern et
al., 1974). Similar studies also showed that such agents could protect
challenged animals against transplanted tumors or increase the survival
time of these animals (Old et al., 1959; 1960).
Further evidence that contributed credibility to the macrophage as
a critical component of the j_n vivo effector response to tumors was later
provided. Macrophages could be made nonspecifical ly cytotoxic to tumor
cells by culturing them in the presence of endotoxin (Alexander and
Evans, 1971), or supernatant fluids from antigen sensitized lymphocyte
cultures (Piessens et al., 1975; Leonard et al., 1978). Alternatively,
macrophages could be rendered specifically cytotoxic or "armed" to a

6
particular tumor by cocultivating them with spleen lymphocytes from an
animal previously inoculated with the tumor (Alexander, 1974). These
macrophages could then be rendered nonspecifical ly cytotoxic (activated)
to different tumors by continued cocultivation with the original
immunizing tumors (Alexander, 1974).
More convincing evidence indicating that macrophages play a
significant role in the overall scheme of tumor defense mechanisms was
revealed by their actual presence within tumors (Evans, 1972; Alexander,
1974; Eccles and Alexander, 1974). Furthermore, tumor regression could
be correlated with macrophage infiltration (Russell et al., 1976).
The Process of Macrophage Activation
The term "activated" as it pertains to the macrophage was
introduced by MacKaness (1970) and it is a term which has received wide
usage by many investigators who study the macrophage. Depending upon
one's interests, the activated macrophage possesses many character!'sties
which distinguish it from a macrophage which is not activated. Allison
and Davies (1975) have described biochemical and biological changes in
macrophages undergoing activation. These include increases in the
following: glucose oxidation, lysosomal enzyme levels, synthesis of
membrane components, pinocytosis, phagocytosis, adhesion, spreading and
motility. In addition, activated macrophages, as opposed to resting
macrophages, can inhibit the proliferation of and have demonstrated
cytotoxicity for tumor cells (Allison and Davies, 1975; Hibbs, 1976;
Keller, 1980).
The cytotoxicity that activated macrophages demonstrate for
transformed cells is most interesting. Aside from being nonspecific

7
as seen by the ability of activated macrophages to kill different types
of tumors from allogeneic, syngeneic or xenogeneic sources (Keller,
1980), most nontra ns formed cells are not killed by activated macrophages
(Hibbs, 1974a; Keller, 1980). Therefore, the activated macrophage
appears to be capable of distinguishing some common feature(s) of the
tumor state from that of normal cells. Currently, the exact distinction
between tumor and nontra ns formed cells made by the activated macrophage
is unknown. However, some evidence suggests the difference lies within
the plasma membrane (Hibbs, 1974a).
The process of macrophage activation is not a simple, absolute,
one-step event, but involves a cascade of transient, intermediary
reactions (Hibbs et al., 1980; Meltzer et al., 1980) dependent upon the
presence of signals in the local environment of the macrophage (Hibbs et
al., 1977). Some of these signals are stimulatory while others are
inhibitory to macrophage activation (Hibbs et al., 1977; Chapman and
Hibbs, 1977). Ruco and Meltzer (1978) have extended this model by
describing it as a series of "priming" signals followed by activation
signal(s).
As mentioned previously, some of the signals that can affect the
state of the macrophage and modulate activation have been identified.
They include endotoxin (Alexander and Evans, 1971), macrophage activating
factor, a T lymphocyte product (Leonard et al., 1978) and type I
interferon (Schultz et al., 1977; Jett et al., 1980). As work in this
field progresses, it is becoming more and more evident that many of these
activation signals are related to one another as part of the macrophage
activation cascade, particularly as priming or activation signals.

8
This is the case concerning macrophage activating factor (MAF) and the
active component of endotoxin, 1 ipopolysaccharide (LPS). It has been
established using highly purified materials that MAF alone cannot
activate macrophages, but merely sensitizes or "primes" them to the
activating capabilities of LPS (Weinberg and Hibbs, 1979; Weinberg and
Hibbs, 1980; Hibbs et al., 1980; Weinberg, 1981; Pace and Russell, 1981).
Earlier studies concerning these two signals in this scheme were
complicated by the presence of small amounts of LPS contaminating the
lymphokine preparations as well as other materi al s (Hibbs et al., 1980).
Aside from clarifying the roles of MAF and LPS in the macrophage
activation scheme, many of these studies demonstrate the importance of
determining the presence of contaminating LPS when working with
macrophages (Levin et al., 1970; Fumarola, 1981).
Despite the apparent clarity of this simple model utilizing MAF (a
priming signal) and LPS (a triggering signal), there are many points
regarding macrophage activation and its associated signals that are still
not clear. First, it is apparent that LPS is not the only triggering
signal. This has been demonstrated in several studies which either
exclude LPS from cultures or utilize macrophages derived from mice that
respond very poorly to LPS (Hibbs, 1976; Schultz and Chirigos, 1980;
Boraschi and Tagliabue, 1981). Secondly, type I interferon has been
described as a priming signal distinct from MAF (Boraschi and Tagliabue,
1981) as well as serving as the endogenous macrophage generated mediator
for LPS induced triggering of macrophage activation (Schultz and Chirigos,
1979). Further studies are needed to determine the identity of other

9
triggering signals as well as the precise role of interferon in the
scheme of macrophage activation.
The Elicitation of Serum Tumor Necrotic
and Cytotoxic Activities
Many of the foregoing mechanisms concerning macrophage activation
are now believed to represent accurate in vitro correlates of host
mediated responses to such agents as BCG, zymosan, Corynebacterium parvum
and LPS. Although the early investigators were not aware of the details
behind the host's response to these agents, it was quite apparent that
they had value in inducing favorable clinical responses in tumor bearing
animals. Ribi et al. ( 1975) gave tumor bearing guinea pigs both BCG and
LPS. The results of this double regimen were rather impressive. Test
animals that had received both BCG and LPS displayed a survival rate of
about 90% compared to a survival rate of about 66% for those receiving
BCG alone.
Carswell et al. (1975) found that they could cause a dramatic
necrosis of transplanted subcutaneous tumors in otherwise unmanipulated
mice that were injected with sera from other mice, rabbits, or rats that
had previously been inoculated intravenously with BCG, Corynebacteriurn
species or zymosan followed by an intravenous injection of LPS 2 weeks
later. They called the agent(s) in the sera responsible for this
phenomenon, tumor necrosis factor (TNF). It was also demonstrated by the
same group (Carswell et al., 1975; Helson et al., 1975; Green et al.,
1976) that crude tumor necrosis serum (TNS) or material that was
partially purified from TNS could kill many but not all types of tumor
cells in vivo and in vitro while being nontoxic for mouse embryo

10
fibroblasts in vitro. Others have reported similar findings for TNS
derived from the mouse (Mannel et al., 1980a; Mannel et al., 1980c) and
rabbit (Matthews and Watkins, 1978; Ostrove and Gifford, 1979). In
addition, some of these as well as other studies demonstrate that neither
tumor necrosis or in vitro cytotoxicity are due to LPS (Carswell et al .,
1975; Oettgen et al., 1980). LPS levels in the serum transferred from
donor animals are substantially below the levels required to cause tumor
necrosis. Furthermore, LPS is not toxic for tumor cells in vitro and has
a profile distinctly different from that of TNF regarding other
biological activities (Oettgen et al., 1980).
Biochemical and Physical Properties of Tumor
Necrosis Factor and Cytotoxins in Tumor
Necrosis Serum
Green et al. (1976) partially purified TNF from mouse TNS and
determined it to have a molecular weight of 125,000 to 150,000 D by gel
filtration. Further studies in the mouse (Mannel et al., 1980a; Mannel
et al., 1980c; Oettgen et al., 1980) demonstrated the molecular weight of
the cytotoxic activity (CT) and TNF respectively (by gel filtration) to
be about 60,000 D, after exposure to high salt concentrations (0.2 M or
greater). This may indicate ionic aggregation of polymeric subunits or
aggregation with some other molecular species found in the serum. Other
properties of mouse TNF and tumor necrosis serum cytotoxin (TNSCT)
include stability to 56° C exposure for 30 minutes, an isoelectric point
of pH 4.8, pronase sensitivity, and the presence of sialic acid and
galactosamine residues (Green et al., 1976; Mannel et al., 1980c; Oettgen
et al., 1980).

11
Studies in the rabbit employing gel filtration have determined the
molecular weight of TNSCT to be 55,000 D (Ruff and Gifford, 1980) and
39,000 D (Matthews et al., 1980). The two groups reported approximately
the same molecular weight (67,000-68,000 D) utilizing polyacrylamide gel
electrophoretic techniques. The consistently higher molecular weight as
determined by electrophoresis over that of gel filtration could possibly
be explained by alteration of configuration of the cytotoxic molecule by
denaturing conditions (Ruff and Gifford, 1980) or interaction of the
molecule with the electrophoretic matrix resulting in retardation of
migration (Matthews et al., 1980). The reasons for the difference between
the two groups concerning the molecular weight as determined by gel
filtration are unclear. Additional characteristics of rabbit TNSCT
include relative stability to 70°C for 20 minutes (Matthews et al.,
1980), an isoelectric point of pH 5.1, and high sensitivity to pronase
and relative insensitivity to trypsin (Ruff and Gifford, 1980; Matthews
et al., 1980). These and other studies (Ruff and Gifford, 1981) suggest
that the rabbit CT molecule does not contain large amounts of exposed
carbohydrate contains little or no carbohydrate, as based upon inability
to bind cytotoxic activity to various plant lectins.
Are Tumor Necrosis Serum-Perived
Necrotic and Cytotoxic Activities
Mediated by the Same Effectors?
It is tempting to speculate from results of many of the
aforementioned studies that the TNS agents responsible for tumor necrosis
(TNF) and in vitro cytotoxicity are one and the same. However, the
complexity of TNS is underscored by the demonstration that a variety of
biological products such as interferon (Sauter and Gifford, 1966), B cell
differentiating factor (Hoffman et al., 1976), bone marrow colony

12
stimulating factor (Hoffman et al., 1977) and lysosomal enzymes (Sauter
and Gifford, 1966; Green et al., 1976; Old, 1976) are released into the
serum under conditions used for induction of tumor necrosis. It is
therefore possible that many mediators may contribute to tumor necrosis
and cytotoxicity and the rnediator(s) responsible for one of these
phenomena may not be responsible for the other.
Initial studies employing crude TNS (Carswell et al., 1975) implied
that a single entity, TNF, could be responsible for both in vivo tumor
necrosis and in vitro cytotoxicity. As a result, many of the studies that
followed exclusively utilized either the in vivo assay for necrosis
Green et al., 1976) or an in vitro assay for cytotoxicity (Matthews et al.,
1980; Mannel et al., 1980d) to study purified effector material. It was
felt that either assay probably reflected the activity of the same
effector. Along these lines, Oettgen (1980) and Kull and Cuatrecasas
(1981), working in the mouse system, were able to partially copurify both
cytotoxic and necrotic activities from TNS. Additional studies
demonstrated that partially purified CT derived frail rabbit TNS could
elicit tumor necrosis (Ruff and Gifford, 1980). The data of Kull and
Cuatrecasas (1981) are particularly interesting because they demonstrated
material with cytotoxic activities having molecular weights of 50,000,
160,000 and 225,000 D, but only the 160,000 D containing fractions also
displayed necrotic activity. In addition, they found that high molecular
weight species persisted after salt fractionation, and gel filtration of
the high molecular weight form in high salt led to loss of activity without
an increase in activity of low molecular weight species (50,000 0). This

13
is contrary to the hypothesis concerning high salt deaggregation of high
molecular weight forms (Mannel et al., 1980a; 1980c).
The results of the aforementioned studies do not definitively
demonstrate whether or not TNS necrotic and cytotoxic activities can be
attributed to a single effector molecule. Further investigation is
needed to clarify this point.
Tumor Necrosis and Tumor Regression
Early studies utilizing animals with transplanted subcutaneous
tumors demonstrated a "hemorrhagic necrosis" of their tumors after being
administered bacterial culture filtrates of such organisms as Escherichia
coli (Gratia and Linz, 1931). Later, Shear and Turner (1943) purified a
polysaccharide containing lipid material from Serratia marcescens (now
known to be LPS) which alone could cause a rapid and dramatic hemorrhagic
necrosis of tumors. Although the mechanism behind this phenomenon
remains unclear at this time, there is evidence suggesting an LPS induced
vasoconstriction and intravascular coagulation within the tumor (Algire
et al., 1952) as well as a need for lymphoreticular cells that are
sensitive to LPS (Mannel et al., 1979). As already mentioned, a similar
phenomenon can be seen in tumor bearing animals administered TNS
(Carswell et al.,1975) or partially purified TNF (Ruff and Gifford,
1980). It is not yet clear (but is circumstantially possible) that TNF
plays a role in the tumor necrosis induced by LPS alone. As rapid (often
less than a day) and dramatic as this phenomenon is, a ring of viable
tumor cells usually survives at the periphery of the neoplasm to grow and
kill the host (Shear, 1943). This demonstrates that tumor necrosis

14
in itself is usually insufficient to lead to total recovery from
neoplastic disease.
Tumor regression, on the other hand, involves all those events
which contribute and ultimately lead to total recovery of the host from
naturally occurring or experimentally induced tumors. It is possible
that tumor necrosis may contribute to or allow subsequent tumor
regression to take place more readily by destroying those parts of the
tumor (the center) which effector mechanisms (such as activated
macrophages) may have difficulty reaching.
As previously discussed, much evidence has been provided concerning
the significance of the macrophage in overall tumor regression (Evans,
1972; Alexander, 1974; Eccles and Alexander, 1974; Russell et al., 1976).
It is possible that in many cases, the macrophage may be directly
involved in tumor regression that has been attributed to other types of
effector cells (natural killer cells, cytotoxic T lymphocytes). As an
example, studies by Berendt et al., (1978a; 1978b) have indicated that
LPS induced tumor regression requires concomitant T cell immunity and
that only those tumors that are immunogenic enough to evoke T cell
immunity will regress. At first inspection it is difficult to envision a
role for macrophages in such a scheme. However, the macrophage
activation scheme involves a series of complex events dependent upon
various signals such as MAF, a T lymphocyte product (Leonard et al.,
1978; Weinberg and Hibbs, 1979). It is quite possible that macrophages
may be "primed" by MAF produced by immunized T cells and subsequently
activated by the administered LPS, thereby allowing them to be
responsible at least in part for LPS induced tumor regression.

15
Activated Macrophages Produce Soluble Cytotoxic
Activity in Vitro
The involvement of the macrophage in the production of tumor
necrotic and cytotoxic serum activities is suggested by the coincident
hyperplasia of the MPS accompanying the administration of many TNS
eliciting agents (Carswell et al., 1975; Old, 1976). In addition,
evidence demonstrating that macrophages can kill tumor cells in vitro
(Alexander, 1974; Hibbs, 1976) prompted studies that sought to better
understand the relationship of TNF and TNSCT to activated macrophages.
Studies in the mouse have demonstrated that LPS sensitive cells are
needed to induce TNF in serum (Mannel et al., 1980d) and that biochemical
and physical properties of serum-derived TNSCT are similar to those of CT
derived from cultures of activated macrophages and macrophage cell lines
(Mannel et al., 1980a, 1980b, 1980d). In addition, these same studies
demonstrated that a rabbit antiserum directed against serum-derived TNSCT
inhibited all soluble cytotoxicity generated in the macrophage cultures.
Working in the rabbit, Matthews (1978) has demonstrated that blood
monocytes produce soluble tumor CT in vitro. This CT has striking
similarity to rabbit TNSCT in terms of selective toxicity for various
tumor cells and biochemical characteristics.
Purpose of These Studies
The aims of these studies are as follows:
1. To study the production of rabbit macrophage CT j_n vitro
utilizing macrophages from different sources (pulmonary alveolar, bone
marrow, and blood mononuclear cells).

16
2. To compare physicochanical and biological properties of these
CT with one another as well as with those of TNSCT.
3. To determine the significance these CT play in macrophage
mediated tumor cytotoxicity.

MATERIALS AND METHODS
Animals
New Zealand white female rabbits, ranging in age from 3 to 5 months
(mean weights of 3 to 5 kilograms) were employed. Mice (CgH/HeN and
C57B1/6, female) used ranged in age from 6 to 12 weeks. All animals
were obtained from commercial sources and housed and fed conventionally.
Cell Lines
L-92 9 cells (a C3H mouse cell line) were grown in Eagle's minimum
essential medium (MEM) supplemented with 10% bovine serum and 250 units of
penicillin/ml and 125 yg of streptomycin/ml. BI6C3 melanoma cells (a
C57B1/6 mouse cell line) were grown in fortified MEM supplemented with
10% fetal bovine serum. Fortified MEM contained 2x concentrations of
essential and nonessential amino acids and vitamins. Glutamine and sodium
pyruvate were also added. Primary mouse embryo fioroblasts (MEF) were
prepared by mincing near term embryos followed by trypsinization for 60
minutes (0.1% trypsin, 0.04% ethylene diamine tetraacetic acid), washing
in Gey's balanced salt solution and culturing in glass bottles containing
FMEM with 10% fetal bovine serum.
Culture Systems
Media
These have been described above (see Cell Lines). For use in
cultures where macrophages were employed, N-2-hydroxyethyl piperazine-N'-
2-ethane sulfonic acid (HEPES buffer, Cal biochan, LaJolla, CA) was added
at .03M.
17

18
Sera
Two lots of bovine sera were used: Gibco (Grand Island, NY), lot
number R290222 and Sterile Systens, Inc. (Logan, UT), lot number 400168.
The latter was certified to contain less than 1 ng lipopolysaccharide/rnl .
Unless otherwise stated, this was the lot used. One lot of fetal bovine
serum was used for all experiments in this study (Kansas City Biological,
Lenexa. KS).
Lipopolysaccharide-Free Culture System
Using a Limulus amebocyte lysate reagent kit (Pyrogent, Mallinckrodt
Inc., St. Louis, MO) to test various media components for contaminating
1ipopolysaccharide (LPS), a culture medium was established which contained
less than 0.025 ng LPS/ml (see Table III for the various materials
screened). This "LPS-free" culture system was used for most of the
studies to be discussed.
Effector Cells (Macrophages)
Rabbit Macrophage Sources
Rabbit macrophages were derived from three sources, pulmonary lavage
cells (PLC), bone marrow cells (BMC), and blood mononuclear cells (BLM).
For harvesting PLC and BMC, rabbits were anesthetized with a combination
general anesthetic consisting of ketamine hydrochloride (Bristol
Laboratories, Syracuse, NY) and xylazine hydrochloride (Cutter
Laboratories, Shawnee, KS) followed by intravenous air embolism sacrifice.
After hair removal and cleansing of the skin with 70% ethanol, the trachea
and/or femurs were exposed. For PLC harvest, the trachea was transected
and the lungs flushed out with six aliquots of 50 ml cold sterile pyrogen-
free saline using a sterile disposable syringe. Cells were immediately
removed from the cold saline by centrifugation and resuspended

19
in cold MEM supplemented with 10% fetal bovine serum and .03M HEPES
buffer. Cells were generally washed twice by centrifugation in medium.
About 20 to 40 x 10® PLC were obtained per rabbit. For BMC, femurs were
removed and trimmed free of as much soft tissue as possible. After
the bones had been rinsed well with sterile, pyrogen-free saline, the ends
were removed with sterilized bone cutters and the contents of the marrow
cavity flushed out with FMEM supplemented with 10% fetal bovine serum and
.03M HEPES buffer, using a 16 gauge needle attached to a 20 ml sterile
disposable syringe. Up to 2 x 10^ bone marrow cells were obtained per
rabbit.
For harvesting BLM, rabbits were anesthetized and bled from the
marginal ear vein into sterile tubes containing pyrogen-free heparin.
After collection, the blood was underlayered with half its volume of
Histopaque-1077 (Sigma Chemical Co., St. Louis, M0) and centrifuged at 400
g for 40 minutes at room temperature. The leukocytes at the interface
were collected, washed 3 times with cold pyrogen-free saline and suspended
in MEM, supplemented as for PLC. Generally, 50 to 100 x 10® BLM were
obtained from one rabbit. Where necessary to minimize effector cell
clumping and to insure a majority of single effector cells (see Plaque
Assay), PLC were pushed through a 19, 23 and finally a 25 gauge hypodermic
needle, using a syringe.
Leake and Myrvik (1968) have described a technique to significantly
increase the yield of PLC from rabbit lungs. When this technique was
modified by intravenous injection of 0.25 ml Freund's complete adjuvant
(Gibco, Grand Island, NY) instead of Mycobacterium bovis, strain BCG
(Leake and Myrvik, 1968), PLC yields were significantly increased up to
about 200 x 10® per rabbit, if cells were harvested 2 to 3 weeks after
the injection. These rabbits will be referred to as "primed" throughout

20
these studies. Those rabbits which were previously unmanipulated prior to
sacrifice will be referred to as "normal."
Mouse Macrophage Source
Mouse macrophages were derived from peritoneal exudate cells (PEC).
CgH/HeN and C57B1/6 mice were injected intraperitoneal ly with 2.5 to
3.0 ml of fluid thioglycollate medium (Bacto, Difco, Detroit, MI). Three
days later, the mice were sacrificed by cervical dislocation and peri¬
toneal exudate cells (PEC) harvested by washing out the peritoneal
cavities with 8 to 10 ml of cold medium. The cells were washed twice by
centrifugation and resuspended in fresh medium. As with rabbit PLC, where
necessary to minimize cell clumping and insure a majority of single cells,
PEC were pushed through 19, 23 and finally 25 gauge hypodermic needles,
using a syringe.
Activation of Macrophages in Culture
LPS (Escherichia col i 055:B5, Westphal, Difco, Detroit, MI) was used
to induce macrophage activation in all cultures. Unless otherwise stated,
culture conditions were as follows: for mouse PEC or rabbit PLC and BLM,
LPS was added at 10 yg/ml in MEM, supplemented with 10% fetal bovine serum
plus .03M HEPES buffer; for rabbit BMC, cells were cultured in FMEM
supplemented with 10% fetal bovine serum plus .03M HEPES buffer. Further¬
more, concerning BMC, the phorbol ester, 12-o-tetradecanoylphorbol
13-acetate (TPA) was added at 0.1 or 1 ug/ml and the cells generally
cultured overnight, after which time they were washed free of TPA (4 to 6
times) and refed with fresh medium containing LPS (see Increasing the
Macrophage Content of Bone Marrow Cultures, under Results). Unless other¬
wise stated, all effector cells were cultured at 1 x 10^ cells/rnl.

21
Viability of all effector cell populations was detennined by trypan
blue dye exclusion. All effector cells used had 95% viability or greater
by this criterion and were always cultured at a density based on the
percentage of viable cells.
Identification of Macrophages in Effector
Cell Populations
Identification of macrophages was based on morphological criteria
(Giemsa's blood stain), phagocytic capability of latex beads (Dow Chemical
Co., Indianapolis, IN) and peroxidase content (Leukocyte Peroxidase,
Histozyme Kit No. 390A, Sigma Chemical Co., St. Louis, MO).
Production of Tumor Necrosis Serum
The basic technique has been described by Carswell et al. (1975).
Rabbits were injected intravenously with viable BCG (Tice strain, 3 x
108 organisms). Two to three weeks later, LPS was injected
intravenously and the rabbits bled 2 to 3 hours later. The blood was
allowed to clot and the serum was withdrawn, further clarified by
centrifugation, and aliquoted. Aliquots were frozen at -20°C until used.
Once thawed, tumor necrosis serum (TNS) could maintain cytotoxicity titer
(see assays) when kept at 4°C for several months.
Production of Rabbit Macrophage Cytotoxins
Rabbit PLC, BLM or TPA-pretreated and washed BMC were cultured in
appropriate medium containing LPS. (Occasionally BMC were cultured in 175
cm8 plastic tissue culture flasks at 2 to 5 x 108 cells/ml to increase
titer yields. Otherwise, PLC and BLM were generally cultured at 1 x
108 cells/ml in 25 cm8 plastic tissue culture flasks or 24 well
plastic tissue culture plates.) After an appropriate time at 37°C,
supernatants were harvested and cells removed by centrifugation.

22
Supernatants were titered and used within 48 hours if stored at 4°C (no
appreciable loss of titer occurred if stored at this temperature for this
time) or frozen at -80°C. Supernatants frozen at -80°C maintained titers
for several months. Cytotoxins (CT) generated by rabbit macrophages are
designated as follows: pulmonary lavage cells (PLCCT), TPA-pretreated and
washed bone marrow cells (BMCCT), and blood mononuclear cells (BLMCT).
Cytotoxicity Assays
Photometric Assay
A basic photometric technique has been previously described (Ruff
and Gifford, 1980). L-929 cells were plated in 96-well tissue culture
trays at 50,000 cell s/0.1 ml to establish a dense monolayer. Dilutions of
CT in 0.1 ml were added along with actinomycin D (Calbiochem, LaJolla, CA)
to give a final concentration of 1 ug/ml and the trays incubated at 37°C
for 18 hours in a humidified 5% COg atmosphere. After the culture
period, the plates were stained for 10 minutes with crystal violet (0.2%
in 2% ethanol), washed with water and allowed to dry.
Destruction of monolayers was evaluated by placing trays on an X-ray
viewing screen and measuring light transmission through monolayers with a
photocell. The reciprocal of the dilution of CT required to kill 50% of
the targets was taken as the 50% endpoint (S5Q). Two kinds of
photocells were used in these studies. A manually operated photocell unit
was constructed using a photoconductive cell (cadmium sulfide, No. 05HL,
Clairex Electronics, Mt. Vernon, NY). The cell was cemented to a small
piece of copper tubing that had an outside diameter approximating the
inside diameter of culture wells. The photocell was attached to a
multipurpose meter and read as a variable resistance at a fixed voltage as
a function of light transmitted through the monolayers. For convenience

23
an automated photocell (Titertek Multiskan, Flow Laboratories, Inc.,
Inglewood, CA) was also used for some of the experiments. Figure 1
compares the two photocells reading the same sample wells. They are
fairly similar, although it appears that the automated photocell is
slightly more sensitive and can detect very small numbers of target cells
retained at lower CT dilutions that the manual photocell cannot.
Nevertheless, since an entire experiment was always read with the same
photocell, and the enhanced sensitivity demonstrated by the automated
photocell was not much greater than the manual one (about 15% greater, see
Figure 1), this difference in photocell sensitivity is insignificant.
This photometric technique was slightly modified to measure effector
cell killing of targets. The only difference from the aforementioned
procedure used to titer CT involved adding actincmycin D at 2 ug/ml for 2
to 3 hours, followed by removal of this drug from the medium by decanting,
washing the monolayer with medium and replacing with fresh medium
containing effector cells. It was previously determined that 2 washings
were sufficient to remove virtually all extracellular actincmycin D
(washings at this point could not inhibit H^-uridine incorporation into
L-929 cells).
Plaque Assay
L-929 cells were grown to monolayers in plastic 24 well flat bottom
tissue culture trays, plating 250,000 cells in 1 ml medium. After the
monolayers were pretreated with actincmycin D (2 ug/ml) for 2 to 3 hours,
they were washed twice with medium. Small numbers of well mixed effector
cells (mouse PEC or rabbit PLC) in 2-fold increments were added carefully
to washed and drained monolayers of targets in a final volume of 0.2 ml
medium. Control target monolayers received only 0.2 ml medium. No agar

Figure 1. Comparison of automated and manual photocell in
the photometric cytotoxicity assay.
Each panel (upper and lower) represents titration of a
single sample by each of the photocells. Each point represents
percent target cell survival of a single culture well.
S = nuntier of cells surviving in cultures, S0 = number
of cells plated.

100
80
60
40
20
100
80
60
40
20
• Automated Photocell
O Mon u o I Photocel I
25
<3
9
O
o
o
o
2
9
o
o
o
i *
40 160 640 2560 0,240
[CYTOTOXIN DILUTION] '*

26
or methyl cellulose overlays were used. LPS was added at a final
concentration of 10 yg/ml to PEC and PLC cultures. Plates were cultured
undisturbed at 37°C for 18 to 20 hours. Supernatant contents were then
decanted and the plates were stained for 10 minutes with crystal violet,
washed with water and allowed to dry. In all cases, at least 95% of the
effector cells plated were single cells and the number plated was based on
number found to be viable (always >95%).
Cr^ Release Assay
L-929 cells were suspended into two 1 ml aliquots of 2 x 10^
cells. One aliquot was treated with actinomycin D for 1 hour. At that
time, 100 yCi sodium^ chromate (^Cr; ICN Chemical, Waltham, MA)
was added to each aliquot and the cells gently mixed by shaking. After 1
hour at 37°C the target cells were washed 5 times by repeated centrifuga¬
tion and resuspension in fresh medium (MEM with 10% fetal bovine serum,
gentamicin and HEPES buffer) to remove unincorporated ^Cr and from
one of the aliquots, actinomycin D. The concentration of the suspension
was adjusted with medium to 3 x 10^ cells/0.1 ml. BI6C3 cells were
labelled in a similar way only in the absence of actinomycin D.
These labelled targets were used to determine cytotoxicity of mouse
PEC (L-929) or CT generated by rabbit macrophage cultures (L-929,
BI6C3).
Mouse PEC were seeded into 96 well flat bottom plastic tissue
culture plates in MEM with 15% fetal bovine serum, gentamicin, and HEPES
buffer at 3 x 10^ cells in 0.1 ml volumes. Plates were centrifuged at
200 g for 30 seconds to sediment the cells to well bottoms. After 2 hours
at 37°C in a humidified air atmosphere containing 5% CO^, the nonad¬
herent cells were removed by decanting the supernatants and washing

27
the adherent PEC monolayers twice gently with warn medium. The great
majority of these adherent PEC, greater than 90%, had macrophage
morphology as revealed by staining with Gieinsa's blood stain and could
phagocytize latex particles. After the second wash, 0.1 ml MEM with 10%
fetal bovine serum, HEPES buffer and gentamicin was added to each well.
In an additional 0.1 ml of similar medium, ^^Cr-labelled targets were
added. LPS was then added. Plates were centrifuged at 200 g for 30
seconds to sediment target cells upon effector monolayers. Plates were
incubated at 37°C, in a humidified air atmosphere containing 5% C0£. At
appropriate times for harvesting the supernatants, 0.05 ml of medium was
added to each well to insure an even distribution of released ^-*-Cr
throughout the cultures. Cr^ labelled targets (L-929 or BI6C3
cells) were added to microtiter wells along with TNSCT, PLCCT, or BMCCT.
Again, at harvest times, 0.05 ml of medium was added to wells to evenly
distribute released Cr^.
For harvest, aliquots of 0.1 ml were ratioved and counted in a gamma
counter (Gamma 300 Radiation Counter, Beckman Instruments, Inc.,
Fullerton, CA). For each time period, the percent specific ^Cr
released was determined by use of the following formula: percent release
= [(E-S -5 (T-S)] x 100, where E = counts released from labelled targets in
test cultures containing effectors and targets, S = spontaneous counts
released from labelled targets cultured alone and T = total releasable
counts from labelled targets, as determined by maximum lysis of targets
with 1% dodecyl sodium sulfate. Appropriate volume correction factors
were employed to calculate counts released in each case.

28
Column Chromatography
Gel Filtration
All gel filtration chromatography was performed using a single,
calibrated coluim of Sephacryl, S-200 (Pharmacia Fine Chemicals,
Piscataway, NJ) of gel bed dimensions 83 x 2.5 cm. Flow rate was 21 ml/hr
using phosphate buffered saline. Samples were concentrated by pressure
dialysis in an Anicon Diaflo ultrafiltration unit (Anicon Corp.,
Lexington, MA) prior to loading 1 ml samples onto the column. Fractions
(2 ml) were collected and assayed for cytotoxicity on L-929 cells.
Ion Exchange Chromatography
Fractions with cytotoxicity for L-929 cells from the Sephacryl S-200
column were pooled and concentrated by pressure dialysis and loaded onto a
column of DEAE-Sephadex (Pharmacia Fine Chemicals, Piscataway, NJ)
previously equilibrated with phosphate buffered saline (pH 7.2, 0.15 M).
The column was washed with 2 column volumes of starting buffer to remove
unbound proteins. A linear salt gradient (0.15 to 0.60 M) was applied to
the column and fractions (4 ml) collected and assayed for cytotoxicity.
Flow rate was 20 ml/hr.
All chromatography was performed at 4°C.
Effect of Trypsin Inhibitors, Phenylmethyl Sulfonyl-
fluoride, Chloroquine and o-Phenanthrolene on
Rabbit Macrophage Cytotoxin Activities
Various drugs were tested in the actinomycin D cytotoxicity assay to
determine their effects on CT activity. The following were tested:
soybean trypsin inhibitor (SBTI, Worthington Biochemical Corp., Freehold,
NJ), bovine pancreatic trypsin inhibitor (BPTI, Sigma Chemical Co., St.
Louis, M0), at 100 ug/ml each; phenylmethyl sulfonylfluoride (PMSF) at
10“^M; chloroquine at 10“^M, and o-phenanthrolene at 10“^M
(last 3 drugs from Sigma Chemical Co., St. Louis, M0).

29
Effects of Prostaglandin Eg on Rabbit Macrophage
Cytotoxin Production
Prostaglandin E2 (PGE2» Sigma Chanical Co., St. Louis, MO) was
dissolved in ethanol and diluted in serum-free medium immediately before
addition to duplicate PLC cultures in serum-free medium. PGE2 was added
at 10"®M, 6 hours after initiation of the cultures with LPS (10
ug/ml). Controls received no PGE2* PGE2 antiserum (0.1 ml of lyoph-
ilized stock reconstituted with 2.5 ml buffer, Sigma Chemical Co., St.
Louis, MO) was added to another set of cultures at the same time as LPS.
Controls for this received no PGE2 antiserum. Tnese latter experiments
were performed in the conventional MEM containing fetal bovine serum.
Effects of Actinomycin D on Rabbit Pulmonary
Lavage Cell Cytotoxin Production
Actinomycin D was added at 1 yg/ml at the same time and at various
times after addition of LPS to PLC cultures. Cultures were incubated for
30 hours, supernatants harvested and assayed.
Effect of Tunicamycin on Molecular Weight of Rabbit
Pulmonary Lavage Cell Cytotoxin
Tunicamycin (Sigma Chemical Co., St. Louis, MO) was added to test
PLC cultures at 0.5 and 5 ug/ml both 2 hours before and at the same time
as LPS addition. Cultures were incubated for 30 hours at 37°C at which
time, supernatants were harvested and titrated for cytotoxic activities.
Cytotoxin was subjected to gel filtration chromatography on Sephacryl
S-200 (previously described).
Freeze-Thawing of Activated Rabbit Pulmonary Lavage Cells
Pulmonary lavage cells were cultured in the presence of LPS (10
ug/ml) and at various times, replicate cultures were terminated. Fran
some, supernatants were harvested and titered as previously described.

30
Other cultures were subjected to five freeze-thaw cycles in a dry
ice-ethanol bath. After freeze-thawing, the culture contents were
centrifuged and both supernatants and pellets titrated for cytotoxicity.
Susceptibility of Tumor Necrosis Serum Cytotoxin
Resistant L-929 Cells to Rabbit Macrophage
Cytotoxins and Pulmonary Lavage Cells
Matthews (1978) has demonstrated that it is possible to select for
TNSCT-resistant L-929 cells by growing L-929 cells in the presence of
small amounts of TNSCT. Using a lot of TNS which had an S^q titer
(See Cytotoxicity Assays, Photometric Assay) in the actinomycin D
cytotoxicity photometric assay of approximately 8000 units, L-929
resistant cells were selected for by growing cells in the presence of a
1:500 dilution of this CT. After about 2 weeks, during which time dead
cells were decanted and viable cells were refed twice a week with fresh
medium containing TNS, resistant cells were trypsinized, washed and plated
into microtiter wells (10,000/well). Conventional L-929 cells were plated
into another set of wells. After allowing cells to adhere overnight in
conventional medium, TNSCT, PLCCT, BMCCT, or PLC (100 or 1000 cells) were
added to test wells (control wells received only medium). In those wells
containing PLC, LPS (10 yg/ml) was added. After 48 hours, plates were
stained and read as previously described.
Time Lapse Cinematography
L-929 cells were plated into 25 cm^ tissue culture flasks and
allowed to adhere and form a monolayer overnight. The cells were
treated with actinomycin D (2 yg/ml) for 2 to 3 hours, then washed
extensively. Rabbit PLC were added in small numbers (10 x 10^ to 50 x
10^) to the washed monolayers in 3 ml medium at which time, LPS was
added (10 yg/ml). Plates were allowed to equilibrate for an hour in a
lucite incubator chamber mounted on an inverted phase contrast microscope

31
connected to a time lapse motion picture camera. A temperature of 37° to
38°C was maintained within the lucite chamber by a heated air curtain
with a thermostat sensing device. An appropriate field was located
(containing as few macrophages as possible). The field of view was
photographed through a green filter. Photography was generally commenced
4 to 6 hours after addition of effector cells and LPS. Exposure rate for
the first 2 to 3 hours of filming was 1 frame/15 seconds. Generally after
that time, exposure rate was increased to 1 frame/8 seconds. Several
films were made at magnifications of 150 and 200 X. Films were viewed at
24 frames/second.

RESULTS
Percent Macrophages in Rabbit Effector Cell Populations
Table I illustrates the percentage of cells that are macrophages
from three sources, in several rabbits, as judged by morphological and
phagocytic criteria. Pulmonary lavage cells represented a population
made up almost exclusively of macrophages (90-95%). Blood mononuclear
cells were 12-20% macrophages while bone marrow cells contained no more
than 0.1-0.5% macrophages.
Increasing the Macrophage Content of Bone
Marrow Cell Cultures
It is obvious from Table I that bone marrow cells provided only
negligible amounts of macrophages. Lotem and Sachs (1979) have demon¬
strated that the phorbol ester, TPA, can cause both mouse and human
myeloid leukemic cells to differentiate into cells with varying degrees
of several macrophage characteristies (adherence to surfaces, Fc and C3
receptors, phagocytic capability, lysozyme activity, and macrophage
morpho logy).
Treatment of rabbit bone marrow cell cultures with TPA (1 yg/ml)
resulted in adherence of many cells to the culture dish by 30 minutes.
By 5 hours, over 90% of the cells were adherent (Table II). In addition,
about one-third of the adherent cells had macrophage morphology and
phagocytized latex beads. Twenty percent stained positive for
peroxidase. (Later studies indicated that 0.1 pg/ml TPA was adequate to
cause similar differentiation changes in rabbit bone marrow cells.)
32

33
TABLE I
RABBIT CELL POPULATIONS UTILIZED AS MACROPHAGE SOURCES
Source
Perce nt
Macrophages3
Pulmonary Lavage Cells
90 - 95
Blood Mononuclear Cells
12 - 20
Bone Marrow Cel Is
<1
aMacrophage morphology (Giemsa's blood stain) and phagocytosis of latex
beads.

34
TABLE II
PERCENT RABBIT BONE MARROW CELLS WITH MACROPHAGE PROPERTIES BEFORE AND
AFTER TPA TREATMENT
Macrophage Property
Before
Percent of Cells
After TPAa
Adherence
<1
ro
cr
Morphology
<1
32c
Phagocytosis (latex beads)
NT*1
35c
Peroxidase positive
NT
20c
a5-28 hours after adding TPA at 1 yg/ml .
^Percent of total bone marrow cells added to culture.
cPercent of total adherent bone marrow cells.
^Not tested.

35
Considering the variable degrees of each property present, this
process can be envisioned as a spectrum of variable differentiation in
the direction of the mature macrophage state.
In Vitro Cytotoxin Production by Rabbit Macrophages
Requirement for Activating Agent
Matthews (1978) has reported on the apparently consistent, spontane¬
ous production of CT by rabbit BLM cultures in the absence of known added
activating agent (e.g., LPS). These studies did not determine the possible
presence of contaminating LPS in the culture medium. This is often a prob¬
lem in studying macrophages and can lead to serious misinterpretation of
experimental results (Fumarola, 1981). Figure 2 demonstrates the results
from experiments employing rabbit PLC and BLM cultures, where no attention
was given to the possibility of contaminating LPS. Depending upon the time
cell supernatants were harvested, the cytotoxin titers in the cultures to
which LPS was added (1 pg/ml) were consistently greater than titers gener¬
ated in cultures to which no LPS was added (3 to 20 times greater for BLM,
10 to 50 times greater for PLC, replicate cultures varied from mean values
by ± 50%).
Using the Limulus amebocyte lysate test (Levin et al., 1970), it was
determined that the culture system used for the experiments demonstrated in
Figure 2 contained LPS (>0.025 ng/ml ). Table III illustrates the various
materials tested. The recycled glassware used to store prepared media
tested positive for LPS. This was probably contaminated with the LPS-
positive bovine serum lot (Gibco) also shown in Table III. Other than the
glassware and Gibco bovine serum, only the trypsin stock concentrate
(Microbiological Associates) and thioglycollate broth base powder (Difco)
tested positive in the Limulus assay.
If no LPS was added to culture systans testing negative for LPS
(<0.025 ng/ml) by the Limulus assay, rabbit PLC generally produced no CT

Figure 2. Rabbit macrophage cytotoxin production with and
without added LPS.
Cytotoxin production by rabbit macrophage cultures in the
presence (1 pg/ml) or absence of added LPS. Culture
supernatants were harvested for assay at times indicated. Blood
monocytes were cultured at 4 x 106/ml , pulmonary lavage cells
at 1 x lO^/rnl.

37
PULMONARY LAVAGE CELLS
LPS ADDED NO LPS ADDED
HOURS

38
TABLE III
ITEMS TESTED WITH LIMULUS AMEBOCYTE LYSATE REAGENT
Test
Item Results3
Bovine calf serum
Gibco; lot No. R290222 +
Sterile Systems, Inc., lot No. 400168 0
Fetal bovine serum
K.C. Biol., lot No. 309447 0
Trypsin
Stock concentrate, Microbiol. Assoc. +
Crystalline, Sigma; bovine, porcine pancreas 0
Eagle's minimum essential medium, base concentrate 0
Glutamine, stock concentrate 0
Gentamicin, stock concentrate 0
HEPES buffer, stock powder 0
Deionized water from ion exchange tap 0
Thioglycollate broth, base powder, Difco +
Reusable autoclaved laboratory glassware^ +
aLimulus lysate test results interpreted as follows: ( + ) = contains at
least 0.025 ng/ml LPS; (0) = contains less than 0.025 ng/ml LPS.
^Sterilized glassware rinsed with pyrogen free water; washings used for
Limulus assay.

39
(Figure 3). However, one out of the ten rabbits sacrificed for these
studies (only four rabbits are shown in Figure 3) had PLC that could
produce detectable levels of CT in LPS-free cultures. Pasturella species
was cultured from lung washings from this rabbit as well as from its PLC
cultures. The presence of a Gram negative infection in the lungs probably
resulted in in vivo activation of PLC. Despite this problem, the great
majority (90%) of the rabbits utilized provided PLC that would not produce
CT unless LPS was added to the culture (Figure 3). As little as 0.1 to 1
ng/ml could induce CT production. As a precaution, PLC ranoved from all
rabbits to be utilized in other experiments (other than to merely generate
CT for physicochemical studies) were tested in LPS-free culture systems for
their ability to produce CT in the absence of added LPS. This insured that
CT was not produced unless LPS was added to the culture systan, allowing
control over PLC activation.
As previously mentioned, many agents such as BCG (Old et al., 1960),
Corynebacteriurn parvum (Hal pern et al., 1974), etc., can cause a general¬
ized macrophage hyperplasia in vivo. Leake and Myrvik (1968) have demon¬
strated this locally in the lungs of rabbits that were injected intraven¬
ously with BCG in mineral oil. They reported up to a 10-fold increase in
the number of PLC obtained from rabbits injected intravenously over 1 week
before sacrifice. They also reported qualitative changes in the PLC (an
increase in cell lysozyme content as well as ultrastructural changes).
Figure 3 shows that PLC from rabbits injected intravenously with complete
Freund's adjuvant 2 to 3 weeks previously, produced no CT in the absence of
added LPS and variable amounts of CT per 1 x 10^ cells cultured in the
presence of different levels of LPS. In addition, the normal rabbit shown
(not administered Freund's adjuvant) produced significantly greater levels
of CT at lower doses of LPS. Other experiments employing single dose
levels of LPS in cultures of PLC demonstrated variable and overlapping

Figure 3. Rabbit macrophage cytotoxin production in the
absence and presence of varying levels of exogenous LPS.
Pulmonary lavage cells frail normal rabbits, or rabbits
primed with Freund's complete adjuvant 2 to 3 weeks previously,
were cultured in the absence or presence of varying levels of
LPS. Supernatants were harvested after 24 hours. Culture
systems contained <0.025 ng/rnl LPS prior to LPS addition.

41
O Normal Rabbit
• Primed Rabbit a
A Primed Rabbit b
B Primed Rabbit c
LPS CONC., ng/ml

42
results for CT production by LPS-activated PLC from normal and primed
rabbits (data not shown). From this, it appears that both primed and
normal rabbit PLC required the presence of LPS to produce CT in vitro. In
addition, CT production by each cell type occurred to a similarly variable
extent. Primed rabbits yielded 4 to 10 times more PLC (up to 200 x 10^
cells) than normal rabbits, 80 to 90% of which had macrophage morphology
(data not shown). In addition, LPS added to PLC cultures from primed or
normal rabbits in amounts exceeding 10 yg/ml produced no more CT than at 10
ug/ml LPS (data not shown).
As with PLC, TPA-pretreated and washed BMC would not produce CT in
LPS-free cultures unless LPS was added. In addition, no CT was produced
from bone marrow cells that were not pretreated with TPA although exposed
to LPS (bone marrow contains <1% macrophages, see Table I). Optimum CT
production resulted when BMC were pretreated with TPA (0.1 and 1 yg/ml
yielded similar results) for a minimum of 7 hours, then washed extensively
(4 to 6 times) prior to activation with LPS (data not shown). This demon¬
strates the prerequisites for production of significant levels of CT by
BMC. These are differentiation of sufficient bone marrow precursor cells
to macrophages and subsequent activation of these macrophages by LPS.
Taking advantage of the strong and efficient adherence of BMC
induced by TPA, BMC were plated at varying densities to determine the most
efficient plating density for CT production (Table IV). Cultures producing
the most CT had cells plated at approximately 0.5 x 10^ and 1.5 x
lO^/cm^. This range of plating densities yielded consistently high CT
titers for PLC as well (data not shown).
It was important that the TPA-pretreated BMC were washed extensively
prior to adding LPS. Initial experiments with BMC were performed by adding
LPS to TPA treated BMC without washing out the TPA. Virtually no CT was

43
TABLE IV
RABBIT BONE MARROW CYTOTOXIN PRODUCTION: OPTIMUM PLATING DENSITY3
Number Cel 1 s
x lO^/cm^
s50Titerb
s50Titerb
per 10 x 10^ cel Is
8.80
2290
104
5.92
2500
169
2.96
2500
338
1.48
2089
564
0.54
933
691
aCells were cultured in MEM with 10% fetal bovine serum and 1 yg/ml TPA
for 12 hours. TPA was removed by washing cultures extensively and LPS
(1 ug/ml) was added.
^30 hour titers.

44
produced by these cultures. In order to determine if TPA could have an
inhibitory effect on the CT, TPA was added to a TNSCT assay (Table V). A
linear relationship was found between the logarithm of the amount of TPA
added to the assay and the decrease in the titer of TNSCT. Pretreating the
target cells (L-929) used in the assay with 100 ng TPA for 18 hours and
then washing out the TPA prior to adding TNSCT to the cultures resulted in
less inhibition of TNSCT titer (21% instead of 36% where only 2.4 ng TPA
was left in the assay with TNSCT). These results demonstrate that TPA
exerts an inhibitory effect on CT activity in general.
Comparison of Cytotoxin Production Capabilities
by Rabbit Macrophages Derived from the
Different Sources
Rabbit PLC, BMC and BLM were cultured under conditions that optim¬
ized CT production for each of the cell types. BMC cultured in a fortified
medium (see Materials and Methods) generated about 4 times more CT than if
cultured in the standard medium used for PLC and BLM. This was done to
determine which of the cell types had the greatest potential for generating
highest CT titers. Based upon 1 x 10^ cells cultured per ml of medium,
PLC were most efficient under the tested conditions (Table VI, 20 to 140
times more CT than BMC or BLM produced). Also as previously seen regarding
sensitivity to LPS, PLC demonstrated much variability (about 7-fold) when
comparing different rabbits with each other. BMC demonstrated much less
variability (about 2-fold).
The overall cell populations used varied greatly in their macrophage
content (Tables I and II). In addition, the BMC demonstrated a broad
range of cells with macrophage characteristics depending upon which prop¬
erty was being utilized to call a cell a macrophage. It was therefore
necessary to normalize the different cell populations for their macro¬
phage content to compare the ability of macrophages from these

45
TABLE V
EFFECT OF TPA ON TUMOR
NECROSIS SERUM (TNS)
TITER
Experiment
TPA(ng)
Percent Inhibition
TNSCT Activity
TNS titrated in presence of TPAa
2.4
36
9.75
48
78
70
TNS titrated on TPA-pretreated and
100
21
washed targets'5
aTPA was added to the standard actinanycin D serum cytotoxin photometric
assay (see Materials and Methods).
^L-929 cells were preincubated with TPA for 18 hours, then washed. Serum
cytotoxin photometric assay was then performed.

46
TABLE VI
CYTOTOXIN PRODUCTION BY RABBIT MACROPHAGES FROM BLOOD MONONUCLEAR, TPA-
PRETREATED BONE MARROW AND PULMONARY LAVAGE CELLS: COMPARISON OF
TITERS PRODUCED PER 1 x 106 CELLS
Cel 1 Type3
Rabbi t
S50Titer
per 1 x 106 Cel Is
Pulmonary lavage*3
1
14,000
2
4,562 ± 526
3
2,200 ± 100
TPA-pretreated bone marrowc
4
70 ± 4d
5
77 ± 6d
6
119 ± 0d
Blood monocytes*3
1
79
aAl1 cells derived from normal rabbits and cultured at 1 x 10^/ml in
medium containing 10% fetal bovine serum, LPS at 10 yg/ml and 3 x 10“^M
HEPES buffer. Plating densities, 0.5-1.5 x 10°/cm .
^Cultured in Eagle's minimum essential medium.
cCultured in Eagle's minimum essential medium fortified with 2 X vita¬
mins and essential amino acids, nonessential amino acids and sodium
pyruvate.
^Corrected for loss of bone marrow cells that do not adhere following
TPA-pretreatment (<10%).

47
different sources to generate CT. Considering the potential ranges of
percent macrophages in each population as well as the extremes of CT
titer produced by each of the cell types, the PLC were still most
efficient at generating CT (see Table VII) on a basis of 1 x 10^
macrophages/ml culture medium (4 to 250 times greater than BMC and 4 to
40 times greater than BLM).
Effects of Bovine and Fetal Bovine Serum on
Rabbit Macrophage Cytotoxin Production
The cytotoxic capabilities of macrophages can be enhanced or
inhibited by various serum components (Hibbs et al., 1977; Chapman and
Hibbs, 1977). Mechanisms which could help explain this phenomenon may
involve production or function of macrophage CT. PLC were cultured in
the absence or presence of varying levels of bovine or fetal bovine serum
(Figure 4). Under the conditions tested, it can be seen that PLC could
generate CT in serum-free medium (PLC from two different rabbits generated
330 to 400 S5Q CT units under the culture conditions described for
Figure 4, panel a). Bovine serum was inhibitory to CT production at levels
generally greater than 4%. Fetal bovine serum enhanced CT production by
PLC up to 3-fold (at 10%). Figure 5 further illustrates that 10% fetal
bovine serum was more effective than lower levels (1%) at potentiating
LPS-induced PLCCT production. This was the case at 3 levels of LPS
concentration. It can also be concluded that to the extent to which they
were tested, LPS at 10 pg/ml with 10% fetal bovine serum were optimal
together for potentiating the activation of PLC by LPS.
Time Kinetics of Macrophage
Cytotoxin Production
Figure 6 demonstrates in vitro production of CT by LPS-activated
PLC with respect to time. Generally, significant levels of CT appeared

48
TABLE VII
DATA FROM TABLE VI CONSIDERING PERCENT MACROPHAGE
CONTENT OF EACH CELL TYPE
Perce nt
Theoretical Range of
Macrophages
Suiter per
Cell Type
Possible Range
1 x 10® Macrophages
Pulmonary lavage
90 - 95
14,737 - 15,556
2,316 - 2,444
TPA-pretreated bone marrow
20 - 100a
70 - 350
119 - 595
Blood monocytes
12 - 20
395 - 658
Considering all macrophage properties of TPA-pretreated and washed
adherent bone marrow cells (e.g. peroxidase staining positive cells
represent 20% of total adherent cell population. See Table I).

Figure 4. Effects of bovine and fetal bovine serum on
pulmonary lavage cell cytotoxin production.
Rabbit pulmonary lavage cells were cultured for 24 hours
in the absence or presence of various levels of bovine or fetal
bovine serum. Experiments employing cells from 2 rabbits are
shown: a. LPS present at 1 pg/ml ; b. LPS present at 10 pg/ml.
Control cultures contained no serum.

50

Figure 5. Comparison of effects of 1% and 10% fetal
bovine serum on cytotoxin production by rabbit pulmonary lavage
cell cultures containing various levels of LPS.
Supernatants were harvested from triplicate cultures that
were incubated for 30 hours.

L P S , fJLQ/ m I
CYTOTOXIN S
c_n
i
50 TITER x ICf3
OJ ■£>
i i
u~l
ro
Fetal Bovine Serum:

Figure 6. Time kinetics of rabbit pulmonary lavage cell
cytotoxin production.
Pulmonary lavage cells from a normal rabbit or rabbits
primed with Freund's complete adjuvant 2 to 3 weeks previously
were cultured in the presence of LPS. Supernatants were
harvested at times indicated.

54
â–¡ PRIMED RABBIT * I
O PRIMED RABBIT #2
a NORMAL RABBIT
HOURS

55
by 3 hours and titers increased significantly between 3 and 5 hours.
Levels remained fairly constant at times tested between 5 to 7 and 18
hours and then increased significantly again after about 18 hours in
culture. Variation of replicate cultures was mean ±20% for the Freund's
adjuvant primed rabbits and mean ±50% for the normal rabbit. Figure 6
also demonstrates the variation among rabbits (normal and primed) in the
CT titers generated by their PLC at any single given time. Other experi¬
ments involving culturing PLC from several normal and primed rabbits under
identical conditions for the same time resulted in overlapping and simi¬
larly variable CT titers for both groups (data not shown).
Time kinetics of CT production by TPA-pretreated and washed, LPS-
activated BMC are given in Figure 7. Detectable levels of CT (20 to 26
S50 units) appeared by 3 hours and increased through 14 hours. Levels
measured at 14 and 30 hours were similar.
Time kinetics of CT production by LPS-activated BLM are included in
Figure 2. Detectable levels of CT were present by 3 hours, increased
between 3 and 7 hours and were similar at 7 and 24 hours. These observa¬
tions are consistent with the data of Matthews (1978). From these observa¬
tions, it is apparent that time kinetics of CT production by PLC, BMC and
BLM are somewhat different after an initial production time during the
first 3 hours. After 30 hours culture, essentially no CT was produced by
PLC and BMC that were refed with fresh LPS-containing medium (data not
shown).
Effects of Actinomycin D on Cytotoxin
Production by Rabbit Pulmonary Lavage Cells
Cameron (1980) has described an actinomycin D-induced inhibition of
cytotoxicity of human macrophages for tumor cells. The data presented

Figure 7. Time kinetics of cytotoxin production by
TPA-pretreated and washed rabuit bone marrow cells.
Rabbit bone marrow cells were plated into 24-well tissue
culture dishes and treated with TPA (0.1 u9/ml) overnight.
After removing TPA from cultures (washed 5 times), fresh medium
containing LPS (10 yg/ml) was added. Supernatants were
harvested at various times.

120
100
80
60
40
20
57
• Rabbi t A
ARabbit B
5 ¡0 15 20 25 30
HOURS

58
in Figure 8 show that actinomycin D (1 yg/ml) consistently inhibited
PLCCT production if added to cultures at the same time as LPS.
(Inhibition of CT titers of test cultures was greater than 95% below
those of control cultures to which no actinomycin D was added.) Delaying
addition of actinanycin D resulted in a decrease of inhibition of CT
titers. This varied considerably among rabbits, especially when compar¬
ing primed to normal rabbits. Fran the data of Figure 8, it is apparent
that in no case was there active CT messenger RNA or active CT in PLC
prior to the addition of LPS to the cultures. In addition, active CT
messenger RNA was fully synthesized by 2 to 6 hours after LPS activation
of primed PLC and about 3 hours after LPS-activation of normal PLC.
Effects of Prostaglandin E (PGE) and Prostaglandin
Antiserum on Cytotoxin Production by Rabbit
Pulmonary Lavage Cells
It has been demonstrated that PGE can be produced by macrophages in
culture and can shut off macrophage cytotoxicity for tumor cells
apparently after a period of macrophage activation (Taffet and Russell,
1981). Figure 9 demonstrates that PGE (10“® M) added to PLC cultures
6 hours after LPS (lOyg/ml) resulted in inhibition of CT production down
to about 30% of control culture levels. In addition, adding PGE anti¬
serum to cultures at the same time as LPS increased CT production by 50%
over the control levels. These data are consistent with the conclusions
of Taffet and Russell (1981) concerning regulation of macrophage cytotox¬
icity by endogenously produced PGE.
Disruption of Rabbit Pulmonary Lavage Cells at Various
Times after LPS Activation: Failure to Demonstrate
Significant Active Intracellular Cytotoxin Pool
Unanue and Kiely (1977) have demonstrated that a macrophage
protein, mitogenic for thymocytes, accumulates within the macrophage in

Figure 8. Effects of actinomycin D on cytotoxin
production by rabbit pulmonary lavage cells.
Actinomycin D (lug/ml) was added to cultures of rabbit
pulmonary lavage cells at the same time and various times after
addition of LPS. Culture supernatants were harvested for ass^y
after 30 hours. Panel a represents 2 primed rabbits, panel b, 2
normal rabbits.

200
150
100
50
150
100
50
60
â–¡ Primed Robbit I
APnmed Robbit 2
â–  Robbit I
A Robbit 2
JRS
I 2 3
ACTINOMYCIN D
4 5 6
ADDED AFTER LPS

Figure 9. Effects of prostaglandin E2 and prostaglandin
antiserum on cytotoxin production by rabbit pulmonary lavage
cel Is.
Rabbit pulmonary lavage cells were cultured in the
presence of LPS at 10yg/ml. To 1 set of cultures in serum-free
FMEM, prostaglandin E2 was added at 10"° M. To another
set of cultures in standard MEM supplemented with 10% fetal
bovine serum, prostaglandin antiserum was added (see Materials
and Methods). Cultures were incubated for 28 hours and
supernatants harvested.

% CONTROL CYTOTOXIN S50 TITER
62

63
the form of an intracellular pool. Only a small portion of this pool may
be secreted by the macrophage depending upon its state of activation. By
repeated freeze-thawing of thioglycollate-stimulated mouse PEC, they
could demonstrate higher levels of mitogenic protein within the cell than
were secreted under certain conditions.
Rabbit PLC were activated by LPS and at various times after LPS
addition (30 minutes, 1, 3, 7 and 24 hours), culture supernatants and
freeze-thawed lysates were analyzed for CT. As previously, supernatant
CT became measurable at 3 hours but at no time, except one (1 hour after
addition of LPS) did freeze-thawed lysate contain measurable CT (data not
shown). At 1 hour after LPS addition to cultures, only about 2 S5Q
units of CT appeared in freeze-thawed lysates while none appeared in the
culture supernatant. Appropriate controls indicated total stability of
PLCCT to repeated freeze-thawing in this culture system. In addition,
assaying the freeze-thawed PLC (none viable by inability to exclude
trypan blue dye) on L-929 cells in the presence of actinomycin D revealed
no cytotoxicity. These studies indicate that no significant levels of
active freeze-thaw-releasable CT were retained by the activated
macrophage.
Physicochemical and Biological Properties of Rabbit
Macrophage Cytotoxins and Tumor Necrosis
Serum Cytotoxin
Gel Filtration Chromatography
All 4 of the cytotoxins were eluted through the same Sephacryl
S-200 column. All eluted as single peaks. The elution volumes for all
corresponded to a molecular weight of about 48,000 D (Figure 10).

Figure 10. Gel filtration of rabbit macrophage cytotoxins
on Sephacryl S-200.
Macrophage culture supernatants were concentrated by
pressure dialysis prior to gel filtration. The column was
calibrated with standards of known molecular weight,
ribonuclease (not shown), chymotrypsin (C), ovalbumin (0V),
bovine serum albumin (BSA).

CYTOTOXIN S Sn TITERS
65
• TNS
OPLC
ABMC
A BLM
Cytotoxic
Activities
ELUTION VOLUME (ml)

66
Ion Exchange Chromatography
The 4 cytotoxins were eluted through freshly poured DEAE-Sephadex
columns with gel beds of approximately similar size. All eluted with the
same linear salt gradient between 0.28 and 0.32 M NaCl (Figure 11).
Effect of Tunicamycin on the Molecular Weight
of Cytotoxin Generated by Rabbit Pulmonary
Lavage Cells
Previous studies failed to demonstrate significant levels of
exposed carbohydrate on rabbit TNSCT (Matthews et al., 1980; Ruff and
Gifford, 1981). These studies drew their conclusions from the inability
to bind TNSCT to a variety of sugars and plant lectins.
Tunicamycin has been shown to inhibit N-glycosylation of
glycoproteins by blocking formation of N-acetylglucosamine-1ipid
intermediates (Hickman et al., 1977). This has been demonstrated for
several types of glycoprotein including interferon (Fujisawa et al.,
1978) and immunoglobulin (Hickman et al., 1977). Rabbit PLC were
cultured with LPS both in the presence and absence of tunicamycin.
Tunicamycin at 1 yg/ml had no effect on production of CT, but at 5 u9/ml ,
CT titers were decreased by 30% when compared to control cultures which
contained no tunicamycin. As high as 5 yg/ml tunicamycin had no effect
on the molecular weight of CT produced as determined by gel filtration
chromatography on Sephacryl S-200 (data not shown). This demonstrates
that CT has no covalently linked carbohydrate or has a covalently linked
carbohydrate of very small molecular weight or could have a carbohydrate
portion of substantial size that is covalently linked to the rest of the
molecule by a tunicamycin resistant mechanism.

Figure 11. Ion exchange chromatography of rabbit
macrophage cytotoxins on DEAE-Sephadex.
Rabbit TNS or pooled cytotoxin containing fractions from
the Sephacryl S-200 column were concentrated by pressure
dialysis and loaded in starting buffer. Unbound proteins were
washed off the column, followed by elution with a linear salt
gradient from 150 to 600 mM phosphate buffered saline.

CYTOTOXIN S50 TITER
68
• TNS
° PLC CYTOTOXIC
FRACTION NUMBER
No Cl CONC. (mM)

69
Temperature Stability of Rabbit
Macrophage Cytotoxins
Each of the cytotoxins were subjected to 56°C and 70°C in separate
experiments (Figure 12). All four of the cytotoxins were stable to 56°C
for 60 minutes. In addition, the titers of all were greater than the
controls, which were maintained at 4°C during the course of the
experiment. This may suggest a heat labile inhibitor of CT present. At
70°C, all cytotoxins demonstrated decay kinetics that were fairly similar
and by 60 minutes, all lost about 80% of activity compared to controls.
Effect of Serine Protease Inhibitors and
o-Phenanthrolene on Cytotoxin Activities
There is evidence in the murine system that serine proteases may
play a role in macrophage mediated tumor cytolysis (Adams, 1980; Adams et
al., 1980). Rabbit PLCCT was not inactivated by phenylmethyl sulfonyl
fluoride (10~^M), an inhibitor of serine proteases (data not shown).
In addition, neither bovine pancreatic trypsin inhibitor nor soybean
trypsin inhibitor inactivated PLCCT or BMCCT activity (data now shown).
None of these enzyme inhibitors inactivated TNSCT either, consistent with
the findings previously reported (Ruff and Gifford, 1981). On the other
hand as this latter group has reported for TNSCT, o-phenanthrolene, a
metal chelator had significant inhibitory effects on the macrophage CT
(Table VIII).
Effect of Chloroquine on Rabbit
Macrophage Cytotoxin Activities
Chloroquine is a drug which can apparently stabilize cell lysozomes
(Lie and Schofield, 1973). Previous work with TNSCT (Ruff and Gifford,
1981) has demonstrated an inhibitory effect of this drug on cytotoxin
activity in the actinomycin D assay. All 4 cytotoxins were tested in the
standard actinomycin D assay with chloroquine (10~4M) and all were
inhibited (Table VIII).

Figure 12. Thermal stability of rabbit TNS cytotoxin and macrophage
cytotoxins.
Rabbit macrophage culture supernatants and TNS diluted with culture
medium were exposed to 56°C for 1 hour or 70°C for 20, 40 or 60 minutes.
Control TNS and culture supernatants were maintained at 4°C for 1 hour.

160
^ â–¡
CYTOTOX I IMS:
TNS
BMC
PLC
BLM

TABLE VIII
72
EFFECTS OF CHLOROQUINE AND o-PHENANTHROLENE ON CYTOTOXIN ACTIVITY
Drug
PLCa
Percent of
BMCb
Control S5QI
BLMC
iter
TNSd
Chloroquine, 10"^M
55
38
37
31
o-Phenanthrolene, 10“^M
46
48
35
50
Cytotoxins from:
aPLC, pulmonary lavage cells
bBMC, TPA-pretreated bone marrow cells
CBLM, blood monocytes
dTNS, tumor necrosis serum

73
Effects of High Arginine Levels on Rabbit
Macrophage Cytotoxin Activities
Many reports indicate that arginase plays some role in macrophage
mediated tumor cytotoxicity (Currie and Basham, 1975, 1978; Currie, 1978;
Chen and Broome, 1980). These same reports indicate inhibition of
macrophage merdiated tumor cytotoxicity by adding excess arginine to the
medium. Arginine added to our cytotoxin assay at 2 x 10"^M (5 to 10
times the level employed by these latter investigators) did not inhibit
cytotoxicity of any of the CT (data not shown). From this, we conclude
that none of our cytotoxins is arginase.
Relative Titers of Rabbit Macrophage Cytotoxins
in the Presence and Absence of Actinomycin D
in the Cytotoxicity Assay
0strove and Gifford (1979) have reported an enhanced sensitivity of
L-929 cells to TNSCT in the presence of actinomycin D. Similar results
were demonstrated with PLCCT and BMCCT. Target cells demonstrated up to
250 times more sensitivity to CT in the presence of actinomycin D than if
actinomycin D was not employed (data not shown).
Target Cell Susceptibility Profiles of Rabbit
Macrophage Cytotoxins and Tumor Necrosis
Serum Cytotoxin
Table IX compares PLCCT and BMCCT with TNSCT target cell
susceptibility. Both macrophage derived cytotoxins are not toxic for
mouse embryo fibroblasts or BI6C3 melanoma, two types of TNSCT-
resistant cell, while L-929 cells, a TNSCT-sensitive cell, is also
sensitive to macrophage CT. Chromium^ release studies demonstrated
L-92 9 sensitivity to PLCCT and BMCCT, while BI6C3 cells were resistant.
L929 cells released up to 18% of their label by 8 hours (taking into
consideration spontaneous release of label by target cells) while BI6C3
cells released none of their label by 21 hours (data not shown).

74
TABLE IX
CELL SUSCEPTIBILITY PROFILES OF RABBIT CYTOTOXINS3
Target Cel 1
Cytotoxin
Source
S50Titerc
L - 92 9
TNSb
>128
PLCb
>128
BMCb
>128
Mouse embryo fibroblasts
TNS
<2
PLC
<2
BMC
<2
B16C3
TNS
<2
PLC
<2
BMC
<2
aPhotometric assay without added actinomycin D was used to titer the
same aliquot of each cytotoxin on the cells indicated.
bTNS, tumor necrosis serum; PLC, pulmonary lavage cells; BMC, bone
marrow cells (TPA pretreated).
cForty-eight hour titers.

75
Table X summarizes biological and physicochemical characteristics
of rabbit macrophage cytotoxins and TNSCT.
Cocultivation of Mouse Peritoneal Exudate Cells or
Rabbit Pulmonary Lavage Cells with Actinomycin D
Pretreated L-929 Targets
Target cells can be rendered increasingly sensitive to TNSCT (a
putative macrophage product) by actinomycin D (Ostrove and Gifford, 1979;
Ruff and Gifford, 1981). In addition, Kunkel and Welsh (1981) have
reported that pretreatment of L-929 cells with actinomycin D followed by
washing rendered them increasingly sensitive to lysis by natural killer
cells. Considering these observations, it was of interest to determine
what effect actinomycin D pretreatment of L-929 cells followed by washing
would have on macrophage mediated killing of L-929 targets. Table XI
illustrates increased sensitivity of L-929 cells to mouse PEC cytotox¬
icity using a ^Cr release assay. By 6 hours, nearly 40% of the
^Cr was released by actinomycin D pretreated targets cocultured with
LPS activated PEC, while by 12 hours, those targets not pretreated with
actinomycin D released less than 10% of their label in the presence of
effector cells. In this experiment, the effector:target cell ratio was
between 1:1 and 10:1 (see Materials and Methods).
Demonstration of Cytotoxicity of Actinomycin D
Pretreated L-929 Cells with Effector:Target Ratios <1
Actinomycin D pretreatment and washing of monolayers was followed
by addition of small numbers of mouse PEC or rabbit PLC activated by the
addition of LPS. Significant levels of killing could be detected by 18
hours using the photometric assay (see Figure 13). If actinomycin D was
omitted, no killing was detected at 18 hours. It was possible to
determine the number of effector cells required to kill 50% of the

TABLE X
SUMMARY: COMPARISON OF PHYSICOCHEMICAL AND BIOLOGICAL PROPERTIES OF RABBIT TUMOR NECROSIS
SERUM CYTOTOXIN (TNSCT) WITH RABBIT MACROPHAGE CYTOTOXINS
Property
TNSCT
Cytotoxin (CT)
PLCa BMCb
BLMC
Molecular weight, gel filtration
48 K
48 K
48 K
48K
Elution from DEAE Sephadex
0.30-0.32M
0.28-0.3 OM
0.30-0.31M
0.32M
Glycoprotein
Nob»e
No^ ?
ND9
ND
Temperature stability: 56°C, 60 min.
All stable
70°C (after 20 minutes)
All unstable
Trypsin inhibitors (soybean and bovine pancreatic)
NIh
NI
NI
ND
Phenylmethyl sul fonylfl uoride
NIe
NI
NO
ND
o-Phenanthrolene
All inhibited
Chloroquine
All inhibited
Arginine, 2 x 10"^M
No Effect
No Effect
No Effect
ND
Cytotoxicity Assay with actinornycin D
All activities enhanced
Target cell cytotoxicity: L-929
Susceptible
to al 1
B16C3
Resistant to all
Mouse embryo fibroblasts
Resistant tc
) all
aPulmonary lavage cells eRuff and Gifford, 1981
bTPA-pretreated bone marrow cells ffunicamycin did not decrease molecular
cBlood monocytes weight of CT produced by PLC
•^Matthews et al. , 1980 9Not determined
bNot inhibited

77
TABLE XI
5lCr RELEASE BY L-929 CELLS COCULTURED WITH MOUSE PERITONEAL
EXUDATE CELLS
Time (hours)
Percent
Actinomycin D
Pretreatment
^Cr Released3»^
No Actinomycin D
Pretreatment
3
1.3 ± 0
0
6
38.3 ± 1.8
1.9 ± 0
12c
46.8 ± 1.3
9.4 ± 0
aPercent specific
^Cr released into supernatants = E - S
T - S
(100)
where E = experimental release, S = spontaneous release and T = total
releasable counts.
S = amount of ^Cr released by target cells cultured alone.
T = total releasable counts by lysis of 3 x 10^ ^Cr labelled
L-929 cells with 1% dodecyl sodium sulfate.
^Samples run in triplicate for determination of experimental and
spontaneous counts released. Samples run in quadruplicate for total
releasable counts.
cMaximum spontaneous counts released = 22% of total releasable counts
by 12 hours.

Figure 13. C^H/HeN mouse peritoneal exude cells on actinomycin D
pretreated L-929 cells: ninety-six well tray.
L-929 monolayers were pretreated with actincmycin D for 2 to 3 hours
and then washed. Mouse peritoneal exudate cells were plated into the wells
in 2-fold increments (numbers below the well columns indicate number of
peritoneal exudate cells plated in 0.2 ml). LPS was added at 10 pg/ml.
Eighteen hours later the plate was stained. Two sets of peritoneal exudate
cells were run in triplicate.

i«r-f
T
t—
’»>• — ^ ~j ’
» -
0 10 20 40 80 160 320 640 1280 2560
C3H peritoneal exudate cells on actinomycin d
PRETREATED L 929 CELLS
i i
II H
/I 'i I
id
—i

80
targets. Figure 14 demonstrates this point. Approximately 17 5 rabbit
PLC and 300 mouse PEC could kill 50% of the actinomycin D pretreated
monolayers. If we take into account the fact that 95% of the rabbit PLC
and 70% of the mouse (C3H/HeN) PEC were macrophages, we can then
determine the average number of targets killed by a single macrophage.
Knowing that approximately 50,000 L-929 cells composed a monolayer, it is
determined that a single rabbit macrophage killed an average of 25,000f
(175x0.95) or 150 targets and a single mouse macrophage killed an average
of 25,0004(300x0.70) or 119 targets. These are only average determin¬
ations, since, as will be shown, not every macrophage necessarily killed
targets, nor were the number of targets killed by a single macrophage the
same.
Controls for these experiments previously determined that the
manual photocell employed for these studies could at no time detect the
small number of effector cells plated. In addition, removal of adherent
cell populations from mouse PEC virtually eliminated their ability to
generate killing. We therefore concluded that the macrophage was the
effector cell in the PEC population.
Demonstration of Single Effector Cell
Cytotoxicity: Plaque Assay
Repeating the same experiment on a larger monolayer surface (24
well plates) resulted in a greater dispersal of the effector cells.
Here, killing was detectable by the formation of plaques (see Figures 15
and 16). If the number of effector cells plated was compared with the
number of plaques that resulted, a linear relationship between the two
was obtained (Figure 17). This dictates that a single effector cell was
capable of forming a single plaque, though not every effector cell

Figure 14. Effector cell cytotoxicity for act i ncmyci n D
pretreated L-929 targets: Determination of Sgg endpoint.
L-929 monolayers were pretreated with actinomycin D
(2ug/ml ) for 2 to 3 hours. After washing monolayers, effector
cells (a, mouse peritoneal exudate cells; b, rabbit pulmonary
lavage cells) were added in 2-fold increments. Control target
monolayers received no effector cells. LPS was added at 10
pg/ml. Target monolayers were stained after 18 hours.

% TARGET CYTOLYSIS
82
NO RABBIT PULMONARY LAVAGE CELLS PLATED

Figure 15. C^H/HeN mouse peritoneal exudate cells on actinomycin D
pretreated L-929 cells: Twenty-four well tray.
Procedure was the same as for peritoneal exudate cells on L-929
targets, 96 well tray (Figure 13). Numbers below the well columns depict
number of peritoneal exudate cells plated in 0.2 ml. Each number of
peritoneal exudate cells run in quadruplicate.

CELLS
40 80 160
ON ACTINOMYCIN D PRETREATED L 929
00

Figure 16. Rabbit pulmonary lavage cells on actincmycin L) pretreated
L-929 cells: Twenty-four well tray.
Procedure as for mouse peritoneal exudate cells (Figures 13 and 15).


Figure 17. Linearity between number of plaques counted
vs. number of mouse peritoneal exudate cells plated.
Each point represents the mean of 4 replicates ± 1
standard deviation. Panel a, represents peritoneal exudate
cells from C57B1/6 mouse; panel b, represents peritoneal exudate
cells from C3H/HeN mouse. Botn lines represent the best fit
by linear regression and nave correlation coefficients of
>0.990.

100
80
60
40
20
100
80
60
40
20
88
NUMBER OF PERITONEAL EXUDATE CELLS PLATED

89
necessarily formed a plaque. Therefore, one plaque represented the
killing events associated with a single macrophage.
For convenience, this assay was developed in mice and then utilized
in the rabbit system. One of the most noticeable observations about the
rabbit system concerns its variability (sensitivity of PLC to LPS when
comparing different rabbits; see Figures 3 and 6). This plaque assay was
performed using PLC from several rabbits. In only about 20% of the
rabbits used did the number of plaques generated conform closely with the
number of PLC plated. This probably demonstrated variation among rabbits
concerning the number of macrophages in the overall PLC population that
could be sufficiently activated to make a plaque. In addition, as in the
mouse system, plaques varied in size, demonstrating variation in the
ability of each macrophage to kill targets.
Other observations made in the rabbit plaque system included the
inability of 2 x 10"^M arginine in the medium to inhibit PLC plaque
formation and the requirement for the addition of LPS to get plaques.
L-929 cells for these latter studies were maintained in a culture system
which tested negative (<0.025 ng/ml) for LPS (see Table III). Finally,
if actinomycin D was added at the same time as PLC and LPS to actinomycin
D pretreated and washed L-929 monolayers, no plaques were produced.
However, if actinomycin D was added to the system 2 hours or more after
the PLC and LPS were added to actinomycin D pretreated and washed
monolayers, the same number of plaques were formed when compared to
controls to which no actinomycin D was added either with or following PLC
(data not shown).
Time Lapse Cinematography of Small Numbers of
Rabbit PLC on Actinomycin D Pretreated L-929 Cells
Using the conditions described in Materials and Methods for
cocultivation of effector cells on actinomycin D pretreated targets, time

90
lapse cinematography of small numbers of rabbit PLC killing L-929 cells
demonstrated three main points. First, the PLC did not undergo much
translational motion. They grazed locally, touching no more than 4 to 6
target cells on the average. Secondly, target cells closest to each PLC
died first (about 6 to 8 hours after PLC and LPS were added to
actinomycin D pretreated and washed targets). Finally, many target cells
could be seen dying that were never touched by a PLC. All of this
implies that single effector cells secreted sufficient CT to kill target
cells and that a visible plaque represented for the most part, secretion
of CT by individual PLC.
Cocultivation of Effector Cells with L-929 Targets
not Pretreated with Actinomycin D
Hibbs (1976) has demonstrated that macrophage mediated target
killing can be seen in 48 to 60 hours in a visual assay similar to our
photometric assay minus the actinomycin D pretreatment. However, he
employed high effector:target ratios (10 to 100:1).
Attempts to demonstrate target killing by low numbers of rabbit PLC
or mouse PEC without pretreating the L-929 cells with actinomycin D have
been unrewarding. At least 2000 to 5000 effector cells were necessary to
produce toxicity for L-929 cells in the 96 well plate assay and in
addition, the results were highly variable and difficult to quantitate.
Furthermore, all attempts to generate plaques over a 48 to 60 hour period
in 24-well plate assays (without actinomycin D pretreatment) have met
with great technical problems. The few times that monolayers could be

91
generated that would survive under these conditions, no plaques were
seen.
Susceptibility of TNSCT-Resistant L-929 Cells to
Rabbit Macrophage Cytotoxins and Pulmonary
Lavage Macrophages
L-929 cells selected for their resistance to TNSCT by being grown
in the presence of the CT displayed significantly greater resistance to
PLCCT, BMCCT and PLC cytotoxicity (Table XII). Most intriguing is the
observation that L-929 cells resistant to the cytotoxins also resisted
killing by macrophages. Similar results were also obtained with BI6C3
cells (data not shown). The implications of this further underscore the
possible significance of the CT in macrophage mediated tumor killing.

92
TABLE XII
EFFECTS OF TNSCT,a pLCCT,b BMCCTc AND PLCd ON TNSCT-
RESISTANTe AND CONVENTIONAL L-929 CELLS
Dilution or
Cytotoxin Effector Cell Percent Survival L-929'
or Cell Number Resistant Conventional
TNSCT
1:20
25
+
8
7
±
2
1:200
75
±
5
48
±
10
PLCCT
1:20
72
±
3
51
±
7
1:200
89
±
4
76
±
4
1:2,000
100
±
3
91
±
13
BMCCT
1:20
66
±
3
51
1:200
99
±
2
83
±
12
PLC
1,000
97
±
3
46
±
8
100
100
±
10
83
±
3
aTumor necrosis serum cytotoxin.
^Pulmonary lavage cell cytotoxin.
cTPA-pretreated bone marrow cell cytotoxin.
^Pulmonary lavage cells.
eGrown in the presence of TNSCT, 1:500 dilution for 10 days.
^Forty-eight hour survival rate compared to control which received no
cytotoxin or PLC; TNSCT-resistant and conventional L-929 cells each
had a set of controls.

DISCUSSION
These studies have demonstrated three major points to be discussed:
1. Macrophages derived from different tissue sources representing
different degrees of maturity all produced a CT in vitro with similar
biological and physicochemical characteristics to one another, as well as
to TNSCT.
2. CT production was dependent upon the presence of an activating
agent (LPS) and was similarly affected by culture conditions that could
alter classical macrophage cytotoxicity (effects of serum, actincmycin D,
prostaglandin). This demonstrated that classical macrophage cytotoxicity
(utilizing the effector cell) correlated with soluble CT production. CT
production may reflect an index of overall macrophage cytotoxicity.
3. Actinomycin D pretreatment of target L-929 cells greatly
enhanced their sensitivity to all macrophage CT tested as well as to the
macrophage. Taking advantage of this, CT production by individual
macrophages could be demonstrated by formation of plaques on target L-929
cel 1 s.
The aforementioned first point clearly demonstrates that activated
macrophages can secrete soluble CT in vitro. This is something which many
investigators have been unable to demonstrate (Hibbs, 1976). There could
be several reasons for failing to detect soluble CT liberated by an
effector cell. The most obvious reasons include using an assay of
93

94
insufficient sensitivity, harvesting and assaying culture super natants
long after labile CT activity is gone and employing an animal species whose
macrophages may not secrete CT to the extent of another. In the system
used in these studies, rabbit macrophages could consistently produce a
stable CT that was detectable in a type of assay previously shown to
enhance the sensitivity of TNSCT (Ruff and Gifford, 1931) and lymphotoxin
(Eifel et al., 1975). The variable nature of the system (outbred animals
and using PLC, cells that are in contact with the environment to some
extent) affected only the sensitivity of effector cells to activating agent
and resulted in CT production of varying titers. Nevertheless, CT
production occurred each time, though to a variable extent.
To the extent examined, it appeared that CT produced by rabbit macro¬
phages from differing sources (representing different degrees of differ¬
entiation) were similar to one another as well as to TNSCT. Although the
data presented did not prove identity of all these CT, they strongly
suggest that this could be the case. If not identical in every respect,
these macrophage CT could represent a family of related molecules demon¬
strating a degree of heterogeneity. There is precedent for this in the
lymphotoxin system (Hiserodt et al., 1976; Hiserodt and Granger, 1977).
On the other hand, concerning the mouse system, there have been some
reports of macrophages producing CT with properties distinct from TNSCT
(MacFarlan and White, 1980). This indicates that the macrophage m^y be
capable of producing groups of CT with broad differences. This may be
necessary in light of the fact that tumors can display resistance to
TNSCT, one particular CT (demonstrated by this dissertation, Tables IX and
XII and Matthews, 1978). By generating more than one type of CT, each with

95
perhaps a different mechanism of action, macrophages may be able to cover
the gamut of resistance displayed by different tumors to particular CT.
The second point of interest generated by these studies concerns the
relevancy of secreted CT to macrophage mediated tumor killing in general.
This too has been the subject of much debate. Some investigators (Keller,
1980) are of the opinion that some secreted soluble CT (e.g., H2O2)
probably play no significant role in extracellular target killing since
the levels generated in vitro are generally too low to be toxic for target
cells. However, this does not rule out the possibility that these same CT
which might be ineffective once released from the effector cell, could be
the mediator of effector cell target killing while still associated with
the effector cell (contact mediated cytolysis or requiring close
apposition of effector to target).
Regardless of the specifics of the mechanism of CT action
(solubilized and extracellular or cell associated), the data presented in
these studies clearly demonstrate that conditions which are known to
affect overall macrophage cytotoxicity for tumors, also affect the
production of CT in culture supernatants. Once again, the variability of
the macrophage system employed (comparing data from different rabbits) was
manifested by differing degrees of CT production under a given set of
culture conditions. However, the overall patterns of activation by LPS,
enhancement by fetal bovine serum, and inhibition by bovine serum, PGE and
actinomycin D were maintained when comparing data from similar experiments
involving different rabbits.
Some specifics about some of these experiments warrant mentioning.
Prostaglandin was added to rabbit PLC cultures 6 hours after addition of
LPS. This was done since it had been determined in the mouse system

96
(Taffet and Russell, 1981) that macrophages require several hours in
culture to become sensitive to the inhibitory effects of PGE. Whether
this is the case for rabbit macrophages has not yet been determined.
Nevertheless, considering the lability of PGE, it was probably wise to add
it at some time after the activation signal and in a serum-free system.
The results of this experiment clearly demonstrated an inhibition of CT
production.
It was demonstrated that in vitro CT production by PLC (and
TPA-pretreated BMC) was completed by 30 hours. Adding fresh medium with
LPS at that time did not result in further production of significant CT
levels, although viability of effectors was high at this time (>95%) and
remained as such for at least 24 additional hours in culture. Whether
this cessation of CT production was mediated by PGE or not was not
determined, but regardless of the mechanism, this observation is important
in demonstrating that macrophages may only have limited temporal
cytotoxicity capabilities despite the continued presence of activating
signal. This may represent another significant reason why tumors, in
vivo, can escape destruction by activated macrophages.
The fact that actinomycin D could inhibit virtually all CT
production if added at the same time as LPS, most likey indicates that no
CT messenger RNA was present in PLC prior to activation (as well as no
active CT). Another possibility may involve an inactive or strongly cell
associated protein precursor which must be activated or released by a
second protein translated from newly transcribed messenger RNA. In any
event, messenger RNA synthesis is required for CT production during
macrophage activation.

97
The third major point to be discussed involves the increased
sensitivity of L-929 cells to PLC cytotoxicity, if the former were
pretreated with actinomycin D, then washed prior to coculturing with
effector cells. This increased target cell sensitivity induced by
actinomycin D pretreatment has also been demonstrated for lymphotoxin
(Eifel et al., 1975), natural killer cells (Kunkel and Welsh, 1981), and
antibody and complement (Segerling et al., 1975) mediated cytotoxicity.
Implicit in all this is the presence of some transcription-dependent
general "repair mechanism(s)," which can aid target cells' survival in the
presence of CT or cytotoxic effector cells.
Taking advantage of the great increase in sensitivity to effector
cell mediated lysis brought about by this regimen, it was demonstrated
that a macrophage could kill many target cells. This was demonstrated
both indirectly (culturing low effector:target ratios in 96 well trays) as
well as directly (plaque assay in 24 well trays).
The plaque assay in itself demonstrated by these studies presents
many benefits to the investigator studying cell mediated cytotoxicity.
Many of the previously described in vitro techniques developed for the
study of effector cell mediated tumor cytotoxicity demonstrate target cell
killing by populations of effector cells. The most commonly used assays
include release of radiolabel led compounds from prelabelled targets
(Brunner et al., 1968; Meltzer et al., 1975), and measuring significant
loss of adherent target cell monolayers (Takasugi and Klein, 1970;
Weinberg et al., 1978). These techniques generally require relatively
large numbers of effector cells while depending upon a high
effector:target ratio (E:T) to detect killing of targets. As a result,

98
they do not allow one to study cytotoxic events brought about by
individual effectors.
Some previously described assays do allow observations of individual
effector cell cytotoxicity. The Jerne plaque assay (Jerne and Nordin,
1963) demonstrates antibody production to a specific antigen by a single B
lymphocyte. Modifications of this assay have allowed the demonstration of
specifically sensitized cytotoxic T lymphocytes. These are depicted as
microscopic plaques on specific target cell monolayers (Bonavida et al.,
1976) or conjugated effector-target cell pairs, many containing a killed
target cell as illustrated by uptake of eosin or trypan blue dye (Grimm
and Bonavida, 1979).
For the study of nonantibody producing cells, these latter assays
have many shortcomings. In particular, among other problems, it is
difficult to distinguish between the effector mediated cytolytic events
(microscopic plaques) and the usual degree of spontaneous dying that cells
can undergo (Bonavida et al., 1976). Aside from being cumbersome, the
single target cell lysis technique depends upon direct contact between
effector and target. This may not be suitable for proper interpretation
of results where significant levels of cytotoxins may be secreted by
effector cells, obviating the need for contact.
Concerning the plaque assay, the linear relationship between the
number of effector cells plated onto target monolayers and number of
plaques that resulted demonstrate that one effector cell (macrophage)
was capable of generating a plaque, thereby detecting cytotoxicity
associated with a single effector cell. This convenient ass^y allows for
enumeration of effectors, can be completed in less than 24 hours and
demonstrates killing that can be directly detected with the unaided eye.

99
This avoids the problem of distinguishing between a more subtle cytotoxic
event mediated by effector cells and spontaneous loss of targets. The
fact that individual effector cells were being detected can allow for the
determination of any heterogeneity which can be present in the effector
cell population, concerning number of effectors in a given population
which can kill target cells and the degree with which each effector can
kill. This was seen in this study. Effector cells (PLC) taken from
different rabbits always demonstrated plaques of varying sizes. In
addition, the number of effector cells in the overall population that were
capable of generating plaques varied from about 30 to 100%. The number of
target cells killed by a single macrophage could be estimated from the
approximate sizes of the plaques and knowing how many target cells
constituted the monolayer (or the calculated area occupied by a single
target cell). This number (100 to 500) is within the same order of
magnitude as that calculated previously for the experiments done in 96
well trays (see Results).
The implication of this is that a plaque, for the most part, must
represent the secreted CT of a single effector cell, since it is unlikely
that a macrophage could have moved so far from its original point of
contact with the monolayer and in such a pattern. The time lapse
cinematography was quite revealing along these lines. Macrophages only
grazed locally, coming into contact with no more than 4 to 6 target
cells. In addition, target cells which at no time were in contact with
macrophages, were seen dying. From this it can be concluded that
formation of a plaque visible to the unaided eye is represented by the
secretion of CT by an individual effector cell.

100
This clearly demonstrates that under the proper culture conditions,
secretion of CT can even be detected at the single effector cell level.
This emphasizes the reality of the cell free CT in this system and its
potential for killing tumor cells. The question which must be raised
concerning this is the reality of such a situation in vivo. Although high
levels of CT and TNF can be demonstrated in the serum of previously
manipulated animals (see Introduction), it must be recalled that the
conditions used to induce this state are incompatible with life. There¬
fore, it is more likely that far more modest levels of CT are usually
generated during more "normal" circumstances. Keeping this in mind, along
with the ability of a particular CT to kill a (sensitive) tumor, one can
envision three conditions that would favor CT concentration in a given
area to remain sufficiently intense to kill tumor cells. They are
adequate macrophage density, sufficient activation of these macrophages,
and the presence of naturally occuring agent(s) that would enhance a
tumor's sensitivity to CT. Macrophages in varying numbers and states of
activation within or near tumors have been described (Eccles and
Alexander, 1974; Russell et al., 1976; 1977). The presence of naturally
occurring factors which sensitize tumor cells to CT has yet to be
described. The advantage this could confer on the tumor bearing host
would be to enhance the "reach" of its macrophages and extend the limits
of the macrophage microenvironment for killing tumor cells. This could be
quite significant for survival of the host.
Another interesting point demonstrated by these studies concerns the
resistance of TNSCT-resistant L-929 cells and BI6C3 cells to PLCCT,
BMCCT, and PLC. The resistance of these cells to the soluble macrophage
CT is consistent with the other data previously presented which

101
demonstrated strong similarity (possible identity) among these CT. More
interesting is the remarkable resistance of the TNSCT-resistant targets to
macrophages (Table XII). Implicit in this is that CT plays a significant
role in overall macrophage mediated target killing and that other possible
mechanisms are no more significant. It is possible that the effector PLC
in these cultures did not secrete much soluble CT, but killed target cells
primarily via a contact mediated mechanism that employed cell associated
CT. This is suggested by the fact that virtually all the TNSCT-resistant
targets survived in the presence of enough PLC (1000) that killed about
half the conventional targets. Soluble PLCCT (as well as other CT tested)
on the other hand, while killing an equivalent amount of conventional cells
(about 50%) at appropriate dilutions, did not spare all the TNSCT-
resistant cells, but killed 25 to 30% of them.
Repeated freeze-thawing of activated PLC did not reveal the presence
of a significant active intracellular pool of CT. In addition, culturing
freeze-thawed cells on L-929 cells in the presence of actinomycin D
revealed no cytotoxicity. This presents a puzzle as to where this CT
resides in the cell, particularly if it can mediate, at least in part,
contact mediated target killing. It is possible that this CT in cell
associated form must be rendered functional by some active effector or
target cell process during macrophage activation or subsequent passage
directly onto or into a target cell during effector:target contact. Hibbs
(1974b) has described such a system where macrophages, while in direct
contact with target cells, demonstrate some active injection process.
More work is needed to clarify these points.

102
Mannel (1981) has drawn similar conclusions in the mouse system
using a rabbit antiserum to TNSCT that could neutralize CT from PEC. In
this mouse system, PEC could be primed in vivo to kill target cells in
vitro without the presence of LPS. Nevertheless, these cytotoxic
macrophages secreted no detectable CT into the medium, but their
cytotoxicity for targets could be inhibited to a variable but never total
extent (33 to 89%) by TNSCT antiserum. This also implied that the CT in
this system was not totally responsible for macrophage mediated tumor
cytotoxicity.
In conclusion, these studies have demonstrated macrophage mediated
tumor cytotoxicity from the point of view of a secreted cytotoxic
molecule. Whereas this may be an oversimplification of a very involved
process due to limited knowledge concerning the intracellular state of
this moiety, it does represent a beginning of sorts in assigning at least
part of a cellular function to a molecular species. By studying how this
CT can distinguish between the tumor and nontrans formed state and by
learning more of its association with the macrophage, we may better
understand why neoplasms can still escape such intricate mechanisms and
contribute much to the frustration accompanying efforts to better
comprehend and treat cancer.

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BIOGRAPHICAL SKETCH
Harvey Fisch was born 17 September, 1946, 3:15 A.M. in Brooklyn,
New York. After graduating from Erasmus Hall High School, Harvey left
Brooklyn to attend Cornell University in frigid Ithaca, New York.
After receiving his Bachelor of Science degree in microbiology in June
of 1968, he remained at Cornell another four years while attending the
New York State College of Veterinary Medicine. After receiving the
Doctor of Veterinary Medicine degree in 1972, he entered private
practice for two years in New Jersey and Florida. From 1974 to 1976,
he served in the U.S. Army as a general veterinary officer and was
stationed at the U.S. Army Environmental Hygiene Agency, working in
laboratory animal medicine and toxicology. After pushing papers for
two years, he left active duty (to be honorably discharged later) and
entered graduate school at the University of Florida, department of
Immunology and Medical Microbiology, in 1976.
For three years, he was a graduate and teaching assistant. He
was then awarded a two year postdoctoral fellowship by the American
Cancer Society. He has just passed his board certification
examination in veterinary microbiology, conferring upon him Diplómate
status in The American College of Veterinary Microbiologists. He
plans to continue his research in his current project for a while
following graduation before pursuing other endeavors.
112

I certify that I
conforms to acceptable
adequate, in scope and
Doctor of Philosophy.
I certify that I
conforms to acceptable
adequate, in scope and
Doctor of Philosophy.
I certify that I
conforms to acceptable
adequate, in scope and
Doctor of Philosophy.
have read this study and that in my opinion it
standards of scholarly presentation and is fully
quality, as a dissertation for the degree of
George E. Gifford, Chairman
Professor of Immunology and
Medical Microbiology
have read this study and that in my opinion it
standards of scholarly presentation and is fully
quality, as a dissertation for the degree of
Kenneth I. Berns
Professor of Immunology and
Medical Microbiology
have read this study and that in my opinion it
standards of scholarly presentation and is fully
quality, as a dissertation for the degree of
Professor of Immunology and
Medical Microbiology

I certify that I
conforms to acceptable
adequate, in scope and
Doctor of Philosophy.
have read this study and that in my opinion it
standards of scholarly presentation and is fully
quality, as a dissertation for the degree of
Uj q.
r* 1 v* ^
Shands, Jr.
’'ofessor 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.
Marón B. Calderwood
Assistant Professor of
Pathology
This dissertation was submitted to the Graduate Faculty of the College
of Medicine and to the Graduate Council, and was accepted as partial
fulfillment of the requirements for the degree of Doctor of
Phil osophy.
December 1981
Dean, College of Medicine
Dean for Graduate Studies and
Research