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Lymphocyte heterogeneity in teleosts and reptiles

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Title:
Lymphocyte heterogeneity in teleosts and reptiles
Creator:
Cuchens, Marvin Agusta, 1948-
Publication Date:
Language:
English
Physical Description:
xii, 162 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Alligators ( jstor )
B lymphocytes ( jstor )
Blood ( jstor )
Cells ( jstor )
Cultured cells ( jstor )
Immunoglobulins ( jstor )
In vitro fertilization ( jstor )
Lymphocytes ( jstor )
Mitogens ( jstor )
Rabbits ( jstor )
Dissertations, Academic -- immunology and medical microbiology -- UF ( mesh )
Fishes ( mesh )
Immunology and Medical Microbiology Thesis Ph.D ( mesh )
Lymphocytes ( mesh )
Reptiles ( mesh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 1977.
Bibliography:
Bibliography: leaves 153-161.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Marvin Agusta Cuchens.

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Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
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.
Resource Identifier:
022942363 ( ALEPH )
25243870 ( OCLC )
AEK1830 ( NOTIS )
AA00006116_00001 ( sobekcm )

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Full Text











LYMPHOCYTE HETEROGENEITY IN
TELEOSTS AND REPTILES


By

MARVIN AGUSTA CUCHENS















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
1977




LYMPHOCYTE HETEROGENEITY IN
TELEOSTS AND REPTILES
By
MARVIN AGUSTA CUCHENS
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
1977


ACKNOWLEDGMENTS
The author wishes to express his appreciation to those who have
helped to make this work possible. I am most appreciative of Dr. L.
W. Clem for his support in this research for his continued encourage-
*
ment and suggestions. Special thanks are also extended to Dr. R. B.
Crandall, Dr. C. A. Crandall, Dr. B. Gebhardt, Dr. J. W. Shands, Jr.,
Dr. P. A. Small, and other members of the department for their assistance
throughout this work.
A very special appreciation is expressed for my mother and father
for their continued support throughout my academic career'and for my
wife for the typing of this manuscript as well as for her patience,
understanding, and love.
n


TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS ii
LIST OF TABLES v
LIST OF FIGURES vii
KEY TO ABBREVIATIONS ix
ABSTRACT x
CHAPTER
I INTRODUCTION 1
II LYMPHOCYTE HETEROGENEITY IN THE BLUEGILL 4
Introduction 4
Materials and Methods 8
Results 18
Discussion 52
III LYMPHOCYTE HETEROGENEITY IN THE ALLIGATOR 59
Introduction 59
Materials and Methods 63
Results 69
Discussion 119
IV MEMBRANE IMMUNOGLOBULINS OF BLUEGILL LYMPHOCYTES ... 128
Introduction 128
Materials and Methods 130
iii


TABLE OF CONTENTS (continued)
CHAPTER Page
IV Results 134
(continued)
Discussion 149
LITERATURE CITED 153
BIOGRAPHICAL SKETCH 162
5
iv


LIST OF TABLES
Table Page
1 White Cell Differentials of Bluegill Whole Blood and
Hypaque-Fic.oll Isolated Blood Cells 22
2 Effect of Dialysis of Plasma Supplements on Mitogenic
Stimulation of Bluegill Anterior Kidney Lymphocytes. 26
*
3 Effect of Maintenance Time of Bluegill in Laboratory
Aquaria on Differential White Cell Counts of
Hypaque-Ficoll Isolated Anterior Kidney Cells. ... 28
4 Effect of Maintenance Time of Bluegill in Laboratory
Aquaria on the Incorporation of Thymidine by
Unstimulated Anterior Kidney Cell Cultures 30
5 Mitogenic Responses of Bluegill Thymus Lymphocytes. . 39
6 Mixed Lymphocyte Responses of Bluegill Anterior Kidney
Lymphocytes 41
7 Mitogen Responses of Bluegill Anterior Kidney Lympho
cytes Treated with Anti-Brain Plus Complement or
Rosette Depleted with Rabbit Red Blood Cells .... 43
8 Rosette Formation of Bluegill Anterior Kidney Lympho
cytes with Red Blood Cells from Heteiologous
Species 44
9 Distribution of Antibody Forming Cells in Various Tissues
of Bluegill Immunized with Sheep Erythrocytes. ... 46
10 Immunoglobulin Producing Cells in the Lymphoid Organs
of the Bluegill 48
11 Primary In. Vitro Immunization of Bluegill Lymphoid Organ
Cell Suspensions with Sheep Red Blood Cells 50
12 White Cell Differentials of Alligator Whole Blood and
Hypaque-Ficoll Isolated Blood Cells 71
13 PHA Responses of Alligator Peripheral Blood Lymphocytes
Cultured with Different Alligator Serum Supplements. 74
v


15
16
17
18
19
20
21
22
23
24
25
26
27
Page
Effect of Sodium Chloride Concentration on Alligator
Peripheral Blood Lymphocytes Cultured with PHA ... 76
3
Effect of H-Thymidine Concentration on Alligator
Peripheral Blood Lymphocytes Cultured with PHA ... 77
3
Effect of Incubation Time with H-Thymidine on PHA
Stimulated Alligator Peripheral Blood Lymphocytes. 78
A Comparison of the Mitogen Responses of Alligator
Blood and Splenic Lymphocytes 95
Mixed Lymphocyte Cultures of Alligator Peripheral
Blood Lymphocytes 97
Combined Effects of Mitogens on Alligator Peripheral
Blood Lymphocytes 98
Mitogen Responses of Peripheral Blood Lymphocytes from
Alligators Maintained at 16C 102
Mitogen Responses of Cell Populations Fractionated on
Glass Wool 106
The Effects of Cytotoxic Treatment with Rabbit Anti-
Alligator Immunoglobulin on the Mitogen Responsive
ness of Alligator Peripheral Blood Lymphocytes . 112
Depletion of LPS and PWM Responsiveness in Alligator
Peripheral Blood Lymphocytes Passed Through an Anti
immunoglobulin Immunoadsorbent 114
9
Cytoplasmic Immunofluorescence Studies of Uncultured
and Cultured Alligator Peripheral Blood Lymphocytes. 116
Primary In Vitro Immunization with Sheep Red Blood Cells
of Alligator Peripheral Blood Lymphocytes 117
Quantitation of Surface Immunoglobulin on Bluegill and
Mouse Lymphocytes 139
Effects of Pronase Digestion on Membrane Associated
Immunoglobulins of Bluegill and Mouse Lymphocytes. 147
vi


LIST OF FIGURES
Figure Page
1 Photomicrographs of representative serial sections
through the gill region of a small bluegill 20
2 Photomicrograph of a representative Hypaque-Ficoll
isolate from bluegill blood 23
¡i
3 Photomicrograph of a representative Hypaque-Ficoll
isolate of bluegill blood after long term
laboratory maintenance of the bluegill 29
4 Correlation of TCA precipitable counts with the number
of autoradiography positive cells from PHA stimulated
bluegill lymphocyte cultures 33
5 Photomicrograph of PHA-stimulated bluegill anterior
kidney lymphocytes 35
6 Temperature effects on mitogenic responses of bluegill
anterior kidney lymphocytes 37
7 Photomicrograph of a representative Hypaque-Ficoll
isolate of alligator peripheral blood 72

8 The effects of temperature on the responsiveness of
alligator peripheral blood lymphocytes to PHA 81
9 Dose and time response of alligator peripheral blood
lymphocytes cultured with PHA 83
10 Dose and time response of alligator peripheral blood
lymphocytes cultured with LPS 85
11 Correlation of TCA precipitable counts with the number
of autoradiography positive cells from PHA
stimulated alligator lymphocyte cultures 88
12 Correlation of TCA precipitable counts with the number
of autoradiography positive cells from LPS
stimulated alligator lymphocyte cultures 90
15 Photomicrograph of PHA-stimulated alligator peripheral
blood lymphocytes 92
vii


Figure Page
14 Photomicrograph of LPS-stimulated alligator peripheral
blood lymphocytes 94
15 A chronological study during the winter months of
alligator peripheral blood lymphocytes 101
16 Diagram of glass wool fractionation procedures 105
17 Effects of increasing the cell density in mitogen
stimulated cultures of alligator peripheral
blood lymphocytes 110
18 Immunoprecipitation of lysates of membrane labeled
bluegill lymphocytes 137
19 Acrylamide gel electrophoresis in sodium dodecyl
sulfate of extensively reduced immune precipitates
of bluegill white blood cell membrane immunoglobu
lins 140
20 Acrylamide gel electrophoresis in sodium dodecyl
sulfate of extensively reduced precipitates of
bluegill spleen and thymus membrane immunoglobu
lins 142
21 Agarose-acrylamide gel electrophoresis and gel
filtration of unreduced immune precipitates of
bluegill white blood cell membrane immunoglobu
lins 144
22 Acrylamide gel electrophoresis in the presence of
sodium dodecyl sulfate of extensively reduced
bluegill white blood cell membrane immunoglobu
lins fractionated by gel filtration 145
viii


KEY TO ABBREVIATIONS
Con A
CPM
DNA
G-MEM
H chain
3H
Ig
L chain
LP3
MFM
MLC
PEC
PHA
PPD
PWM
Ra-BIg
Ra-GL
Ra-M IgM....
RBC
RPMI 1640...
Roswell Park Memorial Institute medium 1640
SDS
SRBC
TCA
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
LYMPHOCYTE HETEROGENEITY IN
TELEOSTS AND REPTILES
By
Marvin Agusta Cuchens
August 1977
Chairman: Dr. William L. Clem
Major Department: Immunology and Medical Microbiology
The purpose of this research was to study the characteristics
of the lymphoid cells from two ectotherms, the bluegill, a repre
sentative teleost, and the Florida alligator, a representative
reptile. The questions approached were 1) whether or not these
animals possessed a heterogeneity of lymphocytes akin to T- and B-
cells of higher animals, 2) whether a cellular basis for the effects
of temperature on the immune response of ectotherms could be ob
tained, and 3) whether there are membrane-associated immunoglobulins
in fish.
Hypaque-Ficoll centrifugation was used as a separation technique
for the isolation of lymphocytes. In vitro mitogenic studies of iso
lated lymphocytes from each species established that homologous serum
was the most satisfactory medium supplement. Bluegill studies demon
strated that the health or physiological state of laboratory maintained
fish appeared to be important in obtaining low background levels of DNA
synthesis. Variables found to be important in alligator lymphocyte
studies were the NaCl concentration in the medium and the age of the
serum donor.
x


Studies of the bluegill have shown that there are at least
two subpopulations of lymphocytes. One population was stimulated by
PHA (and Con A) at 32C and very poorly at 22C and was depleted by
antibrain plus complement treatment. The other population is LPS
responsive at both 32C and 22C, although responsiveness at 22C was
always greater and was depleted by removal of rabbit RBC rosetted
lymphocytes from the total population.
Temperature was also shown to be an important factor in in vitro
antigenic stimulations. In vitro SRBC primed cultures maintained at
32C elicited a very good plaque-forming cell response to SRBC's where
as 22C maintained cells gave no response. The temperature effects on
the in vitro cultures are discussed in reference to the reported in vivo
temperature effects on the teleost immune functions.
Evidence has been presented which argues for the presence of at
least two cell populations of lymphocytes in the alligator. Summarized
these are 1) differences in the magnitude of stimulation with the dif
ferent mitogens* 2) differences in the combined effects of the mitogens,
3) a significant increase in immunoglobulin producing cells in LPS-
stimulated cultures, 4) populations of cells adherent or nonadherent to
glass wool with different responses to LPS and PHA, 5) the depletion of
responsiveness to LPS by cytotoxic treatment with an anti-immunoglobulin
plus complement without reducing the responsiveness to PHA, and 6) the
depletion of responsiveness to LPS by removing immunoglobulin bearing
cells.
Environmental temperature was shown to effect the in vitro mitogenic
responses of cultured alligator lymphocytes. Although there were some
xi


fluctuations in PHA responsiveness, LPS responses dropped significantly
during the winter months or when alligators were housed at 16C.
T-like and B-like designations were assigned to the different
populations in the bluegill and alligator based on arguments by analogy
to bird and mammalian T- and B-lymphocyte characteristics.
Studies of membrane immunoglobulins on bluegill lymphocytes from
blood, anterior kidney, spleen, and thymus revealed that over 90% of the
lymphocytes exhibited membrane immunoglobulin determinants as revealed
by immunofluorescence. The majority of these cells were observed^ to
undergo patching and capping when the membrane proteins were complexed
with antisera to fish immunoglobulins. Lactoperoxidase catalyed radio-
iodination, detergent lysis and immunoprecipitation with appropriate
anitsera wore employed to study the properties of this membrane immuno
globulin. Quantitation indicated the average amount of immunoglobulin
determinants for bluegill lymphocytes to be similar to that present on
mouse B-ceils. Physicochemical characterization of labeled membrane
immunoglobulin from bluegill lymphocytes suggested that only one class
of immunoglobulin heavy chain was present and that about one-half of
this material resembled the monomeric IgM-like proteins present in
bluegill serum.
Xll


CHAPTER I
INTRODUCTION
Immunity in the vertebrates may be defined as a response of an
animal to a foreign substance (an antigen or immunogen) introduced into
its body. The response is specific in that it is directed only to the
antigen introduced and is characteristically more pronounced and occurs
sooner if the same antigen is reintroduced at a later time (43) .
Immune responses in birds and mammals may be cellular (specifically
reactive cells) or humoral (antibody mediated) (43,54,93). Characteris
tic cellular responses are delayed type hypersensitivity reactions, graft
rejection and graft-versus-host reactions. Specific cellular responses
are transferable by lymphocytes. Humoral responses are characterized
by the production of antibody directed to an antigen and the resulting
immunity is transferable by serum.
The lymphocyte has been demonstrated to be the principal cell type
involved in the immune responses of birds and mammals. Although lympho
cytes are morphologically identical, two major subpopulations have been
identified based on ontological origin and functional analysis (43,54,
93). One subpopulation, the T-(thymus derived) lymphocyte, is the func
tional cell in cellular mediated responses. The other subpopulation,
the B-(bursa derived in birds or bursal equivalent in mammals) lympho
cyte, is the functional cell in producing antibodies in humoral responses.
T- and B-lymphocytes have been further characterized on the basis of cell
surface determinants and in vitro responses to mitogens, antigens and
1


2
mixed lymphocyte reactions (43,53,54,93). B-cells respond to different
mitogens (e.g iipopolysaccharide), have demonstrable levels of immuno
globulin on their surfaces, and are stimulated by mitogens or antigens
to synthesize immunoglobulin or antibody. In contrast T-cells prolif
erate in response to different mitogens (e.g. phytohemagglutinin and
concanavalin A), do not have surface immunoglobulin (or at least easily
demonstrable levels), express surface differentiation antigens not found
on B-cells (e.g. Thy-1) and are the responding cells in mixed lymphocyte
reactions. '
A unique feature of the lymphocytes involved in the immune response
is the requirement of T-B cell cooperation in most responses leading to
antibody production, even though the T-cell does not make antibody (49,
52,54,81). This requirement is best illustrated using hapten-carrier
antigen complexes (19,28,54,69) in which carrier recognition by T-cells
is required before B-cells can make antibody to the hapten. T-cells are
also involved in the control of the 19S to 75 switch (IgM to IgG anti
bodies) as well as maturation of the humoral response (increase in anti-
4
body affinity with time after immunization) (19,54,69). It should be
pointed out that except for a few antigens which are structurally very
repetitious (the T-independent antigens capable of reacting directly with
B-cells), both cell types must interact to elicit a response to most
antigens (T-dependent antigens) (28,54,69,82).
The majority of much of the research on the immune systems briefly
described above has been in birds and mammals, both of which are endo
thermic. With the exception of limited studies of the amphibians, stud
ies of the cellular basis of the immune systems in ectotherms have not
been done. Although one could postulate the existence of T-like and


3
B-like cells on the basis of graft rejection and antibody production
(discussed in detail later) direct evidence of lymphocyte heterogeneity
in any ectotherm has not been obtained. Furthermore the molecular or
cellular bases for the commonly observed effects of environmental tem
perature on the immune responses of numerous ectotherms (7,37) have not
been investigated.
The purpose of the research undertaken here was to study the char
acteristics of the lymphoid cells from two ectotherms, the bluegill,
a representative teleost, and the Florida alligator, a representative
reptile. The major questions approached were 1) whether these two ani
mals possessed classes of lymphocytes akin to T- and B-cells of higher
animals, 2) whether a cellular basis for the effects of temperature on
the immune response of ectotherms could be obtained, and 3) whether there
are membrane associated immunoglobulins in fish.


CHAPTER II
LYMPHOCYTE HETEROGENEITY IN THE BLUEGILL
Introduction
Considerable evidence from in vivo studies indicates that teleost
fish can mount a diversity of immune responses. Teleosts are capable
of responding to a wide variety of antigens with both primary and secon
dary responses (4,7,15,32,37,51,112) with the only apparent major dif
ference from mammals being that there is no discernable "IgM -* IgG
switch" in the fish (1, 16,27,62,111, 121). In fact the evidence avail
able to date shows that many species of fish synthesize only 16S tetra-
meric IgM-like immunoglobulin (1,16,27,62,111). In those fish also pos
sessing low molecular weight serum immunoglobulins, the 7S molecules
appear to resemble monomeric forms of the tetramer and hence it seems
that fish are lacking an IgG equivalent (21,29,30,31,55,75). Attempts
to demonstrate IgA-or IgE-like molecules or activities in fish have also
been unsuccessful (31). Thus, in light of these latter deficiencies it
would seem appropriate to speculate that while fish possess cells of B-
like function, their number of immunoglobulin classes is somewhat limit
ed. Several investigators have also demonstrated the ability of fish to
reject both first and second set scale transplants with the second set
rejections occurring more rapidly (12,59,60,61,88). Thus, again arguing
by analogy, it appears that fish have cells with T-like function. Fur
thermore, studies on three different species of fish have revealed the
4


5
existence of the so-called hapten-carrier effect (46,106,121). Since
this "helper effect" is considered to result from T-B cell collaboration
in mammals it appears that fish may also have this coordinated function
in their immune response system.
Since fish are ectothermic animals it is not surprising that numer
ous reports of temperature influences on immune responses have appeared.
The classic studies of Bisset (10), Cushing (40) and Hildemann and Cooper
(61) demonstrated that temperature can have a profound role in these
responses. The more recent studies of Avtalion have served as a basis
for beginning to understand the mechanism of these effects (7). He has
shown that humoral responses in the carp are a two-step process: 1) a
temperature-sensitive step requiring relatively high temperatures for
antigen recognition and 2) a temperature-insensitive step which results
in antibody production. Furthermore, Avtalion suggests that there are
at least three cell types involved; 1) X cells (T-like) which are sensi
tive to low temperatures and are involved in priming and tolerance and
2) Y and Z cells (B-like) which are involved in memory and antibody for
mation respectively. It must be pointed out however that direct proof
for the existence of multiple types of immunocompetent cells in fish is
lacking.
More recently Etlinger et^ al. (46) presented evidence that rainbow
trout have two lymphoid cell types. This evidence is based on responses
of leukocytes isolated from various lymphoid organs to the mammalian T
and B cell mitogens. Thymocytes responded only to Con A (a T-cell mito
gen in mice and man) and anterior kidney leukocytes responded only to
I.PS or PPD (B-cell mitogens). The unique pattern of tissue localization
of cells responsive to mammalian T- and B-lymphocyte mitogens was taken
as evidence for lymphocyte heterogeneity in rainbow trout.


6
Smith et al. (102), Chiller e_t al_. (26), and Pontius and Ambrosius
(89) have studied the cellular responses of teleosts to sheep red blood
cells and have demonstrated antibody-forming cells in the spleen and
anterior kidney. Further studies by Sailendri and Muthukkaruppan have
shorn an appreciable number of antibody-forming cells in the thymus as
well (96,97). One could thus conclude that fish have a B-cell equiva
lent, as defined by the ability of plasma-like cells to produce antibody.
However, the presence of antibody-forming cells in the fish thymus indi
cates that the thymus may not be populated with only T-like cells- as
Etlinger's work suggests. Only in experimentally induced circumstances
are antibody-producing cells (B-cells) found in mammalian thymuses (37).
In addition, > 90% of the cells isolated from thymuses of four different
species of fish have demonstrable levels of immunoglobulin on their
surfaces (44,45,46,116). Although there is some controversy as to
whether or not mammalian T-cells have surface immunoglobulins (to be
discussed further in Chapter IV), the consensus is that if T-cells do
have surface immunoglobulins they are present in very small amounts and
only B-cells have readily demonstrable levels of surface immunoglobulin.
Therefore, in light of the existing data, there is some question as to
whether fish thymocytes are similar to mammalian thymocytes.
It should be pointed out that much of the data supporting the con
cept of two cell types (presumed to be lymphocytes) involved in immune
responses in fish are only inferential and alternative interpretations
may be presented. Indeed the unusual properties of the fish thymus (sur
face immunoglobulin expression and the presence of antibody producing
cells), as well as a lack of maturation in antibody responses (34)


7
and the presence of demonstrable hapten-carrier effects without a 16S
-> 7S switch suggest that if a T-like cell in fish exists it may differ
functionally from higher vertebrate T-cells. Summarizing the current
literature, it appears that direct evidence for two lymphocyte sub
populations in fish is lacking.
The purpose of this portion of the research was to determine in a
direct way if a teleost, the bluegill, has a heterogeneous population
of lymphocytes akin to T- and B-cells in birds and mammals. The ap
proach taken was three fold: 1) to define a separation technique .for
the isolation of relatively pure lymphocytes and to establish appropri
ate In vitro culture conditions, 2) to determine if mitogenic responses
and cell surface determinants employed as T- and B-cell probes in hirds
and mammals are applicable to bluegill lymphocytes as in vitro markers,
and 3) to separate differing subpopulations of lymphocytes on the basis
of differences in mitogenic and cell membrane antigens. Special empha
sis was placed on studying the effects of temperature on bluegill lym
phocytes to determine if a cellular basis for the in vivo temperature
effects on the immune responses in fish exist.


Materials And Methods
Experimental Animals
Bluegill (Lepomis macrochirus), a freshwater teleost, was used
exclusively as a source of lymphocytes in these studies. Sexually
mature male and female specimens, weighing 200-500g, were obtained
from the University of Florida's Lake Alice using cane poles, barbless
hooks and bread as bait. Fish were handled with rubber gloves and kept
in aerated holding tanks until transported to laboratory aquaria. One
hundred twenty-five liter Nalgene tanks filled with dechlorinated water
were used to maintain specimens in the laboratory. A maximum of eight
fish per tank were maintained with continuous aeration and a complete
change of water every 3-4 days. Fish were fed to satiation 2-3 times
each week with TetraMin (Tetra Werke, West Germany). As discussed later,
these holding conditions were less than ideal.
Culture Media
Roswell Park Memorial Institute (RPMI) 1640 was used as a wash
medium and as a supportive medium for in vitro mitogenic studies.
The complete medium used was prepared by dissolving RPMI 1640 instant
tissue culture powder (Grand Island Biological Company [GIBCO], Grand
Island, N.Y.), penicillin (GIBCO; 50 U/ml), streptomycin (GIBCO; 50 ncg/
ml), gentamycin (Schering, Kenilworth, N.J.; 20 mcg/ml), heparin (Sigma,
St. Louis, Mo.; sodium salt, 5 U/ml) and sodium bicarbonate (Mallinckrodt,
8


9
St. Louis, Mo.; 2.2 g/L) in triple-distilled water. The pH was
adjusted to 7.2 with NaOH or HC1, and the solution sterilized by passage
through 0.45y detergent free Swinex-25 millipore filters (Millipore,
Bedford, Mass.).
For in vitro studies of primary immune responses (Mishell-Dutton
type cultures [83]) a medium modified from Click et_ al_. (35) was used.
Modifications of the original technique included exclusion of NaOH and 2-
mercaptoethanol, substitution of RPM1 1640 for Hank's and the addition
of gentamycin (20 mcg/ml), heparin (5 U/ml) and sodium bicarbonate (2.2
g/L, dissolved in the initial media preparation). Concentrations of the
amino acids (GIBCO). nucleic acid precusors (GIBCO), pyruvate (GIBCO),
glutamine (GIBCO), vitamins (GIBCO), penicillin and streptomycin were
added as described by Click et al. (35). The medium was prepared by
dissolving the above ingredients in triple-distilled water, adjusting
the volume and pH and sterilizing as for the preparation of RPMI 1640
(described above).
Medium Supplements
*
Serum and plasma sources which were tested as medium supplements
for in vitro studies were fetal calf serum (GIBCO; Lot # A030113; Inter
national Scientific Ind., Inc., Cary, Ill.; Lot # 7066411), Calf Serum
(GIBCO; Lot # Ro266T), human serum pools (five pools furnished by Dr.
R. Waldman, University of Florida, > 50 normal human sera per pool),
rabbit serum pools (New Zealand White rabbits, two pools, >10 normal
rabbit sera per pool), alligator (Alligator mississippensis) serum
(Silver Springs Reptile Institute, Silver Springs, Fla.; four indivi
dual normal alligator sera), fresh water catfish (Ictaluru cerracanthus)
plasma (heparinized, pool from ten catfish), large mouth bass (Micropterus


10
punctulatus) plasma (five heparinized pools, five normal bass per pool),
giant grouper (Epinephelus itaira) serum (pool from five grouper) and
bream (a collective term for all Lepomis species) plasma (heparinized
pools, > 10 fish per pool). All sera or plasma were heat inactivated
for 30 min at 56C and were sterilized by Millipore filtration (0.45 y)
Preparation of Cell Suspensions and Counting Technique
The sources of cells studied from the bluegill were blood, anterior
kidney (pronephrus or head kidney), thymus and spleen (6,48,68,96,97,102,
117). Heparinized blood, obtained from the caudal vein (108) and all
organs were removed aspectically. Organs were placed in sterile petri
dishes containing cold RPMI 1640. A single cell suspension of each organ
was prepared by gently teasing apart the organ with forceps and pipeting
the teased suspension over a 60-80 mesh steel screen to remove clumps
and connective tissue.
A Hypaque-Ficoll method, adapted from Boyum's Isopaque-Fic.oll tech
nique (14), was used to isolate lymphocyte populations from organ cell
suspensions or heparinized blood. Hypaque-Ficoll solutions were pre
pared by mixing 10 parts of 33.9% Hypaque (Winthrop Laboratories, New
York, N.Y.) with 24 parts or 9% Ficoll (Pharmacia, Piscataway, N.J.),
Densities of prepared solutions were 1.077 0.0005 g/ml (room tempera
ture) as determined by picnometer difference weighings.
A maximum of five ml of a teased organ cell suspension or heparin
ized whole blood diluted 1:4 with RPMI 1640 was gently layered onto five
ml of Hypaque-Ficoll in a 15 ml tube (Falcon, Oxnard, Cal.; 17 x 100 mm).
Tubes were spun at room temperature in a table-top centrifuge (Interna
tional Centrifuge, Boston, Mass.) for 20 min at 1000 RPM. The interface


11
band of cells between the Hypaque-Ficoll and the overlaying suspension
medium was removed using a Pasteur pipet and diluted in cold RPMI 1640.
The suspension was spun for 10 min at 1000 RPM in a refrigerated centri
fuge and the cell pellet washed three times with cold RMPI 1640.
The number of phagocytic cells was assessed using collodial carbon
uptake. India ink was diluted 1:10 with saline, centrifuged for 30 min
at 3500 RPM and millipore filtered (0.45 y) prior to use. One drop was
added to approximately three ml of a cell suspension and the mixture in
cubated for 30 min at 37C. The cells were then washed three times and
May-Grunwald-Giemsa stained cytocentrifuged (Shandon-Elliott Inc.,
Sewickley, Penn.) mounts prepared for quantitation.
Cell counts (109) and viability (13) were determined by diluting
an aliquot of the washed cell suspension in a white blood cell diluting
pipet (Scientific Products, Ocala, Fla.) with 0.1% trypan blue in RPMI
1640 and counting with a Neubaurer hemacytometer (Scientific Products).
Culture Techniques
A laminar flow hood (Abbott Laboratories, Chicago, Ill.) was used
as a sterile environment for all cell culture work.
A microculture method (58,107) was adapted for mitogenic stimulation
and mixed lymphocyte culture assays. For mitogen studies, washed and
pelleted cells were resuspended in serum or plasma supplemented RPMI 1640
and were dispensed into microculture trays (Linbro, Hamden, Conn.; U-
shaped wells) at a cell concentration of 5 x 10^ cells/0.2 ml/well. The
mitogens used consisted of lippolysaccharide (DIFCO Labs, Detroit, Mich.)
from S. typhimurium which was boiled one hr after reconstitution with
triple distilled water, phytohemagglutinin P (DIFCO) and concanavalin A
(Miles Labs, Inc., Kankakee, Ind.; 3x crystallized). Stock solutions


12
were diluted with RPMI 1640 without serum or plasma supplements and
were added to appropriate wells in 20 pi volumes immediately after the
cells were dispensed. Twenty microliters of RPMI 1640 without supple
ment or mitogen was added to control unstimulated wells.
Two-way mixed lymphocyte cultures of cells from two bluegills
were prepared by adding 2.5 x 10^ cells suspended in 0.1 ml of supple
mented RPMI 1640 from each cell preparation (total cell concentration
per well was 5 x 10^/0.2 ml). Five hundred thousand cells/0.2 ml/well
from each source served as controls. ^
Tritiated-thymidine (Schwarz-Mann, Orangeburg, N.Y.; sterile
acqueous solution, pH 7.4, 1.9 Ci/mM, 1.0 mCi/ml), diluted in supple
ment free RPMI 1640, was added to each culture well at a concentration
of 0.5 pCi/10 pl/well at 24 hr prior to harvesting.
Microculture trays were maintained in 5% CO2 95% air, satu
rated-humidity incubators at the temperatures indicated. CO^ content
was routinely measured with a Fryrite CO^ tester (Bacharach Instrument
Company, Pittsburgh, Penn.).
3
Cells, mitogens and H-thymidme were dispensed in microculture
trays using 0.5, 1.0, 5 or 10 ml gas tight syringes (Hamilton, Reno,
Nev.) attached to repeating dispensers (Hamilton) delivering one-
fiftieth of the attached syringe volume.
For .in vitro studies of primary immune responses, single cell
suspensions were prepared from pooled anterior kidney, spleen, and
thymus by teasing apart the organs in RPMI 1640 and seiving through
a 60-80 mesh screen. The cell suspension was centrifuged and the pellet
washed three times. The final cell pellet was resuspended in enriched
RPMI 1640 medium (described above) supplemented with 7% bass plasma.


13
7
White, red, and dead cells were enumerated and 1x10 viable white
cells in three ml of supplemented medium were aliquoted in Falcon 35
x 10 mm tissue culture dishes (Scientific Products).
Sheep red blood cells (SRBC's) used for immunization of the dis
sociated organ suspensions were obtained from a single sheep (Colorado
Serum Comp., Denver, Col.; Sheep # 20, H type antigen). SRBC's were
washed three times with RPMI 1640 and the final pellet suspended in the
enriched RPMI 1640 (without supplement) to 1% of the packed cell volume
Cultures to be immunized received 0.1 ml of the 1% SRBC suspension.
Controls received 0.1 ml of enriched RPMI 1640.
Culture dishes were maintained in 5% CO2 95% air humidified
environments as described above.
Assay for ^H-thymidine Incorporation into DNA
An automatic cell harvester (Otto Hiller Company, Madison, Wis.)
was used to obtain trichloroacetic acid (TCA) precipitable nucleic
acid material from individual wells of cultured cells. Twenty-four
hour pulsed cells were syphoned from the wells onto a glass fiber
fc
filter (Reeve Angel, Whatman, Inc., Clifton, N.J.), rinsed with
saline, precipitated with 10% TCA and methanol dried. Discs, repre
senting individually harvested wells, were punched out of the filter
strip and assayed for using liquid scintillation counting. The
scintillation cocktail used consisted of PPO (Packard, Chicago, Ill.;
16.5 g), POPOP (Packard; 0.3 g), Triton X-100 (Packard; 1.0 L), and
toluene (Maltinckrodt, St. Louis, Mo.; 2.0 L). Samples were counted
in mini-vials (Rochester Scientific, Rochester, N.Y.) using an auto
matic liquid scintillation counter (Beckman Instruments, Fullerton, Cal
h
)
Model LS-100


14
Stimulation Indices and Statistical Analysis
Means and standard deviations were determined for each data group.
An F-test was used for variance analysis. The Student-t test was used
to determine the statistical significance of increases over control
values (20,103). A 95% or greater confidence level (p <_ 0.05) was used
for both the F-test and the t-test.
Stimulation indices were used to express increases of mitogen
stimulated cultures over control cultures or mixed lymphocyte cultures
(MLC's) over controls. Indices for mitogenic studies were determined
using the following equation: Mean CPM of stimulated cultures Indices
Mean CPM of control cultures
for MLC studies were calculated by using the following formula:
Mean CPM of MLC between Fish A and Fish B
(Mean CPM of Fish A Control Culture + Mean CPM of Fish B Control) t 2
Histological and Morphological Techniques
Serial cross sections of paraffin embedded gill regions of bluegill
were kindly prepared by Mr. Melvin Laite (Department of Pathology, J.
Hillis Miller Health Center, Gainesville, Fla.). Sectioned tissues were
*
stained with hematoxylin and eosin.
Cell suspension smears or cytocentrifuge (Shandon-Elliot Inc.)
preparations were stained with May-Grunwald-Giemsa for morphological
examination.
Autoradiography
*7
Cultured cells, pulsed with H-thymidine for 24 hr, were pipeted
from microculture tray wells, washed three times with RPMI 1640 and
cytocentrifuged. Cytocentrifuged preparations were coated with nuclear
track emulsion (Kodak, Inc., Rochester, N.Y.; type NTB3), exposed,


15
developed and fixed as described by Gormus et_ al_ (52). All processed
slides were stained with toluidine blue in order to enhance microscopic
examination of the cells.
Preparation of Rabbit Antisera
A rabbit anti-bluegill brain antiserum was prepared by the proce
dure described by Golub for mouse brain (50). Five brains were homo
genized, using a tissue grinder, diluted 1:2 with PBS and 0.5 ml aliquots
were emulsified w'ith an equal volume of complete Freund's adjuvant (DIFCO)
for each immunization. Sera obtained from the rabbits before immuniza
tion were used as normal rabbit serum controls. The rabbit antiserum
used was obtained from one surviving rabbit which was reimmunized six
times over a three-month period.
The preparation of rabbit anti-bluegill immunoglobulin is described
in Chapter IV.
Cytotoxicity Assay
Complement mediated cytotoxicity of preimmune or immune normal rab
bit serum and rabbit anti-bluegill brain serum on bluegill lymphocytes
7
was accomplished by incubating 1 x 10 cells with 1:5 dilutions of rab
bit sera plus a 1:10 dilution of guinea pig complement (GIBCO, lyophi-
lized). After 1.5 hr at room temperature the cells were washed three
times with RPMI 1640 and cell counts and viability determined.
Rosetting Techniques
The method of Jondal £t_ al. (66) was followed to assess the number
of lymphocytes capable of rosetting with red blood cells (RBC's) from
various animals. Fresh heparinized whole blood obtained from human,
sheep, rabbit, chicken, horse, ferret, guinea pig, mouse, alligator, and


16
bluegill were washed four times with RPMI 1640, and the white buffy
coat was removed after each centrifugation. Winthrop hematocrit tubes
were used to determine percentages of RBC's in each suspension and
dilutions were made accordingly. Controls with only the test RBC's
were routinely assayed to determine the number of white cells con
tributed by the RBC suspension. As a negative control, homologous
RBC's were tested with bluegill lymphocytes.
Hypaque-Ficoll (p = 1.077) centrifugation was used to deplete
rosetted lymphocytes from non-rosetted lymphocytes (41,98). Hypaque-
Ficoll recovered non-arosetting cells were diluted into RPMI 1640, pellet
ed and washed three times.
Immunofluorescence
Immunofluorescent reagents and techniques are described in Chapter
IV.
Hemolytic Plaque Assay
Cells were harvested from tissue culture dishes by gently scrap
ing the bottom of the culture dish with a rubber policeman and pipetting
the cell suspension into a conical centrifuge tube. The plate was rinsed
once with 3 ml RPMI 1640 and the wash medium was added to the cell
suspension. Cells were pelleted and resuspended in RPMI 1640. Viabili
ty and cell recoveries were determined prior to assaying for plaque
forming cells (PFC's).
PFC's (cells producing antibody to SPJBC's) were enumerated using
a slide modification (83) of the Jerne hemolytic plaque assay (65).
Slides were incubated with fresh sucker fish plasma (a plasma pool from
several different species of the Catostomidae family native to the Swanee


17
and Santa Fe Rivers in Florida) diluted 1:20 in RPMI 1640 for 3-5 hr in
a 32C, 5% CO2 95% air incubator. Plaques were routinely examined
microscopically prior to counting on a Quebec colony counter (New
Brunswick Scientific Co., New Brunswick, N.J.).


Results
Lymphoid Organs of the Bluegill
To determine which organis of the bluegill contained lymphoid cells,
smears of blood or organ cell suspensions were stained with May-Grunwald-
Giemsa and examined for the cell types present. Of the tissues examined,
anterior kidney (head kidney or pronephros), spleen, thymus, and blood
were the major sources of lymphocytes. Very few lymphocytes we re found
in the liver, pancreas, gonads, or posterior kidneys. Gut-associated
lymphoid tissue or lymph nodes were not found.
Due to the close proximity of the thymus to the anterior kidney,
serial sections were made through the gill region of a small fish ('v 100 g,
< 1 yr old) and examined histologically. Figure 1 presents photomicro
graphs of representative sections through this region. The anterior kid
ney was seen to be a relatively diffuse organ containing an abundant number
of blood sinuses, had a relatively large number of red blood cells and
contained a heterogeneous mixture of white cells. In contrast, the
thymus contained fewer red blood cells, had few white cells other than
lymphocytes and contained Hassalls corpuscles. Therefore based upon
both the anatomic location and the histologic characteristics, it was
felt that these tissues were in fact anterior kidney and thymus.
Separation and Quantitation of Bluegill Lymphocytes
- Hypaque-Ficoll (p = 1.077) was used to siolate relatively pure
populations of lymphocytes (characterized morphologically) from the
18


Figure 1. Photomicrographs of representative serial sections through
the gill region of a small bluegill. (a) Anterior kidney. Cb) Thymus
Sections were stained with hematoxylin and eosin. Magnification x 100


20


21
blood, spleen, anterior kidney, and thymus of bluegill. Less than 5% of
the total number of cells recovered from Hypaque-Ficoll were RBC's and
the number of lymphocytes recovered represented at least 99% of the
lymphocytes present in unfractionated whole blood or lymphoid organ cell
suspensions.
White cell differentials of whole blood before and after fractiona
tion on Hypaque-Ficoll are presented in Table 1 and illustrate the effi
ciency of this technique in removing other cell types. Figure 2 is a
photomicrograph of the type of lymphocyte preparations routinely obtained
from blood or lymphoid organ cell syspensions. These cell separations
were successful, only if freshly caught fish were used. Another major
cell type, a lymphoblast-like cell, was isolated from Hypaque-Ficoll if
cell suspensions from fish maintained in laboratory aquaria for long
periods of time were used (see Figure 3). The relevance of these blast
like cell isolates and the necessity of using newly acquired fish for
these and subsequent studies is discussed in a later section.
The anterior kidney was the most abundant source of lymphocytes
' 8
(yielding 'u- 2 x 10 cells/fish) whereas spleens and thymuses routinely
7 7
yielded about 5 x 10 and 2 x 10 cells/fish, respectively. Heparinized
blood yielded about 5 x 10^ cells/ml (see Table 10).
Culture Conditions and Assay of Cell Division
As in any study involving in vitro culturing of lymphocytes (or
any other cell type for that matter) there were numerous variables to
be considered. In light of the fact that relatively limited numbers of
cells were available from individual fish and since syngeneic bluegills
were not obtainable it was necessary to approach optimization of culture


22
Table 1
White Cell Differentials of Bluegill Whole Blood
and Hypaque-Ficoll Isolated Blood Cells
Percent of Total*3
a
Blood
Hypaque-Ficoll Isolated
Cell Type
Thrombocyte
255C
0
Granulocyte
304
0
Lymphocyte
455
100
(a) Smears were made of whole blood and Hypaque-Ficoll isolates
of individual samples and were May-Grunwald-Giemsa stained for
quantitation purposes.
(b) Results are expressed as a percent of the total number of
white blood cells counted.
(c) Each value represents the mean of determinations from 6 dif
ferent bluegill samples (>3 determinations per samples) standard
deviations.


23
Figure 2. Photomicrograph of a representative Hypaque-Ficoll isolate
from bluegill blood. A cytocentrifuge preparation stained with May-
Grunwald-Giemsa. Magnification x 400.


24
conditions in a rather "piecemeal" fashion over an extended period of
time. The following commentary is an effort to systhesize the major
observations that enabled the definition of what can be called optimal
conditions. Unless otherwise noted all of these studies were performed
with anterior kidney lymphocytes.
Various sera or plasma were tested to determine which was a suit
able supplement to use with RPMI 1640 for mitogenic studies of cultured
bluegill cells. Ten percent human, calf, fetal calf, rabbit, alligator,
bass, catfish, and grouper sera or plasma and mixtures of 5% humanserum
with 5% calf or fetal calf serum were not supportive in mitogenic stimu
lation studies using PHA (0.1 yl), Con A (10 yg) or LPS (1 or 10 yg) at
either 22, 27 or 32C. Grouper and catfish sera were cytotoxic for
bluegill cells. The other sera gave high TCA precipitable counts in
unstimulated control cultures and stimulation indices for mitogen
stimulated cultures of 1 or < 1. On the other hand, bream (a collective
term for all Lepomis species) serum pools were supportive in the sense
that significant stimulation indicies were obtained with mitogen stimu
lated cultures. '
In the initial experiments 10% bream serum was used. However, due
to the limited supply of bream sera and the difficulty in obtaining
good yields of serum from clotted blood, two modifications were tried
and found satisfactory; 1) heparinized plasma rather than serum was used
and 2) the concentration of supplement was reduced from 10% to 7%.
An additional complication was the observation that not all bream
plasma pools were suitable as supplements in mitogenic assays. Varia
tions in TCA precipitalbe counts of unstimulated control cultures ranged
from < 100 CPM to > 10,000 CPM and stimulation indices varied from 4 to


25
250. One attempt to reduce the high counts of control unstimulated
cultures obtained with some of the supplement pools was to dialyze
the plasma pools against 0.15MNaCl. The data obtained with four
bream plasma pools which elicited high background levels prior to
dialysis are presented in Table 2. In three of the four pools tested
in this experiment the control CPM dropped significantly (p < 0.05)
in the cultures incubated at 22C and thus resulted in increased stimu
lation indices with LPS. With three of four pools used with cells
maintained at 32C the background remained unchanged. In the other
case the background dropped as a result of dialysis and hence the
stimulation index obtained using PHA increased. In conclusion it can
be stated that dialysis of bream plasma did not significantly decrease
the responses in any cultures and in fact in some cases enhanced the
response. Thereafter all bream plasma were dialyzed before use as
culture medium supplements for mitogenic assays.
Dialysis of certain heterologous supplements that elicited high
control CPM was also tried. Dialyzed bass plasma was supportive as a
supplement in mitogenic assays in the sense that significant stimulation
indicies were obtained. However, these indices were never > 10 and
therefore bass plasma was not used routinely. Dialysis of human, calf,
fetal calf, and alligator sera did not improve the situation with re
spect to high levels of background counts.
To summarize the culture conditions discussed thus far, RPMI 1640
supplemented with 7% dialyzed bream plasma was found to be supportive
for iii vitro mitogenic stimulation.
During the course of several experiments involving different fish
it was observed that there were differences both in the types of Hypaque-


26
Table 2
Effect of Dialysis of Plasma Supplements on Mitogenic
Stimulation of Bluegill Anterior Kidney Lymphocytes
Stimulation Indexa
Supplement Plasma
Poolb
PHA (0.1 yl)
- 32C
LPS (1 yg)
- 22C
Undialyzed
Dialyzedc
Undialyzed
Dialyzed
A
1
6.8
1
1
B
4.8
4.0
1
14.0
C
2.5
4.0
5.9
16.4
D
4.8
4.6
1.5
7.4
(a) Triplicate cultures were stimulated with either PHA (0.1 yl) at
32C or LPS (10 yg) at 22C, pulsed on day 6 and harvested on day 7.
(b) Each pool represented the plasma obtained from at least five bream.
(c) Dialysis vas against pyrogen free 0.15 M NaCl.


27
Ficoll isolated cells from anterior kidney cell suspensions and in the
stimulation indices with mitogens. Furthermore these differences ap
peared to be correlated with the length of time the bluegills were main
tained in holding tanks in the laboratory prior to sacrifice. Table 3
presents data on white cell differentials of Hypaque-Ficoll isolated
cells from anterior kidneys of fish sacrificed at either one day or
three weeks after capture. Significant increases in the number of blast
like cells were seen in cell preparations from bluegills maintained for
three weeks. The gross differences in the cell types isolated from
Hypaque-Ficoll can be seen by comparing the cells shown in Figure 2
(from a one day isolate) with those in Figure 3 (a three-week isolate).
An increase in the number of red cells, which would not penetrate the
Hypaque-Ficoll, was also noted in the three-week isolates.
Furthermore, it was also observed that in experiments utilizing
fish maintained in acquaria for long periods of time, TCA precipitable
counts of control unstimulated cultures were high. A composite of data
from five experiments in which bluegill were sacrificed at various
periods of time 'after capture is presented in Table 4. Apparently the
longer a fish is maintained under our laboratory conditions the more
likely it is the animal's lymphocytes will exhibit a high level of
spontaneous thymidine incorporation. It thus seems imperative to use
freshly caught fish as sources of cells for in vitro studies if the
alternative is to keep them under the conditions used here.
To determine if TCA precipitable counts were a valid measure of
cellular events in culture, TCA precipitable counts were correlated
with the actual number of cells containing labeled thymidine. The
technique of autoradiography was used. Antei'ior kidney lymphocytes


28
Table 3
Effect of Maintenance Time of Bluegill in
Laboratory Aquaria on Differential White Cell
Counts of Hypaque-Ficoll Isolated Anterior Kidney Cells
Percent3,
Maintenance Time
Lymphocyte^
Blast-like
1 day
100
0
3 weeks
50-60
50-40
(a) Results are expressed as a percent of the total number of
white cells counted.
(b) Cytocentrifuged preparations of Hypaque-Ficoll separated
blood samples were stained with May-Grunwald-Giemsa for quanti
tation.


29
Figure 3. Photomicrograph of a representative Hypaque-Ficoll isolate
of bluegill blood after long term laboratory maintenance of the blue-
gill. Photomicrograph is of a May-Grunwald-Giemsa stained cytocentri-
fuge preparation of an isolate obtained from a bluegill after three
weeks of laboratory maintenance. Magnification x 400.


30
Table 4
Effect of Maintenance Time of Bluegill in Laboratory
Aquaria on the Incorporation of Thymidine by
Unstimulated Anterior Kidney Cell Cultures
CPM/Culturea
Fish^
Maintenance Timec
22C
32C
A
1 day
508
4518
B
1 day
120114
4416
C
3 weeks
1121137
26451109
D
3 weeks
991168
12311127
E
5 weeks
13,1971580
28381181
(a) Results are expressed as the means of CPM from triplicate
cultures standard deviations.
(b) Cells from individual fish were incubated without mitogens
at 22C or 32C, pulsed with ^H-thymidine on day 2 and harvested
on day 3.
(c) Length of time fish were maintained in laboratory aquaria
before sacrifice.


31
were cultured for seven days with various doses of PHA at 32C.
Tritiated-thymidine was added to all Cultures 24 hr prior to termi
nation. One set of cultures was routinely harvested and assayed for
TCA precipitable counts. Cytocentrifuge preparations were prepared
from cells of a duplicate set of cultures and either processed for
autoradiography and the number of labeled cells (>_ 5 grains) quanti
tated (presented as a percent of the total number counted) or stained
with May-Grunwald-Giemsa for morphological examination. As seen in
Figure 4, increases or decreases in TCA precipitable radioactivity
closely followed changes in the percent of the total number of cells
that contained labeled thymidine.
In cultures stimulated with the optimal concentration of PHA (0.1
yl), 70% of the cells possessed nuclear autoradiographic grains. All
labelled cells examined in toluidine-stained cytocentrifuged preparations
were large b]ast-like cells and were found in clumps or aggregates
(Figure 5a). Figure 5b is a May-Grunwald-Giemsa stained preparation
showing an aggregate of blast-like cells with eccentric nuclei and abun-
dant cytoplasm.
Mitogenic Studies
Experiments were designed to assess the optimal culture conditions
for lymphocytes isolated from the anterior kidney. Variables tested wrere
mitogen doses, time for maximum stimulation and effect of temperature on
the response to the mitogens.
Figure 6 depicts the results of one very large study on the respon
siveness of anterior kidney lymphocytes to LPS, PHA,and Con A under a
variety of conditions. One somewhat surprising result involved the


Figure 4. Correlation of TCA precipitable counts with the number of
autoradiography positive cells from PHA stimulated bluegill lymphocyte
cultures. Cultures were incubated at 32C, pulsed on day 6 and assayed
on day 7.


80-
% OF
TOTAL
NUMBER
POSITIVE
CPM
x!0'3
C/4
04


Figure 5. Photomicrograph of PHA-stimulated bluegill anterior kidney-
lymphocytes. (a) Autoradiograph stained with toluidine blue. (b) May-
Grunwald-Giemsa stained. Magnification x 100.


35


Figure 6. Temperature effects on mitogenic responses of
bluegill anterior kidney lymphocytes.


DAYS IN CULTURE


38
differences in temperature on maximum stimulation with the various
mitogens. Cells stimulated with PHA (0.1 yl) and Con A (50 yg) re
sponded well at 32C (p < 0.01) and very poorly, if at all, at 22C
(p > 0.1), whereas LPS (1 yg) responsiveness was higher at 22C
(p < 0.01). There was, however, a significant response (p < 0.05) to
LPS (10 yg) in 32C incubated cultures which was reproducible. Fifty
micrograms of LPS (not shown) were not stimulatory (stimulation indices
_< 1) at either temperature.
The temperature effects described above were found in ten experi
ments with the only major differences being the magnitude of the re
sponses. These differences may have been due to differences in the
serum supplement pools used as discussed previously.
To summarize the results, optimal mitogen doses at 32C were 0.1 yl,
50 yg, and 10 yg for PHA, Con A, and LPS respectively and. 1.0 yg of LPS
at 22C. PHA and Con A responses were greater at 32C than 22C and
LPS responsiveness was greater at 22C than 32C. Optimal culture times
were 5-7 days for all mitogens with the exception of 10 yg of LPS at
32C where some variations were noted.
Limited experiments with spleen, blood, and thymus lymphocytes indi
cated that all were stimulated by PHA, Con A, and LPS. The mitogenic
responses of thymus lymphocytes are presented in Table 5 to demonstrate
that the temperature effects on mitogenic stimulations were also observed
with cells from this tissue and thus were not limited to anterior kidney
lymphocytes.
Mixed Lymphocyte Cultures
Lymphocytes from anterior kidneys of different bluegills were tested
for their ability to respond in two-way mixed lymphocyte cultures


39
Mitogenic Responses
Table 5
of Bluegill Thymus
Stimulation
Lymphocytes
indexa
Mitogen
32C
22C
LPS (1 yg)
4.7
10
PHA (0.1 yl)
30
4
Con A (10 yg)
53
7
(a) Triplicate cultures were pulsed on day 6 and
harvested on day 7.


40
(both populations capable of responding) at 22C and 32C. Cultures
were initiated with 0.25 x 10^ lymphocytes from each donor fish per
culture well (0.5 x 10^ cells total). Controls in mitogen stimulation
studies for each fish also served as controls for mixed lymphocyte
cultures.
Four of ten two-way mixed lymphocyte cultures exhibited statis
tically significant responses (p < 0.05) and are presented in Table 6.
Significant responses were only obtained at 32C thus mimicking re
sponsiveness to PHA and Con A in temperature sensitivity. Furthermore,
these studies have indicated that maximal stimulation (not shown) in
the mixed lymphocyte cultures occurred at seven days. In this experi
ment all six bluegills studied had significant mitogenic responses,
indicating there is no direct correlation between PHA and LPS responsive
ness and the ability to respond to a mixed lymphocyte culture.
Evidence for Different Populations of Bluegill Lymphocytes
Golub (50) has demonstrated that rabbit anti-mouse brain cross re
acts with mouse thymocytes due to a common antigen on both brain and

thymocytes. In an attempt to elicit antiserum capable of recognizing
antigenic surface determinants on bluegill lymphocytes a rabbit was hy-
perimmunized with bluegill brain homogenates following Golub's immuniza
tion procedures. To determine the specificity of this rabbit anti-brain
serum for bluegill anterior kidney lymphocytes, cells were incubated
with the rabbit serum and guinea pig serum. After appropriate incuba
tion and washing only about 30% of the original number of cells remained
viable (as determined by trypan blue exclusion) in contrast to 100%


41
Table 6
Mixed Lymphocyte Responses of Bluegill
Anterior Kidney Lymphocytes
Stimulation Index3
Mixed
Lymphocyte
Response
Mitogen
Response
22C
32C
Fish
Crossc
22C 32C
LPSb
PHA
LPS
PHA
1
1 + 2
1 25
62
2.3
9
95
2
17.2
7.2
8.9
145
3
3+4
1 12
22
16
3.4
51
4
20
3
2.4
55
3
3+5
1 9
22
16
3.4
51
5
24
2.9
5.3
22
4
4+5
1 19
20
3
2.4
55
5
24
2.9
5.3
22
(a)
Triplicate
cultures were pulsed
on day
6 and
harvested
on day
Results are expressed as stimulation indicies as defined in Materials
and Methods.
(b) Mitogen concentrations were 1 yg and 10 yg of LPS at 22C and
32C, respectively, and 0.1 yl of PHA at 22C and 32C.
(c) Designates the source of cells used in the mixed lymphocyte
cultures.


42
recovery of viable cells when preimmune serum from this rabbit was
employed as a control. When the cells surviving the rabbit antiserum
treatment were assayed for mitogen responsiveness, it was found that
the PIIA response was diminished and the LPS response was intact. Re
sults from two such experiments are presented in Table 7. These data
indicate that cytotoxic treatment of anterior kidney lymphocytes with
anti-brain plus complement may be an effective means of obtaining rela
tively pure LPS responsive cells and that this responsive population,
representing ^30% of the original population, may be a subpopulation of
lymphocytes in the bluegill.
Anterior kidney lymphocytes were tested with heterologous red blood
cells for spontaneous rosette formation. Results are presented in
Table 8. Only rabbit red blood cells were capable of rosetting a sig
nificant portion of the lymphocytes (> 20%).
To determine if the rosetted lymphocytes represented a discreet sub
population of the total with respect to mitogen responsiveness, rosetted
cells were depleted from the non-rosetted ones using Hypaque-Ficoll cen
trifugation. Seventy to 75% of the original number of lymphocytes were
recovered as non-rosette formers and were cultured under optimal mito
genic conditions. The results of two experiments are presented in Table
7. The LPS response was diminished while the PHA response was left intact.
These results indicate that depletion of lymphocytes rosetted with
rabbit red blood cells from non-rosetted lymphocytes may be an effective
means of isolating relatively pure PHA-responsive lymphocytes and that
this responsive population, representing 70-75% of the original popula
tion, may be a subpopulation of lymphocytes in the bluegill.


43
Table 7
Mitogen Responses of Bluegill Anterior Kidney
Lymphocytes Treated with Anti-Brain Plus Complement
or Rosette Depleted with Rabbit Red Blood Cells
Stimulation Index3
Expt
1
Expt
2
Expt
3
Treatment
LPSb
PHA
LPS
PHA
LPS
PHA
Control
4.2
9.8
4
12.4
8
9.9
Anti-Brain + Complement0
30
1.3
26
1.3
ND
ND
Rosette Depletion^
1.8
17
NDe
ND
1
9.0
(a) Triplicate cultures incubated at 32C, pulsed on. day 6 and harvested
on day 7.
(b) Mitogen concentrations were 10 yg and 0.1 yl of LPS and PHA respec
tively.
(c) A 1:5 of rabbit anti-bluegill brain plus a 1:10 of guinea pig com
plement was used in the cytotoxic treatments.
(d) Rabbit red blood cell rosetted cells were depleted by Hypaque-Ficoll
centrifugation.
(e) ND = Not Done.


44
Table 8
Rosette Formation of Bluegill Anterior
Kidney Lymphocytes with Red Blood
Cells from Heterologous Species
RBC Source
% Rosetting'
Bluegill
0
Human
0.65 0.2
Ferret
0
Alligator
0
Rabbit
21 1.5
Guinea Pig
0.75 0.2
Horse
0
Mouse
0
Sheep
1.3 0.6
Chicken
2.5 1.0
(a) Results are expressed as percentages of the total number
of bluegill white cells (total number of white cells minus the
number of white cells in the RBC controls) rosetting with the
red blood cells. Each value represents the mean of triplicate
determinations from three different fish standard deviations.
A white cell in contact with 4 RBC constituted a positive
rosette.


45
In Vivo and In Vitro Studies on Antibody Producing Cells
Prior to in vitro primary immunization studies with cell
suspensions from the bluegill, it was first necessary to determine
which organs contained antibody-producing cells. It was also necessary
to determine if bluegill were responsive In vivo to the test antigen
(SRBC) and to establish a suitable complement source for use in the
hemolytic plaque assay.
Bluegill were immunized intraperitoneally with sheep red blood
cells and sacrificed two weeks later. Cell suspensions were prepkred
from the anterior kidney, spleen, and thymus. Only blood was fraction
ated on Hypaque-Ficoll due to difficulties in assaying samples with a
high ratio of red to white cells. Each cell suspension v/as assayed in
a Jeme hemolytic plaque assay for cells producing antibody to sheep
red blood cells.
Wide variations in responsiveness to sheep red blood cells were
observed in immunized bluegill. Results from two individuals are
presented in Table 9. The spleen, anterior kidney, and thymus each
contained considerable numbers of antibody-producing cells. Each
organ had approximately the same number of plaque-forming cells (PFC)
per 10^ cells. Very few plaque-forming cells were present in blood,
though it should be emphasized that blood was fractionated on Hypaque-
Ficoll prior to assay.
Fresh guinea pig serum, grouper serum, alligator serum, bass plasma,
bream plasma, and sucker plasma were diluted 1:20 and used as sources of
complement in the Jerne assay. Only bream, bass,and sucker sera were
effective sources of complement. Since sucker plasma was not used as
medium supplement and was obtainable in large quantities, it was used
routinely as a complement source.


46
Table 9
Distribution of Antibody Forming Cells in Various Tissues
of Bluegill Immunized with Sheep Erythrocytes
White^
Cells
(xlO-6)
Number of
PFC
Fisha
Tissue
Per 106 Cells
Total
1
Blood (2 ml)
9
4
36
Kidney
100
70
7000
Thymus
18
53
954
Spleen
9
52
468
2
Blood (2 ml)
10
1
10
Kidney
184
3
552
Thymus
30
4
120
Spleen
24
2
48
(a) Bluegill were immunized intraperitoneally with 0.1 ml of 10% SRBC
and were sacrificed after two weeks.
(b) Hypaque-Ficoll fractionated peripheral blood cells and unfractionated
organ cell suspensions were assayed for the number of white cells and the
number of plaque forming cells (PFC).


47
The number of cells in the various lymphoid organs containing
cytoplasmic immunoglobulin were assayed by indirect immunofluorescence.
Smears of washed, unfractionated cell suspensions of anterior kidney,
thymus, spleen, blood, and posterior kidney (as a negative control)
were examined and the number of cells showing positive cytoplasmic
immunoglobulin staining quantitated. The results are presented in
Table 10. The posterior kidney was devoid of any Ig-containing cells.
Anterior kidney, spleen, thymus, and blood demonstrated appreciable
numbers of immunoglobulin containing cells. ,
Preliminary studies were undertaken to determine if bluegill lym
phoid cell suspensions would respond In vitro to an antigenic stimulus.
Several modifications of the culture techniques discussed above for
mitogen studies were employed to enrich the culture media and to ensure
that all necessary cellular components were present.
Undialyzed 7% bass plasma rather than bream plasma was used as a
supplement with an enriched RPMI 1640 medium. Since the hemolytic plaque
assay only measured differences in the number of plaque-forming cells
between control and antigen stimulated cultures, a high nonspecific stimu
lus by bass plasma (see mitogenic studies) was irrelevant as long as an in
crease in plaque-forming cells was attributable to antigenic stimulation.
A pool of unfractionated cell suspensions of anterior kidney', spleen,
and thymus was used for three reasons: 1) to increase the number of
available cells and thus the number of variables that could be tested,
2) to include phagocytic and plasma cells as well as any other cell
types possibly involved in antigen processing and antibody formation,
and 3) to decrease the chance of compartmental effects of individual


48
Table 10
Immunoglobulin Producing Cells
in the Lymphoid Organs of the Bluegill
Organa
% Positive^3
Blood
20 5
Spleen
45 11
Thymus
39 15
Anterior Kidney
40 14
Posterior Kidney
0
(a) Smears of blood and organ cell suspensions were assayed by indirect
immunofluorescence for cytoplasm.ic immunoglobulin.
(b) Results are presented as a percent of the total number of white cells
counted and are means of multiple determinations from three bluegill
standard deviations.


49
organs. All cell suspensions used contained < 30% red blood cells and
'v 7-10% phagocytic cells (determined by colloidal carbon untake).
Control (no SRBC) or immunized (with SRBC) cultures were assayed
for PFC in the Jerne hemolytic plaque assay after incubation at 22C
and 32C for various time periods. Two experiments utilizing unimmunized
"normal" bluegill as cell donors are presented in Table 11. In 32C
incubated cultures there were significant increases in the number of
PFC of immunized cultures over control culture responses. The maximum
PFC response as well as the maximum number of recovered cells frojn
immunized than control cultures occured on day 7. More cells were
recovered from immunized than control cultures and on day 7 more than
the initial (Day 0) number of cells were present in immunized cultures.
Viability in the cultures did not change over the ten-day culture
period.
In contrast to the 32C incubated cultures, cultures maintained at
22C did not show a PFC response. There was no significant difference
between control and immunized cultures and the viability was lower after
ten days. *
One preliminary experiment was done with cells from an immunized
bluegill in order to determine if a secondary immunization in vitro
would increase the number of responsive cells. Unlike cells from normal
fish, the PFC response in this fish was observed to occur only at 22C.
The magnitude of the response measured on day 7 however was much lower
(control = PFC, "boosted" = 18 PFC/Culture) than that seen at 32C
v/ith cells from normal animals. It should be pointed out that the
number of recovered cells in the single experiment conducted was higher
in 22C incubated cultures (22C, 90% for controls, 285% for boosted;


Table 11
Primary In_Vitro Immunization of Bluegill Lymphoid
Organ Cell Suspensions with Sheep Red Blood Cells
PFC/Culture
% Recovered^
% Viable0
Experiment
Culture Days
in Culture
22Ca
32C
22C
32C
22C
32C
1
Control
5
NDd
57
ND
95
ND
89
Immunized
5
ND
660
ND
115
ND
93
Control
7
0
50
72
49
88
96
Immunized
7
0
1045
65
111
94
97
Control
10
ND
38
ND
62
ND
86
Immunized
10
ND
810
ND
101
ND
90
2
Control
7
0
0
83
56
89
91
Immunized
7
0
147
77
109
95
93
Control
10
0
0
36
40
73
94
Immunized
10
0
82
36
68
69
90
(a) Cultures
were maintained at
the indicated
temperatures.
-
(b) Cell recoveries are expressed as a percent of the initial number of cells (Day 0).
(c) Viability was determined by trypan blue exclusion and is expressed as a percent of
the total number of cells recovered from cultures.
(d) ND = Not Done.


32C, 4% for controls, 60% for immunized). It thus seems possible
that a major difference between the in_ vitro primary and secondary
responses to sheep erythrocytes may exist although obviously more
work needs to be done before definitive statements are possible.


Discussion
Effects of Plasma Supplements and Fish Maintenance on Lymphocyte Cultures
Two crucial variables, the medium supplement and the health or
physiological state of the fish appeared to be critical in obtaining
high levels of DNA synthesis (i.e. TCA precipitable counts) in un-r
stimulated lymphocyte cultures. The causative factors in these two
situations are unknown but it would seem appropriate to discuss, in a
speculative way, these two points. The influence of serum factors on
in vitro cultured cells has been well documented in other systems (18,38,
64,85,99,100,101,119,120) and it is conceivable in the studies reported
here that one or more such factors were present in some of the plasma
pools used as supplements. Dialysis experiments suggest that a factor(s)
of < 10,000 molecular weight was responsible for nonspecifically ele
vating unstimulated control TCA precipitable counts. It is also inter
esting that Etlinger's mitogenic studies with rainbow trout leukocytes
(46) also revealed serum effects on stimulation indicies.
Numerous effects on fish subjected to environmental changes or
stress have been reported (23,104). For example, physiologically sig
nificant serum alterations in cortisol, glucose, and free fatty acid
levels as well as morphological changes in adrenocortical, medullary,
and pancreatic tissues occur within minutes in goldfish subjected to
the slight stress of an aquarium transfer. The effects of environmental
factors, other than temperature, on the immune responses of fish have,
52


53
however, not been studied. The data presented here suggest that the
altered cellular-state (an increase in blast-like cells concommitant
with an increase in TCA precipitable counts of unstimulated cultures)
in bluegill maintained for long laboratory periods are caused by envi
ronmental factors in the laboratory aquaria. A likely factor (admit
tedly speculative) might involve endotoxemia resulting from bacterial
infections acquired in the aquarium.
Evidence for Two Subpopulations of Lymphocytes
The studies reported here show that there are at least two sub
populations of lymphocytes in the bluegill. One population is stimu
lated by PHA and Con A at 32C and very poorly at 22C. Although not
proven directly, the cells responding in mixed lymphocyte cultures are
probably a subset of the PHA/Con A responsive population since MLC's
were obtained only at 32C. The other population of lymphocytes is
LPS-responsive at both 32C and 22C although responsiveness at 22C
was usually greater.
The two subpopulatins were shown to be different by anti-brain
\
serum cytotoxicity and rosette depletion experiments. The 32C, PHA
responsive population was depleted from the total population by anti
brain plus complement treatment and left intact by depletion of rabbit
RBC rosetted lymphocytes. The converse was true for the LPS-responsive
population. LPS-responsiveness was depleted by removal of rosetted
lymphocytes from the total population and was unaffected by anti-brain
cytotoxicity treatments.
Comparison of Bluegill and Rainbow Trout Mitogenic Studies
Differences between the results of mitogenic studies presented
here with the bluegill and those of Etlinger et al. (46) with rainbow


54
trout leukocytes suggest that there may be major differences between
different species of fish. Unlike the bluegill, rainbow trout con
tained PHA-responsive cells only in the thymus and LPS-responsive cells
only in the anterior kidney in a manner analogous to the compartmental
localization of T- and B-cells in birds and mammals. However, accurate
comparisons of the rainbow trout and bluegill are tenuous due to experi
mental differences. Unfractionated leukocytes, rather than isolated
lymphocytes, were cultured on]y at 19C in the trout studies. It was
also deemed necessary to switch serum supplements to obtain signifi
cant responses to different mitogens with trout cells. There were also
differences in optimal mitogen doses as well as length of time for
maximum mitogenic stimulation between the two species.
It is thus conceivable that true differences in the lymphoid
systems exist between different species of fish. For example, there
are reports that thymuses of some fish species involute with age while
others do not (37). It is suggested that a third species group may
exist in which the thymus differentiates (or de-differentiates) into a
lymphoid organ similar to the anterior kidney, as apparently is the case
with bluegill.
Differences in environmental temperature tolerances may also effect
the in vitro cellular responses. Rainbow trout live in colder environ
ments, and thus evolutionary pressures may have affected the subpopu
lations of lymphocytes to a point where discernible differences in In
vitro temperature responses may not be recognizable. Further in vitro
studies with other species are necessary before adequate comparisons of
this nature can be made.


55
Are Bluegill Lymphocyte Subpopulations T- and B-Cell Equivalents?
By analogy, the mitogenic and mixed lymphocyte culture responses
of bluegill lymphocytes would support the conclusion that fish have
T- and B-cells. Bird and mammalian T-cells respond to PHA and Con A
(but not LPS) and are reactive in mixed lymphocyte cultures. Similarly,
a bluegill lymphocyte subpopulation (depleted of rabbit RBC rosettes)
responds to PHA or Con A when cultured at 32C. The MLC reactive
cells also responded only at 32C and are probably a subset of the
PHA/Con A reactive cell population. Bluegill lymphocytes of the sub
population unaffected by anti-brain plus complement treatment responded
only to LPS, and B-cell mitogen in birds and mammals. However, such con
clusions should be approached with caution until functional activities
are associated with the two bluegill lymphocyte subpopulations.
It should also be pointed out that the spontaneous rosette forma
tion of the B-like cells with rabbit RBC's is in marked contrast to
all other animal species studied, in which the B-cells do not spontane
ously rosette with any RBC's.
Implications of In Vitro Studies
If one assumes that in vitro studies are valid measures of in vivo
events, several explanations or rationalizations of published in vivo
data are possible in light of the in vitro temperature effects on blue
gill lymphocytes.
Numerous reports on the effects of temperature on the immune responses
in fish to bacterial or protein antigens have been published. Avtalion
et al. (7) have suggested that the effects can be explained by two
populations of lymphoid cells; one is the antigen-reactive population
requiring a higher temperature to process the antigen and the other


56
population is responsible for antibody production at either high or
low temperatures. This may be the case if indeed the PHA-(and Con A)
responsive cell is equivalent to the antigen reactive cell and the LPS-
responsive cell is equivalent to the antibody-producing cell.
The participation of the two defined cell populations and the
temperature effects on immune responses of bluegill should be testable
in vitro. In vitro SRBC primed cultures maintained at 32C elicited a
very good plaque forming cell response to SRBC's whereas cells main
tained at 22C gave no response. If the SRBC is a T-dependent antigen
in the bluegill, as in mammalian systems, then application of depletion
techniques (rosette depletion or anti-brain cytotoxicity) should demon
strate whether cellular cooperation between the two subpopulations is
involved in in vitro antibody production. Further application of in
vitro manipulation techniques to the hapten-carrier effect should also
establish if the two subpopulations are indeed T-like and B-like in
function.
The preliminary study utilizing cells from in vivo primed fish also
were supportive of Avtalion's conclusions that fish can respond to a
secondary antigenic challenge at low temperatures only if they are
primed at a higher temperature. In. vitro "boosted" cells responded at
22C, though with lower numbers of plaque-forming cells. However 32C
cell cultures were not responsive, contrary to in^ vivo primary immuniza
tion studies. This may indicate a secondai-y antigenic stimulus at 32C
which elicits a tolerant state or suppressive factor(s).
Yocum et al. (121) have shown that only 16S IgM-like antibody is
produced in the hapten-carrier effect in a marine fish, the searobin.
Apparently the switch from high molecular weight to low molecular weight


57
antibodies (a T-cell controlled event in mice) associated with the
hapten-carrier effect in mammals does not necessarily occur in fish.
However, Uhr et al. (113) demonstrated that goldfish, when acclimated to
a high temperature (35C), were capable of responding to an antigen with
both 16S and 7S antibodies (as opposed to a response at lower tempera
tures of only 16S antibodies). Though it was not proven that the 7S
antibody vas in fact a de_ novo product and not a degradation product of
the 16S antibody or a shed membrane receptor, a PHA, high temperature
responsive cell type conceivably could be functional in controlling the
switch mechanism at 35C in goldfish.
Temperature effects on lymphocytes may not be confined solely to
bluegill lymphocytes. R. C. Ashman, University of Western Australia,
Nedlands (personal communication) has demonstrated an increase in PHA
responsiveness of human T-cells when cultured at 39C rather than 37C.
Armadillos have body temperatures of < 35C, yet the transformation of
lymphocytes stimulated by PHA was increased approximately 2.6 times
when cultured at 37C rather than 33C (91). Perhaps an evaluation of
mitogenic responses of other mammalian lymphocytes cultured in narrower
temperature ranges (37 2C) is warranted. However experiments done
by J. W. Shands, Jr., University of Florida, Gainesville, Fla. (personal
communication) using mouse spleen lymphocytes cultured with LPS and PHA
at 22, 27, 32, 35, 37 and 39C showed the optimal response to both mito
gens was obtained at 37C.
The Bluegill Lymphocyte as an Experimental Model
Differential responses to mitogens by cells cultured at different
temperatures should provide a valuable method to study functional and
physiochemical properties of the cells involved in immune reactions of


58
fish. One could speculate that the temperature effects on lymphocytes
cultured with mitogens are due to changes in membrane fluidity. Theo
retically a more rigid membrane in a PHA-responsive, 22C cultured
lymphocyte could inhibit capping and membrane events leading to cell
activation, whereas a PHA-responsive, 32C cultured cell with a more
fluid membrane could respond. Changes in membrane fluidity would also
account for changes in optimal doses of LPS required at the different
temperatures. Experiments to chemically alter membrane rigidity would
test the concept of temperature sensitive events at the membrane level.
There are alternative explanations for the temperature effects
demonstrated with bluegill lymphocytes, such as conformational changes
in receptor molecules with changes in temperature or the influence of
temperature on intracellular events involved in cell activation. In
any event, the question of why the two subpopulations differ in respon
siveness at different temperatures is an intriguing one. It would ap
pear that fish may offer a unique approach to dissecting cellular events
in the immune response.


CHAPTER III
LYMPHOCYTE HETEROGENEITY IN THE ALLIGATOR
Introduction
The reptiles are thought to represent a pivotal point in the
'i
phylogeny of the immune system since phylogenetically they are a
common ancestor of the birds and mammals. However, as pointed out
by Cohen (56), immunological studies in the reptiles are severely
lacking. The available data, reviewed in (36,37,59), suggest that
reptiles can mount a diversity of immune responses and arguments by
analogy would suggest they likely have T-like and B-like cells lym
phocytes .
Various antigens have been used to elicit both primary and second
ary humoral responses in various reptilian species (36,37,56,72) with a
switch from 19S IgM-like antibody molecules to 7S IgG-like antibody
molecules occurring during secondary responses (4,56). Unfortunately
relatively little has been done to describe the heavy chain isotypes in
the reptiles and thus IgM and IgG (or IgY) designations are at best
tenuous (31). Cells resembling plasma cells have been detected by
fluorescent antibody techniques, electron microscopy and the Jerne
plaque assay (36) in turtles. Thus on the basis of the ability to
elicit antibody responses as well as the demonstration of plasma-like
cells involved in antibody production, the evidence is rather direct
that reptiles have a B-cell equivalent.
59


60
First and second-set skin allograft rejections (37,59) characteris
tic of T-cell reactions in mammals have also been demonstrated in rep
tiles with an anamnestic second-set response. However there is a major
difference between transplantation reactions of reptiles and mammals, in
that reptilian reactions are typically chronic (36,37) as opposed to the
acute rejections occuring in mammals. These data suggest that T-like
functions may differ from those in mammals. Indeed, graft rejection
sites in turtles and snakes are infiltrated very early not only with
lymphocytes and macrophages, but also with an abundance of plasma., cells
(11). This observation suggests that such chronic graft rejections may
be antibody-mediated rather than cellularly (via T-like cell) mediated.
Responses to haptens conjugated to protein carriers have also been
demonstrated in reptiles (8,73) although the hapten-carrier effect has
apparently not been studied. In brief, data demonstrating that reptiles
can 1) show a 19S to 7S switch, 2) produce anti-hapten antibodies,and 3)
undergo graft rejections are at best only circumstantial evidence for
the existence of a T-like cell in these species. In fact one could
conceivably (although perhaps not too convincingly) argue for the exist
ence of only B-like cells from the same data.
Many of the reports from previous In vivo experiments in which
humoral responses to antigenic challenge were tested conflicted with
one another and in some cases there were questions as to whether rep
tiles could respond to antigenic challenges at all (36). Many of these
discrepancies have since been attributed to differences in the tempera
tures at which the animals were maintained after immunization. As early
as 1901, Metchnikoff demonstrated that the alligator responded to diph
theria toxin by forming antitoxin if the alligators were maintained at


61
32-37C, whereas at 22C they did not respond at all (80). More
recently, Evans has presented evidence that desert lizards maintained
at 35C responded well to sheep red blood cells, but if maintained at
50eC or 40C, temperatures well within physiological temperature ranges,
they did not respond as well (47). Also, an active humoral response to
the antigen was stopped if the animals were moved from 35C to the
lower temperature. Wetherall and Turner (118) observed similar
responses to changes in environmental temperatures in another lizard
species. Environmental temperature is also an important factor in
skin allograft rejections, as shown by Borysenko (11). Snapping
turtles accepted allografts when they were maintained at 10C but
viere able to reject the allografts at 25C, and more rapid rejections
were seen at 35C.
The lymphoid organs of several representative reptilian species
have been examined histologically (36,37). A bursa, thymus, spleen,
and gut associated lymphoid aggregates have been demonstrated. How
ever the functional roles of the various organs are lacking and thus it
cannot be stated whether the "bursa-like" organs are sources of B-like
cells or that the thymocytes are T-like cells as seen in the chicken.
In immunized turtles antibody-forming cells were found in the spleen
but not in the thymus (36), but again, the data are only circumstantial
that the lymphoid organs are compartmentalized into T- and B-cell com
ponents. To summarize the current literature, it would appear that
direct evidence for two cell types in any reptile analogous to T- and
B-lymphocytes in birds and mammals is lacking.
The purpose of this portion of the research was to determine in a
direct way if a reptile, the Florida alligator, has a heterogeneous


62
population of lymphocytes akin to T- and B-cells. The approach taken
was similar to that described previously for the bluegill, i.e. 1) to
define a separation technique for the isolation of relatively pure
lymphocytes and to establish appropriate in vitro culture conditions
for these cells, 2) to determine if mitogen stimulation and cell sur
face antigens employed as T- and B-cell probes and membrane markers in
the bird and mammalian systems are applicable to alligator lymphocytes
as in vitro markers,and 3) to separate differing subpopulations of
lymphocytes on the basis of marker differences. Special emphasis.was
also directed towards studying the effects of temperature on alligator
lymphocytes to determine if a cellular basis for the in vivo temperature
effects on the immune responses in reptiles could be demonstrated.


Materials and Methods
Experimental Animals
Florida alligators (Alligator mississippensis) were obtained from
the Florida Game and Fresh Water Fish Commission. Male and female
alligators., 90-150 cm in length, were used. Accurate age determina
tions were not possible, but were estimated to be betxveen three and
five years. Alligators were individually tagged and housed in a 1.5 m
x 6 m outdoor pen at the University of Florida Animal Quarters. The pen
was designed to provide the alligators with easy access to either water
or a dry platform. The alligators were fed daily with monkey biscuits
(Ralston Purina, St. Louis, Mo.) and to satiation twice each week with
fresh fish (bream).
Culture Media
\
Culture media for in vitro mitogenic and primary immunization
studies were as described in Chapter II with the following modifica
tions: 1) Minimum Essential Medium (MEM) with nonessential amino acids
(GIBCO) was substituted for RPMI 1640 and 2) the NaCl concentration of
the complete media was increased to 0.157 M by dissolving 2.400 g NaCl
in the medium prior to adjusting the final volume to 1.0 L. The pre
pared MEM containing extra NaCl was designated Gator MEM (G-MEM) to
distinguish it from mammalian MEM.
The above modifications were also used in preparing medium used for
in vitro primary immunization studies following the procedure presented
in Chapter II.
63


64
Supplement Sources
Alligator, human, calf, fetal calf, and rabbit sera were tested as
media supplements for in vitro studies. Two alligator serum sources
were used: 1) eight different pools (> 10 individual bleedings, 30-40
ml of serum per animal) were obtained from 1-2 kg alligators (2-3 yr
of age) at Herman Brooks' Alligator Farm (Christmas, Fla.) and 2) sera
(100-250 ml serum per bleed) from individual 100-225 kg alligators
(> 10 yr old) which were maintained at Silver Springs Reptile Institute
(Silver Springs, Fla.). The remaining serum sources are indicated in
Chapter II.
Preparation of Cell Suspensions and Counting Techniques
Lymphoid organs and cell descriptions are described in several
references (25,36,77). Methods for the preparation of organ cell sus
pensions described in Chapter II were followed. Blood was drawn from
the internal jugular vein into a heparinized syringe (50 U heparin/10 ml
blood). This method of obtaining alligator blood was originally de
scribed by Herman Brooks (alligator farmer, Christmas, Fla.) and pub
lished by Olson et al. (86). A maximum of 5 ml of an organ cell
suspension or undiluted heparinized whole blood was layered onto
Hypaque-Ficoll (p = 1.077). Techniques for centrifugation, cell
washes, cell counts, and viability determinations are described in
Chapter II.
Culture Techniques
Culture techniques are described in Chapter II with the following
additions or changes: 1) 10% alligator serum was used as a supplement,
2) two additional mitogens, pokeweed mitogen (DIFCO) and purified


65
protein derivative (a gift from Dr. R. Waldman, University of Florida)
were used,and 3) only peripheral blood lymphocytes were used in in vitro
primary immunization studies.
Assay for %-Thymidine Incorporation into DNA
Assay techniques are described in Chapter II.
Stimulation Indices and Statistical Analysis
Statistical analysis and formulas for calculating stimulation
indices are presented in Chapter II.
Autoradiography
Techniques for autoradiography are presented in Chapter II.
Histological and Morphological Techniques
Serial cross sections of paraffin embedded organs were kindly pre
pared by Mr. Larry J. McCumber (Whitney Marine Laboratory, Marineland,
Fla.). Sectioned tissues, as well as cytocentrifuge preparations of
cell suspensions, were stained with May-Grunwald-Giemsa stain.
Preparation of Rabbit Antisera
The brain of one sacrificed alligator was used for immunization
purposes, following techniques described in Chapter II. Antisera from
two rabbits immunized and boosted eight times over a four month period
were used. Preimmune sera from the same rabbits were used as normal
rabbit serum controls.
Rabbit anti-alligator immunoglobulin was prepared by immunizing
rabbits with immunoglobulins isolated by Sephadex G-200 (Pharmacia)
column chromatography. An ammonium sulfate precipitate of alligator
serurn was applied to the column.


66
Cytotoxicity Assay
The protocol described in Chapter II was followed.
Rosetting Techniques
The method of Jondal et aT. (67) as described in Chapter II was
used to assess the number of peripheral blood lymphocytes capable of
rosetting with sheep red blood cells.
Immunofluoresence
The methods described in Nairn (84) were followed for indirect
immunofluoresent stains of cytocentrifuge preparations of cell suspen
sions normal rabbit serum or rabbit anti-alligator immunoglobulin and a
fluorescein labeled goat anti-rabbit IgG. Immunofluorescent methods for
membrane stains are described in Chapter IV.
Hemolytic Plaque Assay
The techniques for harvesting cultured cells and assaying for
plaque-forming cells are described in Chapter II. Fresh alligator
serum diluted 1:20 was used as a complement source.
Cellular Immunoadsorbents
The method of Chess ejt al. (24) was used for fractionating alliga
tor peripheral blood lymphocytes on cellular immunoadsorbents. Rabbit
anti-alligator immunoglobulin was precipitated with 40% ammonium sulfate,
washed three times and redissolved in 0.15 M NaCl. The immunoglobulin
enriched fraction was then dialyzed against 0.15 M NaCl 0.005 M
Na^B^O^ (pH 8.3) prior to coupling onto CnBr activated Sephadex G-200
(Pharmacia). Preimmune rabbit serum, treated in an identical manner,
was coupled to Sephadex as a control.


67
Affinity columns were prepared as follows: The coupled Sephadex
G-20 preparations were washed with 5% FCS in G-MEM and 8 ml of packed
volume was poured under 1 x g into 12 ml disposable syringes. Two and
one half billion cells in 2.5 ml of 5% FCS in G-MEM were loaded di'op-
wise (10 drops/min) followed by the slow dropwise addition of 5% FCS
in G-MEM. Elutions were monitored periodically until the effluent was
cell free. The nonadherent cells were washed three times with medium
prior to further use.
Glass Wool Fractionation
The method described by Trizio and Cudkowicz (110) was adapted for
use in glass wool and nylon wool column fractionations of alligator
peripheral blood lymphocytes. Glass wool (Corning Glass Works, Corning,
N.Y.) was pretreated by rinsing three times with pyrogen free 0.15 M
NaCl, boiled 1 hr in tripled-distilled water (three changes) and dried
by lyophilization. Twelve milliliter disposable syringes were packed
to the 8 ml mark with the pretreated glass wool and sterilized. Prior to
loading cells on the prepared column, 40 ml of prewarmed (32C) G-MEM
was passed through the column followed by 15 ml of 5% FCS in G-MEM. The
column was then incubated for 30 min at 32C in 5% C02~95% air. One hun
dred million cells in 2 ml of 5% FCS in G-MEM were loaded onto each
column and were washed into the column with 1 ml of 5% FCS in G-MEM.
Loaded columns were incubated in a vertical position at 32C for 1 hr
in 5% C02~95% air. Nonadherent cells were eluted very slowly (20 drops/
min) with 20 ml of 5% FCS in G-MEM (32 C). Fifteen milliliters of warm
5% FCS in G-MEM were then slowly flowed through as a "buffer" between the
nonadherent and the adherent fractions. Care was taken not to gener
ate a fluid head of pressure nor to jar the column during the slow


68
elution of the nonadherent cells or the "buffer" flow through. Ad
herent cells were eluted in a 40 ml volume of G-MEM by generating a
fluid head of pressure as well as mechanically disrupting the glass
wool. Cell fractions were washed three times prior to further
analysis.
A procedure identical to that described in the preceding paragraph
was followed in the preparation and use of nylon wool columns.


Results
Lymphoid Organs of the Alligator
Since the Florida alligator is listed by the Florida Game and Fresh
Water Fish Commission as an endangered species, only a limited number
of alligators were available for experimental purposes. Fortunately,it
was easy to obtain large amounts of blood which was an abundant source
7
of lymphocytes (1-2 x 10 lymphocytes/ml of whole blood) There were no
detrimental effects to the animals. Evidence will be presented in a
subsequent section that the population of lymphocytes isolated from
peripheral blood are representative (on the basis of mitogenic respon
siveness) of the lymphocytic cells isolated from the spleen.
Two of ten alligators obtained from the Florida Game and Fresh
Water Fish Commission were sacrificed (by special permit) for histologi
cal examinations and in vitro mitogenic studies of the lymphoid organs.
The only recognizable lymphoid organs were the thymus and the spleen.
The thymus was a small whitish organ, approximately 2 x 8 mm located
in the throat. Histological examinations of tissue sections showed an
abundance of lymphocytes and signs of thymic involution were seen. Very
few cells were isolated by Hypaque-Ficoll centrifugation from whole
organ cell suspensions (< 5 x 10^). The spleen of the alligator was
a red, kidney-bean shaped organ, located beneath the stomach, and was
surrounded by a thick capsule. Red and white pulp regions were observed
in tissue sections and a heterogeneous population of white cells was
69


70
seen. Only 2-5 x 10 cells ivere isolated from Hypaque-Ficoll isolated
preparations of whole spleen cell suspensions.
Small aggregates of lymphoid cells were present in glandular tis
sues found in the orbital sinus and the area of the cloaca. However
further histological studies are necessary before these tissue can be
defined as lymphoid equivalents of the Harder's Gland or Bursa found
in birds. In vitro studies of these tissues were not possible due to
the very few cells isolated by Hypaque-Ficoll gradient centrifugation.
No gut associated lymphoid tissue or lymph nodes were found.
Separation Technique
Hypaque-Ficoll (p = 1.077) was used to isolate relatively pure
lymphocyte preparations from heparinized whole blood or organ cell sus
pensions. White cell differential counts of fractionated and unfrac
tionated blood are presented in Table 12 and illustrate the efficiency
of the technique for isolating lymphocytes. Hypaque-Ficoll isolates
routinely contained only about 5% granulocytic cells (predominately
basophilic staining cells by May-Grunwald-Giemsa stain), and about 5%
%
red blood cells. Approximately 2-3% of the granulocytes were phagocytic
(assayed by collodial carbon uptake). Examination of the cells recov
ered from the interface and within the Hypaque-Ficoll gradient showed
> 99% of the lymphocytes were present at the interface. A photomicro
graph of a representative isolate is presented in Figure 7.
Culture Conditions
Various sera were tested to determine a suitable supplement with
MEM for in vitro studies. Ten percent alligator, human, calf, fetal
calf, and rabbit sera or all combinations of equimixtures (5% per serum)


71
Table 12
White Cell Differentials of Alligator Whole
Blood and Hypaque-Ficoll Isolated Blood Cells
Percent of Total
b
Cell Typea
Blood
Hypaque-Ficoll Isolated
Thrombocyte
42
0
*
Granulocyte
364
5+5
Lymphocyte
602
955
(a) Smears were made of whole blood and Hypaque-Ficoll isolates
of individual samples and were May-Grunwald-Giemsa stained for
quantitation purposes.
(b) Results are expressed as a percent of the total number of
white blood cells counted.
(c) Each value represents the mean of determinations from 10 dif
ferent alligator samples (> 3 determinations per samples) standard
deviations.


72
Figure 7. Photomicrograph of a representative Hypaque-Ficoll isolate
of alligator peripheral blood. May-Grunwald-Giemsa stained. Magnifi
cation x 400.


73
of any two were tested. Only 10% alligator serum and 5% alligator-5%
fetal calf serum supported in mitogen stimulation of lymphocytes. Al
though significant stimulation was obtained in cultures supplemented
with an equimixture of alligator and fetal calf sera, stimulation in
dices were lower than those obtained from 10% alligator serum supple
mented cultures and therefore 10% alligator serum was used routinely.
Not all alligator sera were supportive as a supplement and it was
necessary to test new alligator supplement sources in mitogenic assays
to determine their suitability. Tests of eight serum pools (> 10.
individual bleedings per pool) obtained from 2-3 yr old alligators
and four individual alligators > 10 yr old are presented in Table 13.
Individual sera from older alligators were more effective than pools of
sera from younger alligators. Since large volumes (200-500 ml) could
be obtained from individual bleedings of 100-225 kg alligators, the
older alligators were used exclusively as sources of serum in subse
quent experiments.
Although statistically significant stimulation of alligator lym
phocytes cultured with mitogens was obtained using 10% alligator serum
supplemented MEM (0.117 M NaCl), severe cell clumping and loss of
viability were noted when cells were suspended in the culture medium.
To determine if the salt concentrations in the medium were appropriate
ly matched to alligator serum levels, three alligator sera (obtained from
individual bleedings) were analyzed by the Blood Chemistry Lab (Depart
ment of Pathology, J. Hillis Miller Health Center). Comparisons of the
chemistry lab reports with the GIBCO MEM formulations revealed a repro
ducible difference in the NaCl concentrations. On the basis of this
finding an experiment in which alligator peripheral blood lymphocytes


74
Table 13
PHA Responses of Alligator Peripheral Blood Lymphocytes
Cultured with Different Alligator Serum Supplements
Supplement
Poolb A
B
C
D
E
F
.G
H
Alligator0 AA
BB
CC
DD
Stimulation Index5
1
36
45
18
18
7
5
19
78
1
90
100
(a) Triplicate cultures were incubated at 32C with or without PHA
(1 ¡J.I), pulsed on day 4 and harvested on day 5.
(b) Each supplement pool is from > 10 individual bleeds of 1-2 kg
alligators 2-3 yrs old.
(c) Individual serum supplements are from bleeds of 100-225 kg alli
gators > 10 yrs old.


75
were stimulated with PHA in different MEM preparations containing various
concentrations of NaCl was conducted. The results of this experiment are
presented in Table 14. TCA precipitable counts of PHA-stimulated cells
cultured in mammalian MEM were significantly increased (p < 0.05) over
control counts. However,stimulation indices of cells cultured with
0.157 M or 0.177 M NaCl concentrations (0.040 M and 0.060 M extra NaCl
respectively) were approximately three times greater than the stimula
tion index of cells cultured in mammalian MEM. Also cell clumping and
loss of viability were no longer evident. Therefore,the NaCl concentra
tion of MEM was routinely increased by 0.04 M to 0.157 M in all media
used in subsequent in vitro studies with alligator lymphocytes.
To determine if optimal conditions for pulsing mammalian cultures
with "*11-thymidine (0.5-1.0 pCi/culture; 24 hr) were applicable for
3
alligator lymphocyte cultures, the effects of H-thymidine concentra
tions used per well and the length of the pulse were examined. The
data presented in Tables 15 and 16 indicate that incubating the cultures
with 0.5 yCi 3H-thymidine for 24 hr prior to culture termination was
optimal for pulsing alligator lymphocyte cultures.
Mitogenic Studies
Since large numbers of lymphocytes could be obtained from single
O
bleedings (4-8 x 10 lymphocytes from 40 ml of blood), large scale
experiments were designed to determine the effects of 1) mitogen dose,
2) length of time in culture, and 3) temperature on the responses of
peripheral blood lymphocytes to phyrtohemagglutinin (PHA), concanavalin
A (Con A), 1ipopolysaccaride (LPS), pokeweed mitogen (PWM), and purified
protein derivative (PPD).


76
Table 14
Effect of Sodium Chloride Concentration on Alligator
Peripheral Blood Lymphocytes Cultured with PHA
a
NaCl Concentration
Stimulation Index^
0.117 Mc
47
0.137 M
85
0.157 M
142
0.177 M
145
0.197 M
59
0.217 M
1.0
(a) Final concentrations of NaCl
in the culture medium.
(b) Cultures were incubated at 32C with or without PHA (1 yl), pulsed
on day 4 and harvested on day 5.
(c) The concentration of NaCl in mammalian MEM was calculated from the
GIBCO formulation to be 0.117 M.


77
Table 15
Effect of H-Thymidine Concentration on Alligator
Peripheral Blood Lymphocytes Cultured with PHA
. 3 ,
yCi H/Culture
Stimulation Index3-
0.05
30
0.1
60 ,
0.25
66
0.5
71
1.0
71
2.0
67
(a) Ceils were incubated at 32C with or without PEA (1 yl), pulsed on
day 4 and harvested on day 5.


78
Table 16
7
Effect of Incubation Time with H-Thymidine on PHA
Stimulated Alligator Peripheral Blood Lymphocytes
Length of Time withc
^H-Thymidine (Hr)
Stimulation Index
24
108
48
119
72
112
96
110
(a) Cells were cultured at 32C with or without PHA (1 pi)
pCi of ^H-thymidine was added at various intervals prior to
cultures. All cultures were terminated on day 5.
Y
Five-tenths
the harvest of


79
The results of one experiment designed to test the effects of tem
perature on responsiveness of alligator lymphocytes to PHA are presented
in Figure 8. Cells were cultured with various doses of PHA at the tem
peratures indicated and the optimal dose and the length of time for max
imum stimulation was determined at each temperature. The results indi
cate that the lower the temperature, the longer the time required for
maximum stimulation (indicated in parenthesis). The response of cells
cultured at 22, 35, 37, and 40C was significantly lower (p < 0.01)
than in cells cultured at 27, 30, and 32C. Although responses pf
cells incubated at 27, 30 and 32C were not significantly different
from each other (p > 0.1), the length of time required for optimal stim
ulation of cultures maintained at 32C was shorter (five days) as com
pared with 27 and 30C maintained cultures (seven days). The optimal
mitogen dose was found to be the same at all temperatures. Typical
responses to various mitogen doses and incubation times of alligator
lymphocytes cultured with PHA and LPS at 32C are presented in Figures 9
and 10 respectively. The response to PHA peaked sharply on day 5 and
decreased slowly, whereas the peak response to LPS remained elevated
after reaching an optimum on day 5. Similar experiments were performed
with each of the other mitogens and the results can be summarized by
stating that the optimal temperature tested was found to be 32C, the
length of time for maximum stimulation was five days and the optimal
mitogen dose was the same at each temperature tested. Optimal doses
per culture of LPS, PPD, PWM, PHA and Con A were 10 yg, 10 yl, 1 yl, and
20 yg respectively. It should be pointed out that responses of cells
cultured with 20 yg of Con A varied in different experiments and was
attributed to changes in the lot numbers of Con A used, as well as the


Figure 8. The effects of temperature on the responsiveness of alligator
peripheral blood lymphocytes to PHA. Cultures were stimulated with 1 pi
of PHA. Numbers within parentheses indicate the length of time (days)
to maximum stimulation at the designated temperature.


00
80
60
40
20
TEMPERATURE (C)


Figure 9. Dose and time response of alligator peripheral blood lymphocytes
cultured with PHA. All cultures were incubated at 32C.


30-
CONTROL
£5 0.0i JJ / culture
LO 0.1
CPM
¡3 1.0
20-
X !0"3
0 5
ES 20
10-
jjL|:2sn_
_ES1
&
*1
1
3
I
I!
i
*
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4i
ik
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DAYS IN
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II
CULTURE


Figure 10. Dose and time response of alligator peripheral blood lymphocyt
cultured with LPS. All cultures were incubated at 32C.


C PM
XI3 3
CONTROL
1 O.i ug / CULTURE
.0
DAYS IN
7 9
CULTURE
II
00
01


86
length of time a mitogen solution was stored at 4C. Responses to PHA
or Con A were always significantly greater (p < 0.01) than responses to
LPS, PIVM, or PPD, with stimulation indices of PHA or Con A ranging from
40-250 and those for LPS, PWM,or PPD stimulated cultures between 1-25.
Assay for In Vitro Cellular Reactions
To determine if TCA precipitable counts were a valid measure of
cellular events in culture, the number of labeled cells stimulated
with various concentrations of LPS or PHA were correlated with the
Y
TCA precipitable counts in experiments (Chapter II). The results are
presented in Figures 11 and 12 and indicate that both LPS-and PHA-stim-
ulated cultures exhibit changes in TCA precipitable counts closely
paralleling those changes in percent of labeled cells identified by
autoradiography. Cells optimally stimulated with PHA (1 yl) were pre
dominately in aggregates and looked like lymphoblasts (Figures 13a and
13b). Cells optimally stimulated with LPS (10 yg) were also morphologi
cally characterized as blast-like but were not clumped (Figures 14a and
14b).
Comparison of Peripheral Blood and Splenic Lymphocyte Mitogen Responses
To assay whether mitogenic responses of peripheral blood lympho
cytes were similar to the mitogenic responses of lymphocytes from other
sources, cell suspensions were prepared from various alligator lymphoid
tissues. Only the spleen cell suspension yielded a sufficient number
of lymphocytes (isolated by Hypaque-Ficoll) to culture in a mitogen
assay. The results obtained from mitogenic stimulations of peripheral
blood and splenic lymphocytes are presented in Table 17. Optimal dose
and time responses of both isolated lymphocyte populations were the same


Figure 11. Correlation of TCA precipitable counts with the number of
autoradiography positive cells from PHA-stimulated alligator lymphocyte
cultures. Cultures were incubated at 32C, pulsed on day 4 and assayed
on day 5.


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INGEST IEID ESZR2BOSN_24HEU7 INGEST_TIME 2017-07-14T22:38:56Z PACKAGE AA00006116_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES


LYMPHOCYTE HETEROGENEITY IN
TELEOSTS AND REPTILES
By
MARVIN AGUSTA CUCHENS
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
1977

ACKNOWLEDGMENTS
The author wishes to express his appreciation to those who have
helped to make this work possible. I am most appreciative of Dr. L.
W. Clem for his support in this research for his continued encourage-
*
ment and suggestions. Special thanks are also extended to Dr. R. B.
Crandall, Dr. C. A. Crandall, Dr. B. Gebhardt, Dr. J. W. Shands, Jr.,
Dr. P. A. Small, and other members of the department for their assistance
throughout this work.
A very special appreciation is expressed for my mother and father
for their continued support throughout my academic career'and for my
wife for the typing of this manuscript as well as for her patience,
understanding, and love.
n

TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS ii
LIST OF TABLES v
LIST OF FIGURES ! vii
KEY TO ABBREVIATIONS ix
ABSTRACT x
CHAPTER
I INTRODUCTION 1
II LYMPHOCYTE HETEROGENEITY IN THE BLUEGILL 4
Introduction 4
Materials and Methods 8
Results 18
Discussion 52
III LYMPHOCYTE HETEROGENEITY IN THE ALLIGATOR 59
Introduction 59
Materials and Methods 63
Results 69
Discussion 119
IV MEMBRANE IMMUNOGLOBULINS OF BLUEGILL LYMPHOCYTES ... 128
Introduction 128
Materials and Methods 130
iii

TABLE OF CONTENTS (continued)
CHAPTER Page
IV Results 134
(continued)
Discussion 149
LITERATURE CITED 153
BIOGRAPHICAL SKETCH 162
5»
iv

LIST OF TABLES
Table Page
1 White Cell Differentials of Bluegill Whole Blood and
Hypaque-Fic.oll Isolated Blood Cells 22
2 Effect of Dialysis of Plasma Supplements on Mitogenic
Stimulation of Bluegill Anterior Kidney Lymphocytes. 26
*
3 Effect of Maintenance Time of Bluegill in Laboratory
Aquaria on Differential White Cell Counts of
Hypaque-Ficoll Isolated Anterior Kidney Cells. ... 28
4 Effect of Maintenance Time of Bluegill in Laboratory
Aquaria on the Incorporation of Thymidine by
Unstimulated Anterior Kidney Cell Cultures 30
5 Mitogenic Responses of Bluegill Thymus Lymphocytes. . . 39
6 Mixed Lymphocyte Responses of Bluegill Anterior Kidney
Lymphocytes 41
7 Mitogen Responses of Bluegill Anterior Kidney Lympho¬
cytes Treated with Anti-Brain Plus Complement or
Rosette Depleted with Rabbit Red Blood Cells .... 43
8 Rosette Formation of Bluegill Anterior Kidney Lympho¬
cytes with Red Blood Cells from Hetei’ologous
Species 44
9 Distribution of Antibody Forming Cells in Various Tissues
of Bluegill Immunized with Sheep Erythrocytes. ... 46
10 Immunoglobulin Producing Cells in the Lymphoid Organs
of the Bluegill 48
11 Primary In. Vitro Immunization of Bluegill Lymphoid Organ
Cell Suspensions with Sheep Red Blood Cells 50
12 White Cell Differentials of Alligator Whole Blood and
Hypaque-Ficoll Isolated Blood Cells 71
13 PHA Responses of Alligator Peripheral Blood Lymphocytes
Cultured with Different Alligator Serum Supplements. 74
v

15
16
17
18
19
20
21
22
23
24
25
26
27
Page
Effect of Sodium Chloride Concentration on Alligator
Peripheral Blood Lymphocytes Cultured with PHA ... 76
3
Effect of H-Thymidine Concentration on Alligator
Peripheral Blood Lymphocytes Cultured with PHA ... 77
3
Effect of Incubation Time with H-Thymidine on PHA
Stimulated Alligator Peripheral Blood Lymphocytes. . 78
A Comparison of the Mitogen Responses of Alligator
Blood and Splenic Lymphocytes 95
Mixed Lymphocyte Cultures of Alligator Peripheral
Blood Lymphocytes ' 97
Combined Effects of Mitogens on Alligator Peripheral
Blood Lymphocytes 98
Mitogen Responses of Peripheral Blood Lymphocytes from
Alligators Maintained at 16°C 102
Mitogen Responses of Cell Populations Fractionated on
Glass Wool 106
The Effects of Cytotoxic Treatment with Rabbit Anti-
Alligator Immunoglobulin on the Mitogen Responsive¬
ness of Alligator Peripheral Blood Lymphocytes . . . 112
Depletion of LPS and PWM Responsiveness in Alligator
Peripheral Blood Lymphocytes Passed Through an Anti¬
immunoglobulin Immunoadsorbent 114
9
Cytoplasmic Immunofluorescence Studies of Uncultured
and Cultured Alligator Peripheral Blood Lymphocytes. 116
Primary In Vitro Immunization with Sheep Red Blood Cells
of Alligator Peripheral Blood Lymphocytes 117
Quantitation of Surface Immunoglobulin on Bluegill and
Mouse Lymphocytes 139
Effects of Pronase Digestion on Membrane Associated
Immunoglobulins of Bluegill and Mouse Lymphocytes. . 147
vi

LIST OF FIGURES
Figure Page
1 Photomicrographs of representative serial sections
through the gill region of a small bluegill 20
2 Photomicrograph of a representative Hypaque-Ficoll
isolate from bluegill blood 23
3 Photomicrograph of a representative Hypaque-Ficoll
isolate of bluegill blood after long term
laboratory maintenance of the bluegill 29
4 Correlation of TCA precipitable counts with the number
of autoradiography positive cells from PHA stimulated
bluegill lymphocyte cultures 33
5 Photomicrograph of PHA-stimulated bluegill anterior
kidney lymphocytes 35
6 Temperature effects on mitogenic responses of bluegill
anterior kidney lymphocytes 37
7 Photomicrograph of a representative Hypaque-Ficoll
isolate of alligator peripheral blood 72
■»
8 The effects of temperature on the responsiveness of
alligator peripheral blood lymphocytes to PHA 81
9 Dose and time response of alligator peripheral blood
lymphocytes cultured with PHA 83
10 Dose and time response of alligator peripheral blood
lymphocytes cultured with LPS 85
11 Correlation of TCA precipitable counts with the number
of autoradiography positive cells from PHA
stimulated alligator lymphocyte cultures 88
12 Correlation of TCA precipitable counts with the number
of autoradiography positive cells from LPS
stimulated alligator lymphocyte cultures 90
15 Photomicrograph of PHA-stimulated alligator peripheral
blood lymphocytes 92
vii

Figure Page
14 Photomicrograph of LPS-stimulated alligator peripheral
blood lymphocytes 94
15 A chronological study during the winter months of
alligator peripheral blood lymphocytes 101
16 Diagram of glass wool fractionation procedures 105
17 Effects of increasing the cell density in mitogen
stimulated cultures of alligator peripheral
blood lymphocytes 110
18 Immunoprecipitation of lysates of membrane labeled
bluegill lymphocytes 137
19 Acrylamide gel electrophoresis in sodium dodecyl
sulfate of extensively reduced immune precipitates
of bluegill white blood cell membrane immunoglobu¬
lins 140
20 Acrylamide gel electrophoresis in sodium dodecyl
sulfate of extensively reduced precipitates of
bluegill spleen and thymus membrane immunoglobu¬
lins 142
21 Agarose-acrylamide gel electrophoresis and gel
filtration of unreduced immune precipitates of
bluegill white blood cell membrane immunoglobu¬
lins 144
22 Acrylamide gel electrophoresis in the presence of
sodium dodecyl sulfate of extensively reduced
bluegill white blood cell membrane immunoglobu¬
lins fractionated by gel filtration 145
viii

KEY TO ABBREVIATIONS
Con A
CPM
DNA
G-MEM
H chain
3H
Ig
L chain
LPS
MFM
MLC
PEC
PHA
PPD
PWM
Ra-BIg
Ra-GL
Ra-M IgM....
RBC
RPMI 1640...
Roswell Park Memorial Institute medium 1640
SDS
SRBC
TCA
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
LYMPHOCYTE HETEROGENEITY IN
TELEOSTS AND REPTILES
By
Marvin Agusta Cuchens
August 1977
Chairman: Dr. William L. Clem
Major Department: Immunology and Medical Microbiology
The purpose of this research was to study the characteristics
of the lymphoid cells from two ectotherms, the bluegill, a repre¬
sentative teleost, and the Florida alligator, a representative
reptile. The questions approached were 1) whether or not these
animals possessed a heterogeneity of lymphocytes akin to T- and B-
cells of higher animals, 2) whether a cellular basis for the effects
of temperature on the immune response of ectotherms could be ob¬
tained, and 3) whether there are membrane-associated immunoglobulins
in fish.
Hypaque-Ficoll centrifugation was used as a separation technique
for the isolation of lymphocytes. In vitro mitogenic studies of iso¬
lated lymphocytes from each species established that homologous serum
was the most satisfactory medium supplement. Bluegill studies demon¬
strated that the health or physiological state of laboratory maintained
fish appeared to be important in obtaining low background levels of DNA
synthesis. Variables found to be important in alligator lymphocyte
studies were the NaCl concentration in the medium and the age of the
serum donor.
x

Studies of the bluegill have shown that there are at least
two subpopulations of lymphocytes. One population was stimulated by
PHA (and Con A) at 32°C and very poorly at 22°C and was depleted by
antibrain plus complement treatment. The other population is LPS
responsive at both 32°C and 22°C, although responsiveness at 22°C was
always greater and was depleted by removal of rabbit RBC rosetted
lymphocytes from the total population.
Temperature was also shown to be an important factor in in_ vitro
antigenic stimulations. In vitro SRBC primed cultures maintained at
32°C elicited a very good plaque-forming cell response to SRBC's where¬
as 22°C maintained cells gave no response. The temperature effects on
the in vitro cultures are discussed in reference to the reported fn vivo
temperature effects on the teleost immune functions.
Evidence has been presented which argues for the presence of at
least two cell populations of lymphocytes in the alligator. Summarized
these are 1) differences in the magnitude of stimulation with the dif¬
ferent mitogens* 2) differences in the combined effects of the mitogens,
3) a significant increase in immunoglobulin producing cells in LPS-
stimulated cultures, 4) populations of cells adherent or nonadherent to
glass wool with different responses to LPS and PHA, 5) the depletion of
responsiveness to LPS by cytotoxic treatment with an anti-immunoglobulin
plus complement without reducing the responsiveness to PHA, and 6) the
depletion of responsiveness to LPS by removing immunoglobulin bearing
cells.
Environmental temperature was shown to effect the in vitro mitogenic
responses of cultured alligator lymphocytes. Although there were some
xi

fluctuations in PHA responsiveness, LPS responses dropped significantly
during the winter months or when alligators were housed at 16°C.
T-like and B-like designations were assigned to the different
populations in the bluegill and alligator based on arguments by analogy
to bird and mammalian T- and B-lymphocyte characteristics.
Studies of membrane immunoglobulins on bluegill lymphocytes from
blood, anterior kidney, spleen, and thymus revealed that over 90% of the
lymphocytes exhibited membrane immunoglobulin determinants as revealed
by immunofluorescence. The majority of these cells were observed^ to
undergo patching and capping when the membrane proteins were complexed
with antisera to fish immunoglobulins. Lactoperoxidase catalyed radio-
iodination, detergent lysis and immunoprecipitation with appropriate
anitsera wore employed to study the properties of this membrane immuno¬
globulin. Quantitation indicated the average amount of immunoglobulin
determinants for bluegill lymphocytes to be similar to that present on
mouse B-ceils. Physicochemical characterization of labeled membrane
immunoglobulin from bluegill lymphocytes suggested that only one class
of immunoglobulin heavy chain was present and that about one-half of
this material resembled the monomeric IgM-like proteins present in
bluegill serum.
Xll

CHAPTER I
INTRODUCTION
Immunity in the vertebrates may be defined as a response of an
animal to a foreign substance (an antigen or immunogen) introduced into
its body. The response is specific in that it is directed only to the
antigen introduced and is characteristically more pronounced and occurs
sooner if the same antigen is reintroduced at a later time (43) .
Immune responses in birds and mammals may be cellular (specifically
reactive cells) or humoral (antibody mediated) (43,54,93). Characteris¬
tic cellular responses are delayed type hypersensitivity reactions, graft
rejection and graft-versus-host reactions. Specific cellular responses
are transferable by lymphocytes. Humoral responses are characterized
by the production of antibody directed to an antigen and the resulting
immunity is transferable by serum.
The lymphocyte has been demonstrated to be the principal cell type
involved in the immune responses of birds and mammals. Although lympho¬
cytes are morphologically identical, two major subpopulations have been
identified based on ontological origin and functional analysis (43,54,
93). One subpopulation, the T-(thymus derived) lymphocyte, is the func¬
tional cell in cellular mediated responses. The other subpopulation,
the B-(bursa derived in birds or bursal equivalent in mammals) lympho¬
cyte, is the functional cell in producing antibodies in humoral responses.
T- and B-lymphocytes have been further characterized on the basis of cell
surface determinants and in vitro responses to mitogens, antigens and
1

2
mixed lymphocyte reactions (43,53,54,93). B-cells respond to different
mitogens (e.g iipopolysaccharide), have demonstrable levels of immuno¬
globulin on their surfaces, and are stimulated by mitogens or antigens
to synthesize immunoglobulin or antibody. In contrast T-cells prolif¬
erate in response to different mitogens (e.g. phytohemagglutinin and
concanavalin A), do not have surface immunoglobulin (or at least easily
demonstrable levels), express surface differentiation antigens not found
on B-cells (e.g. Thy-1) and are the responding cells in mixed lymphocyte
reactions. '
A unique feature of the lymphocytes involved in the immune response
is the requirement of T-B cell cooperation in most responses leading to
antibody production, even though the T-cell does not make antibody (49,
52,54,81). This requirement is best illustrated using hapten-carrier
antigen complexes (19,28,54,69) in which carrier recognition by T-cells
is required before B-cells can make antibody to the hapten. T-cells are
also involved in the control of the 19S to 75 switch (IgM to IgG anti¬
bodies) as well as maturation of the humoral response (increase in anti-
â– 4
body affinity with time after immunization) (19,54,69). It should be
pointed out that except for a few antigens which are structurally very
repetitious (the T-independent antigens capable of reacting directly with
B-cells), both cell types must interact to elicit a response to most
antigens (T-dependent antigens) (28,54,69,82).
The majority of much of the research on the immune systems briefly
described above has been in birds and mammals, both of which are endo¬
thermic. With the exception of limited studies of the amphibians, stud¬
ies of the cellular basis of the immune systems in ectotherms have not
been done. Although one could postulate the existence of T-like and

3
B-like cells on the basis of graft rejection and antibody production
(discussed in detail later) direct evidence of lymphocyte heterogeneity
in any ectotherm has not been obtained. Furthermore the molecular or
cellular bases for the commonly observed effects of environmental tem¬
perature on the immune responses of numerous ectotherms (7,37) have not
been investigated.
The purpose of the research undertaken here was to study the char¬
acteristics of the lymphoid cells from two ectotherms, the bluegill,
a representative teleost, and the Florida alligator, a representative
reptile. The major questions approached were 1) whether these two ani¬
mals possessed classes of lymphocytes akin to T- and B-cells of higher
animals, 2) whether a cellular basis for the effects of temperature on
the immune response of ectotherms could be obtained, and 3) whether there
are membrane associated immunoglobulins in fish.

CHAPTER II
LYMPHOCYTE HETEROGENEITY IN THE BLUEGILL
Introduction
Considerable evidence from in vivo studies indicates that teleost
fish can mount a diversity of immune responses. Teleosts are capable
of responding to a wide variety of antigens with both primary and secon¬
dary responses (4,7,15,32,37,51,112) with the only apparent major dif¬
ference from mammals being that there is no discernable "IgM -*â–  IgG
switch" in the fish (1, 16,27,62,111, 121). In fact the evidence avail¬
able to date shows that many species of fish synthesize only 16S tetra-
meric IgM-like immunoglobulin (1,16,27,62,111). In those fish also pos¬
sessing low molecular weight serum immunoglobulins, the 7S molecules
appear to resemble monomeric forms of the tetramer and hence it seems
that fish are lacking an IgG equivalent (21,29,30,31,55,75). Attempts
to demonstrate IgA-or IgE-like molecules or activities in fish have also
been unsuccessful (31). Thus, in light of these latter deficiencies it
would seem appropriate to speculate that while fish possess cells of B-
like function, their number of immunoglobulin classes is somewhat limit¬
ed. Several investigators have also demonstrated the ability of fish to
reject both first and second set scale transplants with the second set
rejections occurring more rapidly (12,59,60,61,88). Thus, again arguing
by analogy, it appears that fish have cells with T-like function. Fur¬
thermore, studies on three different species of fish have revealed the
4

5
existence of the so-called hapten-carrier effect (46,106,121). Since
this "helper effect" is considered to result from T-B cell collaboration
in mammals it appears that fish may also have this coordinated function
in their immune response system.
Since fish are ectothermic animals it is not surprising that numer¬
ous reports of temperature influences on immune responses have appeared.
The classic studies of Bisset (10), Cushing (40) and Hildemann and Cooper
(61) demonstrated that temperature can have a profound role in these
responses. The more recent studies of Avtalion have served as a basis
for beginning to understand the mechanism of these effects (7). He has
shown that humoral responses in the carp are a two-step process: 1) a
temperature-sensitive step requiring relatively high temperatures for
antigen recognition and 2) a temperature-insensitive step which results
in antibody production. Furthermore, Avtalion suggests that there are
at least three cell types involved; 1) X cells (T-like) which are sensi¬
tive to low temperatures and are involved in priming and tolerance and
2) Y and Z cells (B-like) which are involved in memory and antibody for¬
mation respectively. It must be pointed out however that direct proof
for the existence of multiple types of immunocompetent cells in fish is
lacking.
More recently Etlinger et^ al. (46) presented evidence that rainbow
trout have two lymphoid cell types. This evidence is based on responses
cf leukocytes isolated from various lymphoid organs to the mammalian T
and B cell mitogens. Thymocytes responded only to Con A (a T-cell mito¬
gen in mice and man) and anterior kidney leukocytes responded only to
I.PS or PPD (B-cell mitogens). The unique pattern of tissue localization
of cells responsive to mammalian T- and B-lymphocyte mitogens was taken
as evidence for lymphocyte heterogeneity in rainbow trout.

6
Smith et al. (102), Chiller et al_. (26), and Pontius and Ambrosius
(89) have studied the cellular responses of teleosts to sheep red blood
cells and have demonstrated antibody-forming cells in the spleen and
anterior kidney. Further studies by Sailendri and Muthukkaruppan have
shorn an appreciable number of antibody-forming cells in the thymus as
well (96,97). One could thus conclude that fish have a B-cell equiva¬
lent, as defined by the ability of plasma-like cells to produce antibody.
However, the presence of antibody-forming cells in the fish thymus indi¬
cates that the thymus may not be populated with only T-like cells- as
Etlinger's work suggests. Only in experimentally induced circumstances
are antibody-producing cells (B-cells) found in mammalian thymuses (37).
In addition, > 90% of the cells isolated from thymuses of four different
species of fish have demonstrable levels of immunoglobulin on their
surfaces (44,45,46,116). Although there is some controversy as to
whether or not mammalian T-cells have surface immunoglobulins (to be
discussed further in Chapter IV), the consensus is that if T-cells do
have surface immunoglobulins they are present in very small amounts and
only B-cells have readily demonstrable levels of surface immunoglobulin.
Therefore, in light of the existing data, there is some question as to
whether fish thymocytes are similar to mammalian thymocytes.
It should be pointed out that much of the data supporting the con¬
cept of two cell types (presumed to be lymphocytes) involved in immune
responses in fish are only inferential and alternative interpretations
may be presented. Indeed the unusual properties of the fish thymus (sur¬
face immunoglobulin expression and the presence of antibody producing
cells), as well as a lack of maturation in antibody responses (34)

7
and the presence of demonstrable hapten-carrier effects without a 16S
-> 7S switch suggest that if a T-like cell in fish exists it may differ
functionally from higher vertebrate T-cells. Summarizing the current
literature, it appears that direct evidence for two lymphocyte sub¬
populations in fish is lacking.
The purpose of this portion of the research was to determine in a
direct way if a teleost, the bluegill, has a heterogeneous population
of lymphocytes akin to T- and B-cells in birds and mammals. The ap¬
proach taken was three fold: 1) to define a separation technique .for
the isolation of relatively pure lymphocytes and to establish appropri¬
ate In vitro culture conditions, 2) to determine if mitogenic responses
and cell surface determinants employed as T- and B-cell probes in hirds
and mammals are applicable to bluegill lymphocytes as in vitro markers,
and 3) to separate differing subpopulations of lymphocytes on the basis
of differences in mitogenic and cell membrane antigens. Special empha¬
sis was placed on studying the effects of temperature on bluegill lym¬
phocytes to determine if a cellular basis for the in vivo temperature
effects on the immune responses in fish exist.

Materials And Methods
Experimental Animals
Bluegill (Lepomis macrochirus), a freshwater teleost, was used
exclusively as a source of lymphocytes in these studies. Sexually
mature male and female specimens, weighing 200-500g, were obtained
from the University of Florida's Lake Alice using cane poles, barbless
hooks and bread as bait. Fish were handled with rubber gloves and kept
in aerated holding tanks until transported to laboratory aquaria. One
hundred twenty-five liter Nalgene tanks filled with dechlorinated water
were used to maintain specimens in the laboratory. A maximum of eight
fish per tank were maintained with continuous aeration and a complete
change of water every 3-4 days. Fish were fed to satiation 2-3 times
each week with TetraMin (Tetra Werke, West Germany). As discussed later,
these holding conditions were less than ideal.
Culture Media
Roswell Park Memorial Institute (RPMI) 1640 was used as a wash
medium and as a supportive medium for in vitro mitogenic studies.
The complete medium used was prepared by dissolving RPMI 1640 instant
tissue culture powder (Grand Island Biological Company [GIBCO], Grand
Island, N.Y.), penicillin (GIBCO; 50 U/ml), streptomycin (GIBCO; 50 ncg/
ml), gentamycin (Schering, Kenilworth, N.J.; 20 mcg/ml), heparin (Sigma,
St. Louis, Mo.; sodium salt, 5 U/ml) and sodium bicarbonate (Mallinckrodt,
8

9
St. Louis, Mo.; 2.2 g/L) in triple-distilled water. The pH was
adjusted to 7.2 with NaOH or HC1, and the solution sterilized by passage
through 0.45y detergent free Swinex-25 millipore filters (Millipore,
Bedford, Mass.).
For in vitro studies of primary immune responses (Mishell-Dutton
type cultures [83]) a medium modified from Click et_ al_. (35) was used.
Modifications of the original technique included exclusion of NaOH arid 2-
mercaptoethanol, substitution of RPM1 1640 for Hank's and the addition
of gentamycin (20 mcg/ml), heparin (5 U/ml) and sodium bicarbonate (2.2
g/L, dissolved in the initial media preparation). Concentrations of the
amino acids (GIBCO). nucleic acid precusors (GIBCO), pyruvate (GIBCO),
glutamine (GIBCO), vitamins (GIBCO), penicillin and streptomycin were
added as described by Click et al. (35). The medium was prepared by
dissolving the above ingredients in triple-distilled water, adjusting
the volume and pH and sterilizing as for the preparation of RPMI 1640
(described above).
Medium Supplements
*
Serum and plasma sources which were tested as medium supplements
for in vitro studies were fetal calf serum (GIBCO; Lot # A030113; Inter¬
national Scientific Ind., Inc., Cary, Ill.; Lot # 7066411), Calf Serum
(GIBCO; Lot # Ro266T), human serum pools (five pools furnished by Dr.
R. Waldman, University of Florida, > 50 normal human sera per pool),
rabbit serum pools (New Zealand White rabbits, two pools, >10 normal
rabbit sera per pool), alligator (Alligator mississippensis) serum
(Silver Springs Reptile Institute, Silver Springs, Fla.; four indivi¬
dual normal alligator sera), fresh water catfish (Ictaluru cerracanthus)
plasma (heparinized, pool from ten catfish), large mouth bass (Micropterus

10
punctulatus) plasma (five heparinized pools, five normal bass per pool),
giant grouper (Epinephelus itaira) serum (pool from five grouper) and
bream (a collective term for all Lepomis species) plasma (heparinized
pools, > 10 fish per pool). All sera or plasma were heat inactivated
for 30 min at 56°C and were sterilized by Millipore filtration (0.45 y)
Preparation of Cell Suspensions and Counting Technique
The sources of cells studied from the bluegill were blood, anterior
kidney (pronephrus or head kidney), thymus and spleen (6,48,68,96,97,102,
117). Heparinized blood, obtained from the caudal vein (108) and all
organs were removed aspectically. Organs were placed in sterile petri
dishes containing cold RPMI 1640. A single cell suspension of each organ
was prepared by gently teasing apart the organ with forceps and pipeting
the teased suspension over a 60-80 mesh steel screen to remove clumps
and connective tissue.
A Hypaque-Ficoll method, adapted from Boyum's Isopaque-Fic.oll tech¬
nique (14), was used to isolate lymphocyte populations from organ cell
suspensions or heparinized blood. Hypaque-Ficoll solutions were pre¬
pared by mixing 10 parts of 33.9% Hypaque (Winthrop Laboratories, New
York, N.Y.) with 24 parts or 9% Ficoll (Pharmacia, Piscataway, N.J.),
Densities of prepared solutions were 1.077 ± 0.0005 g/ml (room tempera¬
ture) as determined by picnometer difference weighings.
A maximum of five ml of a teased organ cell suspension or heparin¬
ized whole blood diluted 1:4 with RPMI 1640 was gently layered onto five
ml of Hypaque-Ficoll in a 15 ml tube (Falcon, Oxnard, Cal.; 17 x 100 mm).
Tubes were spun at room temperature in a table-top centrifuge (Interna¬
tional Centrifuge, Boston, Mass.) for 20 min at 1000 RPM. The interface

11
band of cells between the Hypaque-Ficoll and the overlaying suspension
medium was removed using a Pasteur pipet and diluted in cold RPMI 1640.
The suspension was spun for 10 min at 1000 RPM in a refrigerated centri¬
fuge and the cell pellet washed three times with cold RMPI 1640.
The number of phagocytic cells was assessed using collodial carbon
uptake. India ink was diluted 1:10 with saline, centrifuged for 30 min
at 3500 RPM and millipore filtered (0.45 y) prior to use. One drop was
added to approximately three ml of a cell suspension and the mixture in¬
cubated for 30 min at 37°C. The cells were then washed three times and
May-Grunwald-Giemsa stained cytocentrifuged (Shandon-Elliott Inc.,
Sewickley, Penn.) mounts prepared for quantitation.
Cell counts (109) and viability (13) were determined by diluting
an aliquot of the washed cell suspension in a white blood cell diluting
pipet (Scientific Products, Ocala, Fla.) with 0.1% trypan blue in RPMI
1640 and counting with a Neubaurer hemacytometer (Scientific Products).
Culture Techniques
A laminar flow hood (Abbott Laboratories, Chicago, Ill.) was used
as a sterile environment for all cell culture work.
A microculture method (58,107) was adapted for mitogenic stimulation
and mixed lymphocyte culture assays. For mitogen studies, washed and
pelleted cells were resuspended in serum or plasma supplemented RPMI 1640
and were dispensed into microculture trays (Linbro, Hamden, Conn.; U-
shaped wells) at a cell concentration of 5 x 10^ cells/0.2 ml/well. The
mitogens used consisted of lippolysaccharide (DIFCO Labs, Detroit, Mich.)
from S_. typhimurium which was boiled one hr after reconstitution with
triple distilled water, phytohemagglutinin P (DIFCO) and concanavalin A
(Miles Labs, Inc., Kankakee, Ind.; 3x crystallized). Stock solutions

12
were diluted with RPMI 1640 without serum or plasma supplements and
were added to appropriate wells in 20 pi volumes immediately after the
cells were dispensed. Twenty microliters of RPMI 1640 without supple¬
ment or mitogen was added to control unstimulated wells.
Two-way mixed lymphocyte cultures of cells from two bluegills
were prepared by adding 2.5 x 10^ cells suspended in 0.1 ml of supple¬
mented RPMI 1640 from each cell preparation (total cell concentration
per well was 5 x 10^/0.2 ml). Five hundred thousand cells/0.2 ml/well
from each source served as controls. ^
Tritiated-thymidine (Schwarz-Mann, Orangeburg, N.Y.; sterile
acqueous solution, pH 7.4, 1.9 Ci/mM, 1.0 mCi/ml), diluted in supple¬
ment free RPMI 1640, was added to each culture well at a concentration
of 0.5 yCi/10 pl/well at 24 hr prioi' to harvesting.
Microculture trays were maintained in 5% CO2 - 95% air, satu¬
rated-humidity incubators at the temperatures indicated. CO^ content
was routinely measured with a Fryrite CO^ tester (Bacharach Instrument
Company, Pittsburgh, Penn.).
3
Cells, mitogens and H-thymidme were dispensed in microculture
trays using 0.5, 1.0, 5 or 10 ml gas tight syringes (Hamilton, Reno,
Nev.) attached to repeating dispensers (Hamilton) delivering one-
fiftieth of the attached syringe volume.
For .in vitro studies of primary immune responses, single cell
suspensions were prepared from pooled anterior kidney, spleen, and
thymus by teasing apart the organs in RPMI 1640 and seiving through
a 60-80 mesh screen. The cell suspension was centrifuged and the pellet
washed three times. The final cell pellet was resuspended in enriched
RPMI 1640 medium (described above) supplemented with 7% bass plasma.

13
7
White, red, and dead cells were enumerated and 1x10 viable white
cells in three ml of supplemented medium were aliquoted in Falcon 35
x 10 mm tissue culture dishes (Scientific Products).
Sheep red blood cells (SRBC's) used for immunization of the dis¬
sociated organ suspensions were obtained from a single sheep (Colorado
Serum Comp., Denver, Col.; Sheep # 20, H type antigen). SRBC's were
washed three times with RPMI 1640 and the final pellet suspended in the
enriched RPMI 1640 (without supplement) to 1% of the packed cell volume
Cultures to be immunized received 0.1 ml of the 1% SRBC suspension.
Controls received 0.1 ml of enriched RPMI 1640.
Culture dishes were maintained in 5% CO2 - 95% air humidified
environments as described above.
Assay for ^H-thymidine Incorporation into DNA
An automatic cell harvester (Otto Hiller Company, Madison, Wis.)
was used to obtain trichloroacetic acid (TCA) precipitable nucleic
acid material from individual wells of cultured cells. Twenty-four
hour pulsed cells were syphoned from the wells onto a glass fiber
filter (Reeve Angel, Whatman, Inc., Clifton, N.J.), rinsed with
saline, precipitated with 10% TCA and methanol dried. Discs, repre¬
senting individually harvested wells, were punched out of the filter
strip and assayed for using liquid scintillation counting. The
scintillation cocktail used consisted of PPO (Packard, Chicago, Ill.;
16.5 g), POPOP (Packard; 0.3 g), Triton X-100 (Packard; 1.0 L), and
toluene (Maitinckrodt, St. Louis, Mo.; 2.0 L). Samples were counted
in mini-vials (Rochester Scientific, Rochester, N.Y.) using an auto¬
matic liquid scintillation counter (Beckman Instruments, Fullerton, Cal
h
) •
Model LS-100

14
Stimulation Indices and Statistical Analysis
Means and standard deviations were determined for each data group.
An F-test was used for variance analysis. The Student-t test was used
to determine the statistical significance of increases over control
values (20,103). A 95% or greater confidence level (p <_ 0.05) was used
for both the F-test and the t-test.
Stimulation indices were used to express increases of mitogen
stimulated cultures over control cultures or mixed lymphocyte cultures
(MLC's) over controls. Indices for mitogenic studies were determined
using the following equation: Mean CPM of stimulated cultures _ Indices
Mean CPM of control cultures
for MLC studies were calculated by using the following formula:
Mean CPM of MLC between Fish A and Fish B
(Mean CPM of Fish A Control Culture + Mean CPM of Fish B Control) t 2
Histological and Morphological Techniques
Serial cross sections of paraffin embedded gill regions of bluegill
were kindly prepared by Mr. Melvin Laite (Department of Pathology, J.
Hillis Miller Health Center, Gainesville, Fla.). Sectioned tissues were
stained with hematoxylin and eosin.
Cell suspension smears or cytocentrifuge (Shandon-Elliot Inc.)
preparations were stained with May-Grunwald-Giemsa for morphological
examination.
Autoradiography
*7
Cultured cells, pulsed with H-thymidine for 24 hr, were pipeted
from microculture tray wells, washed three times with RPMI 1640 and
cytocentrifuged. Cytocentrifuged preparations were coated with nuclear
track emulsion (Kodak, Inc., Rochester, N.Y.; type NTB3), exposed,

15
developed and fixed as described by Gormus et_ al_ (52). All processed
slides were stained with toluidine blue in order to enhance microscopic
examination of the cells.
Preparation of Rabbit Antisera
A rabbit anti-bluegill brain antiserum was prepared by the proce¬
dure described by Golub for mouse brain (50). Five brains were homo¬
genized, using a tissue grinder, diluted 1:2 with PBS and 0.5 ml aliquots
were emulsified w'ith an equal volume of complete Freund's adjuvant (DIFCO)
for each immunization. Sera obtained from the rabbits before immuniza¬
tion were used as normal rabbit serum controls. The rabbit antiserum
used was obtained from one surviving rabbit which was reimmunized six
times over a three-month period.
The preparation of rabbit anti-bluegill immunoglobulin is described
in Chapter IV.
Cytotoxicity Assay
Complement mediated cytotoxicity of preimmune or immune normal rab¬
bit serum and rabbit anti-bluegill brain serum on bluegill lymphocytes
7
was accomplished by incubating 1 x 10 cells with 1:5 dilutions of rab¬
bit sera plus a 1:10 dilution of guinea pig complement (GIBCO, lyophi-
lized). After 1.5 hr at room temperature the cells were washed three
times with RPMI 1640 and cell counts and viability determined.
Rosetting Techniques
The method of Jondal £t_ al. (66) was followed to assess the number
of lymphocytes capable of rosetting with red blood cells (RBC's) from
various animals. Fresh heparinized whole blood obtained from human,
sheep, rabbit, chicken, horse, ferret, guinea pig, mouse, alligator, and

16
bluegill were washed four times with RPMI 1640, and the white buffy
coat was removed after each centrifugation. Winthrop hematocrit tubes
were used to determine percentages of RBC's in each suspension and
dilutions were made accordingly. Controls with only the test RBC's
were routinely assayed to determine the number of white cells con¬
tributed by the RBC suspension. As a negative control, homologous
RBC's were tested with bluegill lymphocytes.
Hypaque-Ficoll (p = 1.077) centrifugation was used to deplete
rosetted lymphocytes from non-rosetted lymphocytes (41,98). Hypaque-
Ficoll recovered nonarosetting cells were diluted into RPMI 1640, pellet¬
ed and washed three times.
Immunofluorescence
Immunofluorescent reagents and techniques are described in Chapter
IV.
Hemolytic Plaque Assay
Cells were harvested from tissue culture dishes by gently scrap¬
ing the bottom of the culture dish with a rubber policeman and pipetting
the cell suspension into a conical centrifuge tube. The plate was rinsed
once with 3 ml RPMI 1640 and the wash medium was added to the cell
suspension. Cells were pelleted and resuspended in RPMI 1640. Viabili¬
ty and cell recoveries were determined prior to assaying for plaque¬
forming cells (PFC's).
PFC's (cells producing antibody to SPJBC's) were enumerated using
a slide modification (83) of the Jerne hemolytic plaque assay (65).
Slides were incubated with fresh sucker fish plasma (a plasma pool from
several different species of the Catostomidae family native to the Swanee

17
and Santa Fe Rivers in Florida) diluted 1:20 in RPMI 1640 for 3-5 hr in
a 32°C, 5% CO2 - 95% air incubator. Plaques were routinely examined
microscopically prior to counting on a Quebec colony counter (New
Brunswick Scientific Co., New Brunswick, N.J.).

Results
Lymphoid Organs of the Bluegill
To determine which organis of the bluegill contained lymphoid cells,
smears of blood or organ cell suspensions were stained with May-Grunwald-
Giemsa and examined for the cell types present. Of the tissues examined,
anterior kidney (head kidney or pronephros), spleen, thymus, and blood
were the major sources of lymphocytes. Very few lymphocytes were 'found
in the liver, pancreas, gonads, or posterior kidneys. Gut-associated
lymphoid tissue or lymph nodes were not found.
Due to the close proximity of the thymus to the anterior kidney,
serial sections were made through the gill region of a small fish ('v 100 g,
< 1 yr old) and examined histologically. Figure 1 presents photomicro¬
graphs of representative sections through this region. The anterior kid¬
ney was seen to be a relatively diffuse organ containing an abundant number
of blood sinuses, had a relatively large number of red blood cells and
contained a heterogeneous mixture of white cells. In contrast, the
thymus contained fewer red blood cells, had few white cells other than
lymphocytes and contained Hassall’s corpuscles. Therefore based upon
both the anatomic location and the histologic characteristics, it was
felt that these tissues were in fact anterior kidney and thymus.
Separation and Quantitation of Bluegill Lymphocytes
- Hypaque-Ficoll (p = 1.077) was used to siolate relatively pure
populations of lymphocytes (characterized morphologically) from the
18

Figure 1. Photomicrographs of representative serial sections through
the gill region of a small bluegill. (a) Anterior kidney. Cb) Thymus
Sections were stained with hematoxylin and eosin. Magnification x 100

20

21
blood, spleen, anterior kidney, and thymus of bluegill. Less than 5% of
the total number of cells recovered from Hypaque-Ficoll were RBC's and
the number of lymphocytes recovered represented at least 99% of the
lymphocytes present in unfractionated whole blood or lymphoid organ cell
suspensions.
White cell differentials of whole blood before and after fractiona¬
tion on Hypaque-Ficoll are presented in Table 1 and illustrate the effi¬
ciency of this technique in removing other cell types. Figure 2 is a
photomicrograph of the type of lymphocyte preparations routinely obtained
from blood or lymphoid organ cell syspensions. These cell separations
were successful, only if freshly caught fish were used. Another major
cell type, a lymphoblast-like cell, was isolated from Hypaque-Ficoll if
cell suspensions from fish maintained in laboratory aquaria for long
periods of time were used (see Figure 3). The relevance of these blast¬
like cell isolates and the necessity of using newly acquired fish for
these and subsequent studies is discussed in a later section.
The anterior kidney was the most abundant source of lymphocytes
' 8
(yielding ^ 2 x 10 cells/fish) whereas spleens and thymuses routinely
7 7
yielded about 5 x 10 and 2 x 10 cells/fish, respectively. Heparinized
blood yielded about 5 x 10^ cells/ml (see Table 10).
Culture Conditions and Assay of Cell Division
As in any study involving in vitro culturing of lymphocytes (or
any other cell type for that matter) there were numerous variables to
be considered. In light of the fact that relatively limited numbers of
cells were available from individual fish and since syngeneic bluegills
were not obtainable it was necessary to approach optimization of culture

22
Table 1
White Cell Differentials of Bluegill Whole Blood
and Hypaque-Ficoll Isolated Blood Cells
Percent of Total*3
a
Blood
Hypaque-Ficoll Isolated
Cell Type
Thrombocyte
25±5C
0
Granulocyte
30±4
0
Lymphocyte
45±5
100
(a) Smears were made of whole blood and Hypaque-Ficoll isolates
of individual samples and were May-Grunwald-Giemsa stained for
quantitation purposes.
(b) Results are expressed as a percent of the total number of
white blood cells counted.
(c) Each value represents the mean of determinations from 6 dif¬
ferent bluegill samples (>3 determinations per samples) ± standard
deviations.

23
Figure 2. Photomicrograph of a representative Hypaque-Ficoll isolate
from bluegill blood. A cytocentrifuge preparation stained with May-
Grunwald-Giemsa. Magnification x 400.

24
conditions in a rather "piecemeal" fashion over an extended period of
time. The following commentary is an effort to systhesize the major
observations that enabled the definition of what can be called optimal
conditions. Unless otherwise noted all of these studies were performed
with anterior kidney lymphocytes.
Various sera or plasma were tested to determine which was a suit¬
able supplement to use with RPMI 1640 for mitogenic studies of cultured
bluegill cells. Ten percent human, calf, fetal calf, rabbit, alligator,
bass, catfish, and grouper sera or plasma and mixtures of 5% human’serum
with 5% calf or fetal calf serum were not supportive in mitogenic stimu¬
lation studies using PHA (0.1 yl), Con A (10 yg) or LPS (1 or 10 yg) at
either 22, 27 or 32°C. Grouper and catfish sera were cytotoxic for
bluegill cells. The other sera gave high TCA precipitable counts in
unstimulated control cultures and stimulation indices for mitogen
stimulated cultures of 1 or < 1. On the other hand, bream (a collective
term for all Lepomis species) serum pools were supportive in the sense
that significant stimulation indicies were obtained with mitogen stimu¬
lated cultures. '
In the initial experiments 10% bream serum was used. However, due
to the limited supply of bream sera and the difficulty in obtaining
good yields of serum from clotted blood, two modifications were tried
and found satisfactory; 1) heparinized plasma rather than serum was used
and 2) the concentration of supplement was reduced from 10% to 7%.
An additional complication was the observation that not all bream
plasma pools were suitable as supplements in mitogenic assays. Varia¬
tions in TCA precipitalbe counts of unstimulated control cultures ranged
from < 100 CPM to > 10,000 CPM and stimulation indices varied from 4 to

25
250. One attempt to reduce the high counts of control unstimulated
cultures obtained with some of the supplement pools was to dialyze
the plasma pools against 0.15MNaCl. The data obtained with four
bream plasma pools which elicited high background levels prior to
dialysis are presented in Table 2. In three of the four pools tested
in this experiment the control CPM dropped significantly (p < 0.05)
in the cultures incubated at 22°C and thus resulted in increased stimu¬
lation indices with LPS. With three of four pools used with cells
maintained at 32°C the background remained unchanged. In the other
case the background dropped as a result of dialysis and hence the
stimulation index obtained using PHA increased. In conclusion it can
be stated that dialysis of bream plasma did not significantly decrease
the responses in any cultures and in fact in some cases enhanced the
response. Thereafter all bream plasma were dialyzed before use as
culture medium supplements for mitogenic assays.
Dialysis of certain heterologous supplements that elicited high
control CPM was also tried. Dialyzed bass plasma was supportive as a
X
supplement in mitogenic assays in the sense that significant stimulation
indicies were obtained. However, these indices were never > 10 and
therefore bass plasma was not used routinely. Dialysis of human, calf,
fetal calf, and alligator sera did not improve the situation with re¬
spect to high levels of background counts.
To summarize the culture conditions discussed thus far, RPMI 1640
supplemented with 7% dialyzed bream plasma was found to be supportive
for iii vitro mitogenic stimulation.
During the course of several experiments involving different fish
it was observed that there were differences both in the types of Hypaque-

26
Table 2
Effect of Dialysis of Plasma Supplements on Mitogenic
Stimulation of Bluegill Anterior Kidney Lymphocytes
Stimulation Indexa
Supplement Plasma
Poolb
PHA (0.1 yl)
- 32°C
LPS (1 yg)
- 22°C
Undialyzed
Dialyzedc
Undialyzed
Dialyzed
A
1
6.8
1
1
B
4.8
4.0
1
14.0
C
2.5
4.0
5.9
16.4
D
4.8
4.6
1.5
7.4
(a) Triplicate cultures were stimulated with either PHA (0.1 yl) at
32°C or LPS (10 yg) at 22°C, pulsed on day 6 and harvested on day 7.
(b) Each pool represented the plasma obtained from at least five bream.
(c) Dialysis vías against pyrogen free 0.15 M NaCl.

27
Ficoll isolated cells from anterior kidney cell suspensions and in the
stimulation indices with mitogens. Furthermore these differences ap¬
peared to be correlated with the length of time the bluegills were main¬
tained in holding tanks in the laboratory prior to sacrifice. Table 3
presents data on white cell differentials of Hypaque-Ficoll isolated
cells from anterior kidneys of fish sacrificed at either one day or
three weeks after capture. Significant increases in the number of blast¬
like cells were seen in cell preparations from bluegills maintained for
three weeks. The gross differences in the cell types isolated from
Hypaque-Ficoll can be seen by comparing the cells shown in Figure 2
(from a one day isolate) with those in Figure 3 (a three-week isolate).
An increase in the number of red cells, which would not penetrate the
Hypaque-Ficoll, was also noted in the three-week isolates.
Furthermore, it was also observed that in experiments utilizing
fish maintained in acquaria for long periods of time, TCA precipitable
counts of control unstimulated cultures were high. A composite of data
from five experiments in which bluegill were sacrificed at various
periods of time 'after capture is presented in Table 4. Apparently the
longer a fish is maintained under our laboratory conditions the more
likely it is the animal's lymphocytes will exhibit a high level of
spontaneous thymidine incorporation. It thus seems imperative to use
freshly caught fish as sources of cells for in vitro studies if the
alternative is to keep them under the conditions used here.
To determine if TCA precipitable counts were a valid measure of
cellular events in culture, TCA precipitable counts were correlated
with the actual number of cells containing labeled thymidine. The
technique of autoradiography was used. Anterior kidney lymphocytes

28
Table 3
Effect of Maintenance Time of Bluegill in
Laboratory Aquaria on Differential White Cell
Counts of Hypaque-Ficoll Isolated Anterior Kidney Cells
Percent3,
Maintenance Time
Lymphocyte^
Blast-like
1 day
100
0
3 weeks
50-60
50-40
(a) Results are expressed as a percent of the total number of
white cells counted.
(b) Cytocentrifuged preparations of Hypaque-Ficoll separated
blood samples were stained with May-Grunwald-Giemsa for quanti¬
tation.

29
Figure 3. Photomicrograph of a representative Hypaque-Ficoll isolate
of bluegill blood after long term laboratory maintenance of the blue-
gill. Photomicrograph is of a May-Grunwald-Giemsa stained cytocentri-
fuge preparation of an isolate obtained from a bluegill after three
weeks of laboratory maintenance. Magnification x 400.

30
Table 4
Effect of Maintenance Time of Bluegill in Laboratory
Aquaria on the Incorporation of Thymidine by
Unstimulated Anterior Kidney Cell Cultures
CPM/Culturea
Fish^
Maintenance Timec
22°C
32°C
A
1 day
50±8
4518
B
1 day
120114
4416
C
3 weeks
1121137
26451109
D
3 weeks
991168
12311127
E
5 weeks
13,1971580
28381181
(a) Results are expressed as the means of CPM from triplicate
cultures ± standard deviations.
(b) Cells from individual fish were incubated without mitogens
at 22°C or 32°C, pulsed with ^H-thymidine on day 2 and harvested
on day 3.
(c) Length of time fish were maintained in laboratory aquaria
before sacrifice.

31
were cultured for seven days with various doses of PHA at 32°C.
Tritiated-thymidine was added to all Cultures 24 hr prior to termi¬
nation. One set of cultures was routinely harvested and assayed for
TCA precipitable counts. Cytocentrifuge preparations were prepared
from cells of a duplicate set of cultures and either processed for
autoradiography and the number of labeled cells (>_ 5 grains) quanti¬
tated (presented as a percent of the total number counted) or stained
with May-Grunwald-Giemsa for morphological examination. As seen in
Figure 4, increases or decreases in TCA precipitable radioactivity
closely followed changes in the percent of the total number of cells
that contained labeled thymidine.
In cultures stimulated with the optimal concentration of PHA (0.1
yl), 70% of the cells possessed nuclear autoradiographic grains. All
labelled cells examined in toluidine-stained cytocentrifuged preparations
were large blast-like cells and were found in clumps or aggregates
(Figure 5a). Figure 5b is a May-Grunwald-Giemsa stained preparation
showing an aggregate of blast-like cells with eccentric nuclei and abun-
dant cytoplasm.
Mitogenic Studies
Experiments were designed to assess the optimal culture conditions
for lymphocytes isolated from the anterior kidney. Variables tested wrere
mitogen doses, time for maximum stimulation and effect of temperature on
the response to the mitogens.
Figure 6 depicts the results of one very large study on the respon¬
siveness of anterior kidney lymphocytes to LPS, PHA,and Con A under a
variety of conditions. One somewhat surprising result involved the

Figure 4. Correlation of TCA precipitable counts with the number of
autoradiography positive cells from PHA stimulated bluegill lymphocyte
cultures. Cultures were incubated at 32°C, pulsed on day 6 and assayed
on day 7.

80-
% OF
TOTAL
NUMBER
POSITIVE
CPM
x!0'3
C/4
04

Figure 5. Photomicrograph of PHA-stimulated bluegill anterior kidney-
lymphocytes. (a) Autoradiograph stained with toluidine blue. (b) May-
Grunwald-Giemsa stained. Magnification x 100.

35

Figure 6. Temperature effects on mitogenic responses of
bluegill anterior kidney lymphocytes.

DAYS IN CULTURE

38
differences in temperature on maximum stimulation with the various
mitogens. Cells stimulated with PHA (0.1 yl) and Con A (50 yg) re¬
sponded well at 32°C (p < 0.01) and very poorly, if at all, at 22°C
(p > 0.1), whereas LPS (1 yg) responsiveness was higher at 22°C
(p < 0.01). There was, however, a significant response (p < 0.05) to
LPS (10 yg) in 32°C incubated cultures which was reproducible. Fifty
micrograms of LPS (not shown) were not stimulatory (stimulation indices
_< 1) at either temperature.
The temperature effects described above were found in ten experi¬
ments with the only major differences being the magnitude of the re¬
sponses. These differences may have been due to differences in the
serum supplement pools used as discussed previously.
To summarize the results, optimal mitogen doses at 32°C were 0.1 yl,
50 yg, and 10 yg for PHA, Con A, and LPS respectively and. 1.0 yg of LPS
at 22°C. PHA and Con A responses were greater at 32°C than 22°C and
LPS responsiveness was greater at 22°C than 32°C. Optimal culture times
were 5-7 days for all mitogens with the exception of 10 yg of LPS at
32°C where some variations were noted.
Limited experiments with spleen, blood, and thymus lymphocytes indi¬
cated that all were stimulated by PHA, Con A, and LPS. The mitogenic
responses of thymus lymphocytes are presented in Table 5 to demonstrate
that the temperature effects on mitogenic stimulations were also observed
with cells from this tissue and thus were not limited to anterior kidney
lymphocytes.
Mixed Lymphocyte Cultures
Lymphocytes from anterior kidneys of different bluegills were tested
for their ability to respond in two-way mixed lymphocyte cultures

39
Mitogenic Responses
Table 5
of Bluegill Thymus
Stimulation
Lymphocytes
indexa
Mitogen
32°C
22°C
LPS (1 yg)
4.7
10
PHA (0.1 yl)
30
4
Con A (10 yg)
53
7
(a) Triplicate cultures were pulsed on day 6 and
harvested on day 7.

40
(both populations capable of responding) at 22°C and 32°C. Cultures
were initiated with 0.25 x 10^ lymphocytes from each donor fish per
culture well (0.5 x 10^ cells total). Controls in mitogen stimulation
studies for each fish also served as controls for mixed lymphocyte
cultures.
Four of ten two-way mixed lymphocyte cultures exhibited statis¬
tically significant responses (p < 0.05) and are presented in Table 6.
Significant responses were only obtained at 32°C thus mimicking re¬
sponsiveness to PHA and Con A in temperature sensitivity. Furthermore,
these studies have indicated that maximal stimulation (not shown) in
the mixed lymphocyte cultures occurred at seven days. In this experi¬
ment all six bluegills studied had significant mitogenic responses,
indicating there is no direct correlation between PHA and LPS responsive¬
ness and the ability to respond to a mixed lymphocyte culture.
Evidence for Different Populations of Bluegill Lymphocytes
Golub (50) has demonstrated that rabbit anti-mouse brain cross re¬
acts with mouse thymocytes due to a common antigen on both brain and
»
thymocytes. In an attempt to elicit antiserum capable of recognizing
antigenic surface determinants on bluegill lymphocytes a rabbit was hy-
perimmunized with bluegill brain homogenates following Golub's immuniza¬
tion procedures. To determine the specificity of this rabbit anti-brain
serum for bluegill anterior kidney lymphocytes, cells were incubated
with the rabbit serum and guinea pig serum. After appropriate incuba¬
tion and washing only about 30% of the original number of cells remained
viable (as determined by trypan blue exclusion) in contrast to 100%

41
Table 6
Mixed Lymphocyte Responses of Bluegill
Anterior Kidney Lymphocytes
Stimulation Index3
Mixed
Lymphocyte
Response
Mitogen
Response
22°C
32°C
Fish
Crossc
22°C 32°C
LPSb
PHA
LPS
PHA
1
1 + 2
1 25
62
2.3
9
95
2
17.2
7.2
8.9
145
3
3+4
1 12
22
16
3.4
51
4
20
3
2.4
55
3
3+5
1 9
22
16
3.4
51
5
24
2.9
5.3
22
4
4+5
1 19
20
3
2.4
55
5
24
2.9
5.3
22
(a)
Triplicate
cultures were pulsed
on day
6 and
harvested
on day
Results are expressed as stimulation indicies as defined in Materials
and Methods.
(b) Mitogen concentrations were 1 yg and 10 yg of LPS at 22°C and
32°C, respectively, and 0.1 yl of PHA at 22°C and 32°C.
(c) Designates the source of cells used in the mixed lymphocyte
cultures.

42
recovery of viable cells when preimmune serum from this rabbit was
employed as a control. When the cells surviving the rabbit antiserum
treatment were assayed for mitogen responsiveness, it was found that
the PIIA response was diminished and the LPS response was intact. Re¬
sults from two such experiments are presented in Table 7. These data
indicate that cytotoxic treatment of anterior kidney lymphocytes with
anti-brain plus complement may be an effective means of obtaining rela¬
tively pure LPS responsive cells and that this responsive population,
representing ^30% of the original population, may be a subpopulation of
lymphocytes in the bluegill.
Anterior kidney lymphocytes were tested with heterologous red blood
cells for spontaneous rosette formation. Results are presented in
Table 8. Only rabbit red blood cells were capable of rosetting a sig¬
nificant portion of the lymphocytes (A* 20%).
To determine if the rosetted lymphocytes represented a discreet sub¬
population of the total with respect to mitogen responsiveness, rosetted
cells were depleted from the non-rosetted ones using Hypaque-Ficoll cen¬
trifugation. Seventy to 75% of the original number of lymphocytes were
recovered as non-rosette formers and were cultured under optimal mito¬
genic conditions. The results of two experiments are presented in Table
7. The LPS response was diminished while the PHA response was left intact.
These results indicate that depletion of lymphocytes rosetted with
rabbit red blood cells from non-rosetted lymphocytes may be an effective
means of isolating relatively pure PHA-responsive lymphocytes and that
this responsive population, representing 70-75% of the original popula¬
tion, may be a subpopulation of lymphocytes in the bluegill.

43
Table 7
Mitogen Responses of Bluegill Anterior Kidney
Lymphocytes Treated with Anti-Brain Plus Complement
or Rosette Depleted with Rabbit Red Blood Cells
Stimulation Index3
Expt
1
Expt
2
Expt
3
Treatment
LPSb
PHA
LPS
PHA
LPS
PHA
Control
4.2
9.8
4
12.4
8
9.9
Anti-Brain + Complement0
30
1.3
26
1.3
ND
ND
Rosette Depletion^
1.8
17
NDe
ND
1
9.0
(a) Triplicate cultures incubated at 32°C, pulsed on. day 6 and harvested
on day 7.
(b) Mitogen concentrations were 10 yg and 0.1 yl of LPS and PHA respec¬
tively.
(c) A 1:5 of rabbit anti-bluegill brain plus a 1:10 of guinea pig com¬
plement was used in the cytotoxic treatments.
(d) Rabbit red blood cell rosetted cells were depleted by Hypaque-Ficoll
centrifugation.
(e) ND = Not Done.

44
Table 8
Rosette Formation of Bluegill Anterior
Kidney Lymphocytes with Red Blood
Cells from Heterologous Species
RBC Source
% Rosetting
Bluegill
0
Human
0.65 ± 0.2
Ferret
0
Alligator
0
Rabbit
21 ± 1.5
Guinea Pig
0.75 ± 0.2
Horse
0
Mouse
0
Sheep
1.3 ± 0.6
Chicken
2.5 ± 1.0
(a) Results are expressed as percentages of the total number
of bluegill white cells (total number of white cells minus the
number of white cells in the RBC controls) rosetting with the
red blood cells. Each value represents the mean of triplicate
determinations from three different fish ± standard deviations
A white cell in contact with >_ 4 RBC constituted a positive
rosette.

45
In Vivo and In Vitro Studies on Antibody Producing Cells
Prior to in vitro primary immunization studies with cell
suspensions from the bluegill, it was first necessary to determine
which organs contained antibody-producing cells. It was also necessary
to determine if bluegill were responsive In vivo to the test antigen
(SRBC) and to establish a suitable complement source for use in the
hemolytic plaque assay.
Bluegill were immunized intraperitoneally with sheep red blood
cells and sacrificed two weeks later. Cell suspensions were prepkred
from the anterior kidney, spleen, and thymus. Only blood was fraction¬
ated on Hypaque-Ficoll due to difficulties in assaying samples with a
high ratio of red to white cells. Each cell suspension v/as assayed in
a Jeme hemolytic plaque assay for cells producing antibody to sheep
red blood cells.
Wide variations in responsiveness to sheep red blood cells were
observed in immunized bluegill. Results from two individuals are
presented in Table 9. The spleen, anterior kidney, and thymus each
contained considerable numbers of antibody-producing cells. Each
organ had approximately the same number of plaque-forming cells (PFC)
per 10^ cells. Very few plaque-forming cells were present in blood,
though it should be emphasized that blood was fractionated on Hypaque-
Ficoll prior to assay.
Fresh guinea pig serum, grouper serum, alligator serum, bass plasma,
bream plasma, and sucker plasma were diluted 1:20 and used as sources of
complement in the Jerne assay. Only bream, bass,and sucker sera were
effective sources of complement. Since sucker plasma was not used as
medium supplement and was obtainable in large quantities, it was used
routinely as a complement source.

46
Table 9
Distribution of Antibody Forming Cells in Various Tissues
of Bluegill Immunized with Sheep Erythrocytes
White^
Cells
(xlO-6)
Number of
PFC
Fisha
Tissue
Per 106 Cells
Total
1
Blood (2 ml)
9
4
36
Kidney
100
70
7000
Thymus
18
53
954
Spleen
9
52
468
2
Blood (2 ml)
10
1
10
Kidney
â–  184
3
552
Thymus
30
4
120
Spleen
24
2
48
(a) Bluegill were immunized intraperitoneally with 0.1 ml of 10% SRBC
and were sacrificed after two weeks.
(b) Hypaque-Ficoll fractionated peripheral blood cells and unfractionated
organ cell suspensions were assayed for the number of white cells and the
number of plaque forming cells (PFC).

47
The number of cells in the various lymphoid organs containing
cytoplasmic immunoglobulin were assayed by indirect immunofluorescence.
Smears of washed, unfractionated cell suspensions of anterior kidney,
thymus, spleen, blood, and posterior kidney (as a negative control)
were examined and the number of cells showing positive cytoplasmic
immunoglobulin staining quantitated. The results are presented in
Table 10. The posterior kidney was devoid of any Ig-containing cells.
Anterior kidney, spleen, thymus, and blood demonstrated appreciable
numbers of immunoglobulin containing cells. •,
Preliminary studies were undertaken to determine if bluegill lym¬
phoid cell suspensions would respond in vitro to an antigenic stimulus.
Several modifications of the culture techniques discussed above for
mitogen studies were employed to enrich the culture media and to ensure
that all necessary cellular components were present.
Undialyzed 7% bass plasma rather than bream plasma was used as a
supplement with an enriched RPMI 1640 medium. Since the hemolytic plaque
assay only measured differences in the number of plaque-forming cells
between control and antigen stimulated cultures, a high nonspecific stimu¬
lus by bass plasma (see mitogenic studies) was irrelevant as long as an in¬
crease in plaque-forming cells was attributable to antigenic stimulation.
A pool of unfractionated cell suspensions of anterior kidney', spleen,
and thymus was used for three reasons: 1) to increase the number of
available cells and thus the number of variables that could be tested,
2) to include phagocytic and plasma cells as well as any other cell
types possibly involved in antigen processing and antibody formation,
and 3) to decrease the chance of compartmental effects of individual

48
Table 10
Immunoglobulin Producing Cells
in the Lymphoid Organs of the Bluegill
Organa
% Positive^3
Blood
20 ± 5
Spleen
45 ± 11
Thymus
39 ± 15
Anterior Kidney
40 ± 14
Posterior Kidney
0
(a) Smears of blood and organ cell suspensions were assayed by indirect
immunofluorescence for cytoplasm.ic immunoglobulin.
(b) Results are presented as a percent of the total number of white cells
counted and are means of multiple determinations from three bluegill
± standard deviations.

49
organs. All cell suspensions used contained < 30% red blood cells and
'v 7-10% phagocytic cells (determined by colloidal carbon untake).
Control (no SRBC) or immunized (with SRBC) cultures were assayed
for PFC in the Jerne hemolytic plaque assay after incubation at 22°C
and 32°C for various time periods. Two experiments utilizing unimmunized
"normal" bluegill as cell donors are presented in Table 11. In 32°C
incubated cultures there were significant increases in the number of
PFC of immunized cultures over control culture responses. The maximum
PFC response as well as the maximum number of recovered cells frojn
immunized than control cultures occured on day 7. More cells were
recovered from immunized than control cultures and on day 7 more than
the initial (Day 0) number of cells were present in immunized cultures.
Viability in the cultures did not change over the ten-day culture
period.
In contrast to the 32°C incubated cultures, cultures maintained at
22°C did not show a PFC response. There was no significant difference
between control and immunized cultures and the viability was lower after
ten days. *
One preliminary experiment was done with cells from an immunized
bluegill in order to determine if a secondary immunization in vitro
would increase the number of responsive cells. Unlike cells from normal
fish, the PFC response in this fish was observed to occur only at 22°C.
The magnitude of the response measured on day 7 however was much lower
(control = 0 PFC, "boosted" = 18 PFC/Culture) than that seen at 32°C
v/ith cells from normal animals. It should be pointed out that the
number of recovered cells in the single experiment conducted was higher
in 22°C incubated cultures (22°C, 90% for controls, 285% for boosted;

Table 11
Primary In_Vitro Immunization of Bluegill Lymphoid
Organ Cell Suspensions with Sheep Red Blood Cells
PFC/Culture
% Recovered^
% Viable0
Experiment
Culture
Days in Culture
22°Ca
32°C
22°C
32°C
22°C
32°C
1
Control
5
NDd
57
ND
95
ND
89
Immunized
5
ND
660
ND
115
ND
93
Control
7
0
50
72
49
88
96
Immunized
7
0
1045
65
177
94
97
Control
10
ND
38
ND
62
ND
86
Immunized
10
ND
810
ND
101
ND
90
2
Control
7
0
0
83
56
89
91
Immunized
7
0
147
77
109
95
93
Control
10
0
0
36
40
73
94
Immunized
10
0
82
36
68
69
90
(a) Cultures were maintained at the indicated temperatures.
(b) Cell recoveries are expressed as a percent of the initial number of cells (Day 0).
(c) Viability was determined by trypan blue exclusion and is expressed as a percent of
the total number of cells recovered from cultures.
(d) ND = Not Done.

32°C, 4% for controls, 60% for immunized). It thus seems possible
that a major difference between the in_ vitro primary and secondary
responses to sheep erythrocytes may exist although obviously more
work needs to be done before definitive statements are possible.

Discussion
Effects of Plasma Supplements and Fish Maintenance on Lymphocyte Cultures
Two crucial variables, the medium supplement and the health or
physiological state of the fish appeared to be critical in obtaining
high levels of DNA synthesis (i.e. TCA precipitable counts) in un-r
stimulated lymphocyte cultures. The causative factors in these two
situations are unknown but it would seem appropriate to discuss, in a
speculative way, these two points. The influence of serum factors on
in vitro cultured cells has been well documented in other systems (18,38,
64,85,99,100,101,119,120) and it is conceivable in the studies reported
here that one or more such factors were present in some of the plasma
pools used as supplements. Dialysis experiments suggest that a factor(s)
of < 10,000 molecular weight was responsible for nonspecifically ele¬
vating unstimula’ted control TCA precipitable counts. It is also inter¬
esting that Etlinger's mitogenic studies with rainbow trout leukocytes
(46) also revealed serum effects on stimulation indicies.
Numerous effects on fish subjected to environmental changes or
stress have been reported (23,104). For example, physiologically sig¬
nificant serum alterations in cortisol, glucose, and free fatty acid
levels as well as morphological changes in adrenocortical, medullary,
and pancreatic tissues occur within minutes in goldfish subjected to
the slight stress of an aquarium transfer. The effects of environmental
factors, other than temperature, on the immune responses of fish have,
52

53
however, not been studied. The data presented here suggest that the
altered cellular-state (an increase in blast-like cells concommitant
with an increase in TCA precipitable counts of unstimulated cultures)
in bluegill maintained for long laboratory periods are caused by envi¬
ronmental factors in the laboratory aquaria. A likely factor (admit¬
tedly speculative) might involve endotoxemia resulting from bacterial
infections acquired in the aquarium.
Evidence for Two Subpopulations of Lymphocytes
The studies reported here show that there are at least two sub¬
populations of lymphocytes in the bluegill. One population is stimu¬
lated by PHA and Con A at 32°C and very poorly at 22°C. Although not
proven directly, the cells responding in mixed lymphocyte cultures are
probably a subset of the PHA/Con A responsive population since MLC's
were obtained only at 32°C. The other population of lymphocytes is
LPS-responsive at both 32°C and 22°C although responsiveness at 22°C
was usually greater.
The two subpopulatins were shown to be different by anti-brain
\
serum cytotoxicity and rosette depletion experiments. The 32°C, PHA
responsive population was depleted from the total population by anti¬
brain plus complement treatment and left intact by depletion of rabbit
RBC rosetted lymphocytes. The converse was true for the LPS-responsive
population. LPS-responsiveness was depleted by removal of rosetted
lymphocytes from the total population and was unaffected by anti-brain
cytotoxicity treatments.
Comparison of Bluegill and Rainbow Trout Mitogenic Studies
Differences between the results of mitogenic studies presented
here with the bluegill and those of Etlinger et al. (46) with rainbow

54
trout leukocytes suggest that there may be major differences between
different species of fish. Unlike the bluegill, rainbow trout con¬
tained PHA-responsive cells only in the thymus and LPS-responsive cells
only in the anterior kidney in a manner analogous to the compartmental
localization of T- and B-cells in birds and mammals. However, accurate
comparisons of the rainbow trout and bluegill are tenuous due to experi¬
mental differences. Unfractionated leukocytes, rather than isolated
lymphocytes, were cultured on]y at 19°C in the trout studies. It was
also deemed necessary to switch serum supplements to obtain signifi¬
cant responses to different mitogens with trout cells. There were also
differences in optimal mitogen doses as well as length of time for
maximum mitogenic stimulation between the two species.
It is thus conceivable that true differences in the lymphoid
systems exist between different species of fish. For example, there
are reports that thymuses of some fish species involute with age while
others do not (37). It is suggested that a third species group may
exist in which the thymus differentiates (or de-differentiates) into a
lymphoid organ similar to the anterior kidney, as apparently is the case
with bluegill.
Differences in environmental temperature tolerances may also effect
the in vitro cellular responses. Rainbow trout live in colder environ¬
ments, and thus evolutionary pressures may have affected the subpopu¬
lations of lymphocytes to a point where discernible differences in In
vitro temperature responses may not be recognizable. Further in vitro
studies with other species are necessary before adequate comparisons of
this nature can be made.

55
Are Bluegill Lymphocyte Subpopulations T- and B-Cell Equivalents?
By analogy, the mitogenic and mixed lymphocyte culture responses
of bluegill lymphocytes would support the conclusion that fish have
T- and B-cells. Bird and mammalian T-cells respond to PHA and Con A
(but not LPS) and are reactive in mixed lymphocyte cultures. Similarly,
a bluegill lymphocyte subpopulation (depleted of rabbit RBC rosettes)
responds to PHA or Con A when cultured at 32°C. The MLC reactive
cells also responded only at 32°C and are probably a subset of the
PHA/Con A reactive cell population. Bluegill lymphocytes of the sub¬
population unaffected by anti-brain plus complement treatment responded
only to LPS, and B-cell mitogen in birds and mammals. However, such con¬
clusions should be approached with caution until functional activities
are associated with the two bluegill lymphocyte subpopulations.
It should also be pointed out that the spontaneous rosette forma¬
tion of the B-like cells with rabbit RBC's is in marked contrast to
all other animal species studied, in which the B-cells do not spontane¬
ously rosette with any RBC's.
Implications of In Vitro Studies
If one assumes that in vitro studies are valid measures of in vivo
events, several explanations or rationalizations of published in vivo
data are possible in light of the ini vitro temperature effects on blue¬
gill lymphocytes.
Numerous reports on the effects of temperature on the immune responses
in fish to bacterial or protein antigens have been published. Avtalion
et al. (7) have suggested that the effects can be explained by two
populations of lymphoid cells; one is the antigen-reactive population
requiring a higher temperature to process the antigen and the other

56
population is responsible for antibody production at either high or
low temperatures. This may be the case if indeed the PHA-(and Con A)
responsive cell is equivalent to the antigen reactive cell and the LPS-
responsive cell is equivalent to the antibody-producing cell.
The participation of the two defined cell populations and the
temperature effects on immune responses of bluegill should be testable
in vitro. In vitro SRBC primed cultures maintained at 32°C elicited a
very good plaque forming cell response to SRBC's whereas cells main¬
tained at 22°C gave no response. If the SRBC is a T-dependent antigen
in the bluegill, as in mammalian systems, then application of depletion
techniques (rosette depletion or anti-brain cytotoxicity) should demon¬
strate whether cellular cooperation between the two subpopulations is
involved in in vitro antibody production. Further application of in
vitro manipulation techniques to the hapten-carrier effect should also
establish if the two subpopulations are indeed T-like and B-like in
function.
The preliminary study utilizing cells from in vivo primed fish also
were supportive of Avtalion's conclusions that fish can respond to a
secondary antigenic challenge at low temperatures only if they are
primed at a higher temperature. In. vitro "boosted" cells responded at
22°C, though with lower numbers of plaque-forming cells. However 32°C
cell cultures were not responsive, contrary to iia vivo primary immuniza¬
tion studies. This may indicate a secondai-y antigenic stimulus at 32°C
which elicits a tolerant state or suppressive factor(s).
Yocum et al. (121) have shown that only 16S IgM-like antibody is
produced in the hapten-carrier effect in a marine fish, the searobin.
Apparently the switch from high molecular weight to low molecular weight

57
antibodies (a T-cell controlled event in mice) associated with the
hapten-carrier effect in mammals does not necessarily occur in fish.
However, Uhr et al. (113) demonstrated that goldfish, when acclimated to
a high temperature (35°C), were capable of responding to an antigen with
both 16S and 7S antibodies (as opposed to a response at lower tempera¬
tures of only 16S antibodies). Though it was not proven that the 7S
antibody was in fact a de_ novo product and not a degradation product of
the 16S antibody or a shed membrane receptor, a PHA, high temperature
responsive cell type conceivably could be functional in controlling the
switch mechanism at 35°C in goldfish.
Temperature effects on lymphocytes may not be confined solely to
bluegill lymphocytes. R. C. Ashman, University of Western Australia,
Nedlands (personal communication) has demonstrated an increase in PHA
responsiveness of human T-cells when cultured at 39°C rather than 37°C.
Armadillos have body temperatures of < 35°C, yet the transformation of
lymphocytes stimulated by PHA was increased approximately 2.6 times
when cultured at 37°C rather than 33°C (91). Perhaps an evaluation of
mitogenic responses of other mammalian lymphocytes cultured in narrower
temperature ranges (37° ± 2°C) is warranted. However experiments done
by J. W. Shands, Jr., University of Florida, Gainesville, Fla. (personal
communication) using mouse spleen lymphocytes cultured with LPS and PHA
at 22, 27, 32, 35, 37 and 39°C showed the optimal response to both mito¬
gens was obtained at 37°C.
The Bluegill Lymphocyte as an Experimental Model
Differential responses to mitogens by cells cultured at different
temperatures should provide a valuable method to study functional and
physiochemical properties of the cells involved in immune reactions of

58
fish. One could speculate that the temperature effects on lymphocytes
cultured with mitogens are due to changes in membrane fluidity. Theo¬
retically a more rigid membrane in a PHA-responsive, 22°C cultured
lymphocyte could inhibit capping and membrane events leading to cell
activation, whereas a PHA-responsive, 32°C cultured cell with a more
fluid membrane could respond. Changes in membrane fluidity would also
account for changes in optimal doses of LPS required at the different
temperatures. Experiments to chemically alter membrane rigidity would
test the concept of temperature sensitive events at the membrane level.
There are alternative explanations for the temperature effects
demonstrated with bluegill lymphocytes, such as conformational changes
in receptor molecules with changes in temperature or the influence of
temperature on intracellular events involved in cell activation. In
any event, the question of why the two subpopulations differ in respon¬
siveness at different temperatures is an intriguing one. It would ap¬
pear that fish may offer a unique approach to dissecting cellular events
/
in the immune response.

CHAPTER III
LYMPHOCYTE HETEROGENEITY IN THE ALLIGATOR
Introduction
The reptiles are thought to represent a pivotal point in the
'i
phylogeny of the immune system since phylogenetically they are a
common ancestor of the birds and mammals. However, as pointed out
by Cohen (56), immunological studies in the reptiles are severely
lacking. The available data, reviewed in (36,37,59), suggest that
reptiles can mount a diversity of immune responses and arguments by
analogy would suggest they likely have T-like and B-like cells lym¬
phocytes .
Various antigens have been used to elicit both primary and second¬
ary humoral responses in various reptilian species (36,37,56,72) with a
switch from 19S IgM-like antibody molecules to 7S IgG-like antibody
molecules occurring during secondary responses (4,56). Unfortunately
relatively little has been done to describe the heavy chain isotypes in
the reptiles and thus IgM and IgG (or IgY) designations are at best
tenuous (31). Cells resembling plasma cells have been detected by
fluorescent antibody techniques, electron microscopy and the Jerne
plaque assay (36) in turtles. Thus on the basis of the ability to
elicit antibody responses as well as the demonstration of plasma-like
cells involved in antibody production, the evidence is rather direct
that reptiles have a B-cell equivalent.
59

60
First and second-set skin allograft rejections (37,59) characteris¬
tic of T-cell reactions in mammals have also been demonstrated in rep¬
tiles with an anamnestic second-set response. However there is a major
difference between transplantation reactions of reptiles and mammals, in
that reptilian reactions are typically chronic (36,37) as opposed to the
acute rejections occuring in mammals. These data suggest that T-like
functions may differ from those in mammals. Indeed, graft rejection
sites in turtles and snakes are infiltrated very early not only with
lymphocytes and macrophages, but also with an abundance of plasma., cells
(11). This observation suggests that such chronic graft rejections may
be antibody-mediated rather than cellularly (via T-like cell) mediated.
Responses to haptens conjugated to protein carriers have also been
demonstrated in reptiles (8,73) although the hapten-carrier effect has
apparently not been studied. In brief, data demonstrating that reptiles
can 1) show a 19S to 7S switch, 2) produce anti-hapten antibodies,and 3)
undergo graft rejections are at best only circumstantial evidence for
the existence of a T-like cell in these species. In fact one could
conceivably (although perhaps not too convincingly) argue for the exist¬
ence of only B-like cells from the same data.
Many of the reports from previous In vivo experiments in which
humoral responses to antigenic challenge were tested conflicted with
one another and in some cases there were questions as to whether rep¬
tiles could respond to antigenic challenges at all (36). Many of these
discrepancies have since been attributed to differences in the tempera¬
tures at which the animals were maintained after immunization. As early
as 1901, Metchnikoff demonstrated that the alligator responded to diph¬
theria toxin by forming antitoxin if the alligators were maintained at

61
32-37°C, whereas at 22°C they did not respond at all (80). More
recently, Evans has presented evidence that desert lizards maintained
at 35°C responded well to sheep red blood cells, but if maintained at
50eC or 40°C, temperatures well within physiological temperature ranges,
they did not respond as well (47). Also, an active humoral response to
the antigen was stopped if the animals were moved from 35°C to the
lower temperature. Wetherall and Turner (118) observed similar
responses to changes in environmental temperatures in another lizard
species. Environmental temperature is also an important factor in
skin allograft rejections, as shown by Borysenko (11). Snapping
turtles accepted allografts when they were maintained at 10°C but
viere able to l'eject the allografts at 25°C, and more rapid rejections
were seen at 35°C.
The lymphoid organs of several representative reptilian species
have been examined histologically (36,37). A bursa, thymus, spleen,
and gut associated lymphoid aggregates have been demonstrated. How¬
ever the functional roles of the various organs are lacking and thus it
cannot be stated whether the "bursa-like" organs are sources of B-like
cells or that the thymocytes are T-like cells as seen in the chicken.
In immunized turtles antibody-forming cells were found in the spleen
but not in the thymus (36), but again, the data are only circumstantial
that the lymphoid organs are compartmentalized into T- and B-cell com¬
ponents. To summarize the current literature, it would appear that
direct evidence for two cell types in any reptile analogous to T- and
B-lymphocytes in birds and mammals is lacking.
The purpose of this portion of the research was to determine in a
direct way if a reptile, the Florida alligator, has a heterogeneous

62
population of lymphocytes akin to T- and B-cells. The approach taken
was similar to that described previously for the bluegill, i.e. 1) to
define a separation technique for the isolation of relatively pure
lymphocytes and to establish appropriate in vitro culture conditions
for these cells, 2) to determine if mitogen stimulation and cell sur¬
face antigens employed as T- and B-cell probes and membrane markers in
the bird and mammalian systems are applicable to alligator lymphocytes
as in vitro markers,and 3) to separate differing subpopulations of
lymphocytes on the basis of marker differences. Special emphasis.was
also directed towards studying the effects of temperature on alligator
lymphocytes to determine if a cellular basis for the in vivo temperature
effects on the immune responses in reptiles could be demonstrated.

Materials and Methods
Experimental Animals
Florida alligators (Alligator mississippensis) were obtained from
the Florida Game and Fresh Water Fish Commission. Male and female
alligators., 90-150 cm in length, were used. Accurate age determina¬
tions were not possible, but were estimated to be between three and
five years. Alligators were individually tagged and housed in a 1.5 m
x 6 m outdoor pen at the University of Florida Animal Quarters. The pen
was designed to provide the alligators with easy access to either water
or a dry platform. The alligators were fed daily with monkey biscuits
(Ralston Purina, St. Louis, Mo.) and to satiation twice each week with
fresh fish (bream).
Culture Media
\
Culture media for in vitro mitogenic and primary immunization
studies were as described in Chapter II with the following modifica¬
tions: 1) Minimum Essential Medium (MEM) with nonessential amino acids
(GIBCO) was substituted for RPMI 1640 and 2) the NaCl concentration of
the complete media was increased to 0.157 M by dissolving 2.400 g NaCl
in the medium prior to adjusting the final volume to 1.0 L. The pre¬
pared MEM containing extra NaCl was designated Gator MEM (G-MEM) to
distinguish it from mammalian MEM.
The above modifications were also used in preparing medium used for
in vitro primary immunization studies following the procedure presented
in Chapter II.
63

64
Supplement Sources
Alligator, human, calf, fetal calf, and rabbit sera were tested as
media supplements for ini vitro studies. Two alligator serum sources
were used: 1) eight different pools (> 10 individual bleedings, 30-40
ml of serum per animal) were obtained from 1-2 kg alligators (2-3 yr
of age) at Herman Brooks' Alligator Farm (Christmas, Fla.) and 2) sera
(100-250 ml serum per bleed) from individual 100-225 kg alligators
(> 10 yr old) which were maintained at Silver Springs Reptile Institute
(Silver Springs, Fla.). The remaining serum sources are indicated in
Chapter II.
Preparation of Cell Suspensions and Counting Techniques
Lymphoid organs and cell descriptions are described in several
references (25,36,77). Methods for the preparation of organ cell sus¬
pensions described in Chapter II were followed. Blood was drawn from
the internal jugular vein into a heparinized syringe (50 U heparin/10 ml
blood). This method of obtaining alligator blood was originally de¬
scribed by Herman Brooks (alligator farmer, Christmas, Fla.) and pub¬
lished by Olson et al. (86). A maximum of 5 ml of an organ cell
suspension or undiluted heparinized whole blood was layered onto
Hypaque-Ficoll (p = 1.077). Techniques for centrifugation, cell
washes, cell counts, and viability determinations are described in
Chapter II.
Culture Techniques
Culture techniques are described in Chapter II with the following
additions or changes: 1) 10% alligator serum was used as a supplement,
2) two additional mitogens, pokeweed mitogen (DIFCO) and purified

65
protein derivative (a gift from Dr. R. Waldman, University of Florida)
were used,and 3) only peripheral blood lymphocytes were used in in vitro
primary immunization studies.
Assay for %-Thymidine Incorporation into DNA
Assay techniques are described in Chapter II.
Stimulation Indices and Statistical Analysis
Statistical analysis and formulas for calculating stimulation
indices are presented in Chapter II.
Autoradiography
Techniques for autoradiography are presented in Chapter II.
Histological and Morphological Techniques
Serial cross sections of paraffin embedded organs were kindly pre¬
pared by Mr. Larry J. McCumber (Whitney Marine Laboratory, Marineland,
Fla.). Sectioned tissues, as well as cytocentrifuge preparations of
cell suspensions, were stained with May-Grunwald-Giemsa stain.
Preparation of Rabbit Antisera
The brain of one sacrificed alligator was used for immunization
purposes, following techniques described in Chapter II. Antisera from
two rabbits immunized and boosted eight times over a four month period
were used. Preimmune sera from the same rabbits were used as normal
rabbit serum controls.
Rabbit anti-alligator immunoglobulin was prepared by immunizing
rabbits with immunoglobulins isolated by Sephadex G-200 (Pharmacia)
column chromatography. An ammonium sulfate precipitate of alligator
serum was applied to the column.

66
Cytotoxicity Assay
The protocol described in Chapter II was followed.
Rosetting Techniques
The method of Jondal _et aT. (67) as described in Chapter II was
used to assess the number of peripheral blood lymphocytes capable of
rosetting with sheep red blood cells.
Immunofluoresence
The methods described in Nairn (84) were followed for indirect
immunofluoresent stains of cytocentrifuge preparations of cell suspen¬
sions normal rabbit serum or rabbit anti-alligator immunoglobulin and a
fluorescein labeled goat anti-rabbit IgG. Immunofluorescent methods for
membrane stains are described in Chapter IV.
Hemolytic Plaque Assay
The techniques for harvesting cultured cells and assaying for
plaque-forming cells are described in Chapter II. Fresh alligator
serum diluted 1:20 was used as a complement source.
Cellular Immunoadsorbents
The method of Chess ejt al. (24) was used for fractionating alliga¬
tor peripheral blood lymphocytes on cellular immunoadsorbents. Rabbit
anti-alligator immunoglobulin was precipitated with 40% ammonium sulfate,
washed three times and redissolved in 0.15 M NaCl. The immunoglobulin
enriched fraction was then dialyzed against 0.15 M NaCl - 0.005 M
Na^B^O^ (pH 8.3) prior to coupling onto CnBr activated Sephadex G-200
(Pharmacia). Preimmune rabbit serum, treated in an identical manner,
was coupled to Sephadex as a control.

67
Affinity columns were prepared as follows: The coupled Sephadex
G-2Ü0 preparations were washed with 5% FCS in G-MEM and 8 ml of packed
volume was poured under 1 x g into 12 ml disposable syringes. Two and
one half billion cells in 2.5 ml of 5% FCS in G-MEM were loaded di'op-
wise (10 drops/min) followed by the slow dropwise addition of 5% FCS
in G-MEM. Elutions were monitored periodically until the effluent was
cell free. The nonadherent cells were washed three times with medium
prior to further use.
Glass Wool Fractionation
The method described by Trizio and Cudkowicz (110) was adapted for
use in glass wool and nylon wool column fractionations of alligator
peripheral blood lymphocytes. Glass wool (Corning Glass Works, Corning,
N.Y.) was pretreated by rinsing three times with pyrogen free 0.15 M
NaCl, boiled 1 hr in tripled-distilled water (three changes) and dried
by lyophilization. Twelve milliliter disposable syringes were packed
to the 8 ml mark with the pretreated glass wool and sterilized. Prior to
loading cells on the prepared column, 40 ml of prewarmed (32°C) G-MEM
was passed through the column followed by 15 ml of 5% FCS in G-MEM. The
column was then incubated for 30 min at 32°C in 5% C02~95% air. One hun¬
dred million cells in 2 ml of 5% FCS in G-MEM were loaded onto each
column and were washed into the column with 1 ml of 5% FCS in G-MEM.
Loaded columns were incubated in a vertical position at 32°C for 1 hr
in 5% C02~95% air. Nonadherent cells were eluted very slowly (20 drops/
min) with 20 ml of 5% FCS in G-MEM (32 C). Fifteen milliliters of warm
5% FCS in G-MEM were then slowly flowed through as a "buffer" between the
nonadherent and the adherent fractions. Care was taken not to gener¬
ate a fluid head of pressure nor to jar the column during the slow

68
elution of the nonadherent cells or the "buffer" flow through. Ad¬
herent cells were eluted in a 40 ml volume of G-MEM by generating a
fluid head of pressure as well as mechanically disrupting the glass
wool. Cell fractions were washed three times prior to further
analysis.
A procedure identical to that described in the preceding paragraph
was followed in the preparation and use of nylon wool columns.

Results
Lymphoid Organs of the Alligator
Since the Florida alligator is listed by the Florida Game and Fresh
Water Fish Commission as an endangered species, only a limited number
of alligators were available for experimental purposes. Fortunately,it
was easy to obtain large amounts of blood which was an abundant source
7
of lymphocytes (1-2 x 10 lymphocytes/ml of whole blood) . There were no
detrimental effects to the animals. Evidence will be presented in a
subsequent section that the population of lymphocytes isolated from
peripheral blood are representative (on the basis of mitogenic respon¬
siveness) of the lymphocytic cells isolated from the spleen.
Two of ten alligators obtained from the Florida Game and Fresh
Water Fish Commission were sacrificed (by special permit) for histologi¬
cal examinations and in vitro mitogenic studies of the lymphoid organs.
The only recognizable lymphoid organs were the thymus and the spleen.
The thymus was a small whitish organ, approximately 2 x 8 mm located
in the throat. Histological examinations of tissue sections showed an
abundance of lymphocytes and signs of thymic involution were seen. Very
few cells were isolated by Hypaque-Ficoll centrifugation from whole
organ cell suspensions (< 5 x 10^). The spleen of the alligator was
a red, kidney-bean shaped organ, located beneath the stomach, and was
surrounded by a thick capsule. Red and white pulp regions were observed
in tissue sections and a heterogeneous population of white cells was
69

70
seen. Only 2-5 x 10 cells were isolated from Hypaque-Ficoll isolated
preparations of whole spleen cell suspensions.
Small aggregates of lymphoid cells were present in glandular tis¬
sues found in the orbital sinus and the area of the cloaca. However
further histological studies are necessary before these tissue can be
defined as lymphoid equivalents of the Harder's Gland or Bursa found
in birds. In vitro studies of these tissues were not possible due to
the very few cells isolated by Hypaque-Ficoll gradient centrifugation.
No gut associated lymphoid tissue or lymph nodes were found.
Separation Technique
Hypaque-Ficoll (p = 1.077) was used to isolate relatively pure
lymphocyte preparations from heparinized whole blood or organ cell sus¬
pensions. White cell differential counts of fractionated and unfrac¬
tionated blood are presented in Table 12 and illustrate the efficiency
of the technique for isolating lymphocytes. Hypaque-Ficoll isolates
routinely contained only about 5% granulocytic cells (predominately
basophilic staining cells by May-Grunwald-Giemsa stain), and about 5%
%
red blood cells. Approximately 2-3% of the granulocytes were phagocytic
(assayed by collodial carbon uptake). Examination of the cells recov¬
ered from the interface and within the Hypaque-Ficoll gradient showed
> 99% of the lymphocytes were present at the interface. A photomicro¬
graph of a representative isolate is presented in Figure 7.
Culture Conditions
Various sera were tested to determine a suitable supplement with
MEM for in vitro studies. Ten percent alligator, human, calf, fetal
calf, and rabbit sera or all combinations of equimixtures (5% per serum)

71
Table 12
White Cell Differentials of Alligator Whole
Blood and Hypaque-Ficoll Isolated Blood Cells
Percent of Total
b
Cell Typea
Blood
Hypaque-Ficoll Isolated
Thrombocyte
4±2
0
*
Granulocyte
36±4
5+5
Lymphocyte
60±2
95±5
(a) Smears were made of whole blood and Hypaque-Ficoll isolates
of individual samples and were May-Grunwald-Giemsa stained for
quantitation purposes.
(b) Results are expressed as a percent of the total number of
white blood cells counted.
(c) Each value represents the mean of determinations from 10 dif¬
ferent alligator samples (> 3 determinations per samples) ± standard
deviations.

72
Figure 7. Photomicrograph of a representative Hypaque-Ficoll isolate
of alligator peripheral blood. May-Grunwald-Giemsa stained. Magnifi¬
cation x 400.

73
of any two were tested. Only 10% alligator serum and 5% alligator-5%
fetal calf serum supported in mitogen stimulation of lymphocytes. Al¬
though significant stimulation was obtained in cultures supplemented
with an equimixture of alligator and fetal calf sera, stimulation in¬
dices were lower than those obtained from 10% alligator serum supple¬
mented cultures and therefore 10% alligator serum was used routinely.
Not all alligator sera were supportive as a supplement and it was
necessary to test new alligator supplement sources in mitogenic assays
to determine their suitability. Tests of eight serum pools (> 10.
individual bleedings per pool) obtained from 2-3 yr old alligators
and four individual alligators > 10 yr old are presented in Table 13.
Individual sera from older alligators were more effective than pools of
sera from younger alligators. Since large volumes (200-500 ml) could
be obtained from individual bleedings of 100-225 kg alligators, the
older alligators were used exclusively as sources of serum in subse¬
quent experiments.
Although statistically significant stimulation of alligator lym¬
phocytes cultured with mitogens was obtained using 10% alligator serum
supplemented MEM (0.117 M NaCl), severe cell clumping and loss of
viability were noted when cells were suspended in the culture medium.
To determine if the salt concentrations in the medium were appropriate¬
ly matched to alligator serum levels, three alligator sera (obtained from
individual bleedings) were analyzed by the Blood Chemistry Lab (Depart¬
ment of Pathology, J. Hillis Miller Health Center). Comparisons of the
chemistry lab reports with the GIBCO MEM formulations revealed a repro¬
ducible difference in the NaCl concentrations. On the basis of this
finding an experiment in which alligator peripheral blood lymphocytes

74
Table 13
PHA Responses of Alligator Peripheral Blood Lymphocytes
Cultured with Different Alligator Serum Supplements
Supplement
Poolb A
B
C
D
E
F
.G
H
Alligator0 AA
BB
CC
DD
Stimulation Index5
1
36
45
18
18
7
5
19
78
1
90
100
(a) Triplicate cultures were incubated at 32°C with or without PHA
(1 ¡J.I), pulsed on day 4 and harvested on day 5.
(b) Each supplement pool is from > 10 individual bleeds of 1-2 kg
alligators 2-3 yrs old.
(c) Individual serum supplements are from bleeds of 100-225 kg alli¬
gators > 10 yrs old.

75
were stimulated with PHA in different MEM preparations containing various
concentrations of NaCl was conducted. The results of this experiment are
presented in Table 14. TCA precipitable counts of PHA-stimulated cells
cultured in mammalian MEM were significantly increased (p < 0.05) over
control counts. However,stimulation indices of cells cultured with
0.157 M or 0.177 M NaCl concentrations (0.040 M and 0.060 M extra NaCl
respectively) were approximately three times greater than the stimula¬
tion index of cells cultured in mammalian MEM. Also cell clumping and
loss of viability were no longer evident. Therefore,the NaCl concentra¬
tion of MEM was routinely increased by 0.04 M to 0.157 M in all media
used in subsequent in vitro studies with alligator lymphocytes.
To determine if optimal conditions for pulsing mammalian cultures
with "*11-thymidine (0.5-1.0 pCi/culture; 24 hr) were applicable for
3
alligator lymphocyte cultures, the effects of H-thymidine concentra¬
tions used per well and the length of the pulse were examined. The
data presented in Tables 15 and 16 indicate that incubating the cultures
with 0.5 yCi 3H-thymidine for 24 hr prior to culture termination was
optimal for pulsing alligator lymphocyte cultures.
Mitogenic Studies
Since large numbers of lymphocytes could be obtained from single
O
bleedings (4-8 x 10 lymphocytes from 40 ml of blood), large scale
experiments were designed to determine the effects of 1) mitogen dose,
2) length of time in culture, and 3) temperature on the responses of
peripheral blood lymphocytes to phyrtohemagglutinin (PHA), concanavalin
A (Con A), 1ipopolysaccaride (LPS), pokeweed mitogen (PWM), and purified
protein derivative (PPD).

76
Table 14
Effect of Sodium Chloride Concentration on Alligator
Peripheral Blood Lymphocytes Cultured with PHA
a
NaCl Concentration
Stimulation Index^
0.117 Mc
47
0.137 M
85
0.157 M
142
0.177 M
145
0.197 M
59
0.217 M
1.0
(a) Final concentrations of NaCl
in the culture medium.
(b) Cultures were incubated at 32°C with or without PHA (1 yl), pulsed
on day 4 and harvested on day 5.
(c) The concentration of NaCl in mammalian MEM was calculated from the
GIBCO formulation to be 0.117 M.

77
Table 15
•z
Effect of H-Thymidine Concentration on Alligator
Peripheral Blood Lymphocytes Cultured with PHA
. 3 ,
yCi H/Culture
Stimulation Index3-
0.05
30
0.1
60 ,
0.25
66
0.5
71
1.0
71
2.0
67
(a) Ceils were incubated at 32°C with or without PEA (1 yl), pulsed on
day 4 and harvested on day 5.

78
Table 16
â– 7
Effect of Incubation Time with H-Thymidine on PHA
Stimulated Alligator Peripheral Blood Lymphocytes
Length of Time withc
•^H-Thymidine (Hr)
Stimulation Index
24
108
48
119
72
112
96
110
(a) Cells were cultured at 32°C with or without PHA (1 pi)
pCi of ^H-thymidine was added at various intervals prior to
cultures. All cultures were terminated on day 5.
Y
Five-tenths
the harvest of

79
The results of one experiment designed to test the effects of tem¬
perature on responsiveness of alligator lymphocytes to PHA are presented
in Figure 8. Cells were cultured with various doses of PHA at the tem¬
peratures indicated and the optimal dose and the length of time for max¬
imum stimulation was determined at each temperature. The results indi¬
cate that the lower the temperature, the longer the time required for
maximum stimulation (indicated in parenthesis). The response of cells
cultured at 22°, 35°, 37°, and 40°C was significantly lower (p < 0.01)
than in cells cultured at 27°, 30°, and 32°C. Although responses pf
cells incubated at 27°, 30° and 32°C were not significantly different
from each other (p > 0.1), the length of time required for optimal stim¬
ulation of cultures maintained at 32°C was shorter (five days) as com¬
pared with 27° and 30°C maintained cultures (seven days). The optimal
mitogen dose was found to be the same at all temperatures. Typical
responses to various mitogen doses and incubation times of alligator
lymphocytes cultured with PHA and LPS at 32°C are presented in Figures 9
and 10 respectively. The response to PHA peaked sharply on day 5 and
decreased slowly, whereas the peak response to LPS remained elevated
after reaching an optimum on day 5. Similar experiments were performed
with each of the other mitogens and the results can be summarized by
stating that the optimal temperature tested was found to be 32°C, the
length of time for maximum stimulation was five days and the optimal
mitogen dose was the same at each temperature tested. Optimal doses
per culture of LPS, PPD, PWM, PHA and Con A were 10 yg, 10 yl, 1 yl, and
20 yg respectively. It should be pointed out that responses of cells
cultured with 20 yg of Con A varied in different experiments and was
attributed to changes in the lot numbers of Con A used, as well as the

Figure 8. The effects of temperature on the responsiveness of alligator
peripheral blood lymphocytes to PHA. Cultures were stimulated with 1 pi
of PHA. Numbers within parentheses indicate the length of time (days)
to maximum stimulation at the designated temperature.

00
80
60
40
20
TEMPERATURE (C°)

Figure 9. Dose and time response of alligator peripheral blood lymphocytes
cultured with PHA. All cultures were incubated at 32°C.

30-
â–¡ CONTROL
23 0.01 JÜ 1 / culture
e o.i
CPM
0 1.0
20-
X !0"3
0 5
£2 20
10-
imm
«■T.wM
£
*1
1
3
I
'.'1'
I
*
I
*X
J
■'¿S
xS
US
DAYS IN
CULTURE

Figure 10. Dose and time response of alligator peripheral blood lymphocyt
cultured with LPS. All cultures were incubated at 32°C.

C PM
XIÓ3 3
â–¡ CONTROL
1 O.i ug / CULTURE
.0
DAYS IN
00
Ol

86
length of time a mitogen solution was stored at 4°C. Responses to PHA
or Con A were always significantly greater (p < 0.01) than responses to
LPS, PWM, or PPD, with stimulation indices of PHA or Con A ranging from
40-250 and those for LPS, PWM,or PPD stimulated cultures between 1-25.
Assay for In Vitro Cellular Reactions
To determine if TCA precipitable counts were a valid measure of
cellular events in culture, the number of labeled cells stimulated
with various concentrations of LPS or PHA were correlated with the
Y
TCA precipitable counts in experiments (Chapter II). The results are
presented in Figures 11 and 12 and indicate that both LPS-and PHA-stim-
ulated cultures exhibit changes in TCA precipitable counts closely
paralleling those changes in percent of labeled cells identified by
autoradiography. Cells optimally stimulated with PHA (1 yl) were pre¬
dominately in aggregates and looked like lymphoblasts (Figures 13a and
13b). Cells optimally stimulated with LPS (10 yg) were also morphologi¬
cally characterized as blast-like but were not clumped (Figures 14a and
14b).
Comparison of Peripheral Blood and Splenic Lymphocyte Mitogen Responses
To assay whether mitogenic responses of peripheral blood lympho¬
cytes were similar to the mitogenic responses of lymphocytes from other
sources, cell suspensions were prepared from various alligator lymphoid
tissues. Only the spleen cell suspension yielded a sufficient number
of lymphocytes (isolated by Hypaque-Ficoll) to culture in a mitogen
assay. The results obtained from mitogenic stimulations of peripheral
blood and splenic lymphocytes are presented in Table 17. Optimal dose
and time responses of both isolated lymphocyte populations were the same

Figure 11. Correlation of TCA precipitable counts with the number of
autoradiography positive cells from PHA-stimulated alligator lymphocyte
cultures. Cultures were incubated at 32°C, pulsed on day 4 and assayed
on day 5.

40
30
20
10
MITOGEN CONCENTRATION (pi)
oo
Cc

Figure 12. Correlation of TCA precipitable counts with the number of
autoradiography positive cells from LPS-stimulated alligator lymphocyte
cultures. Cultures were incubated at 32°C, pulsed on day 4 and assayed
on day 5.

20-1
% OF
TOTAL
NUMBER
POSITIVE
o —o
LPS
CONCENTRATION
LO
O

Figure 13. Photomicrograph of PHA-stimulated alligator peripheral blood
lymphocytes. (a) Autoradiograph stained with toluidine blue. (b) May-
Grunwald-Giemsa stained. Magnification x 1000.

92

Figure 14. Photomicrograph of LPS-stimulated alligator peripheral blood
lymphocytes. (a) Autoradiograph stained with toluidine blue. (b) May-
Grunwald-Giemsa stained. Magnification x 1000.

94

95
Table 17
A Comparison of the Mitogen Responses of
Alligator Blood and Splenic Lymphocytes
Stimulation Index
Mitogen
Blood Spleen
LPS (10 yg)
10 1 '
PWM (10 yg)
3 6
PPD (10 yg)
2 6
PHA (1 yl)
192 142
Con A (10 yg) 49 47
(a) Cell suspensions of blood and spleen were separated on Hypaque-Ficoli
Control and mitogen stimulated cultures of Hypaque-Ficoli isolates were
incubated at 32°C, pulsed on day 4 and harvested on day 5.
%

96
for each mitogen, with the exception of the splenic lymphocyte response
to LPS, in which case no response was observed. PHA and Con A responses
in the two preparations were comparable. Responses to PWM and PPD were
similar (p > 0.1) for splenic and peripheral blood lymphocytes.
Mixed Lymphocytes Cultures
Peripheral blood lymphocytes from different alligators were test¬
ed for their ability to respond in two-way mixed lymphocyte cultures
(MLC's). Eight alligators were bled and the lymphocytes isolated by
>»
Hypaque-Ficoll. The results of all possible two way MLC's as well as
PHA stimulation indices (from individual animals) are presented in
Table 18. Optimal MLC responses were obtained on day 5 and remained
high through day 7. There was a wide spectrum of responses, expressed
as stimulation index, ranging from 1 (no response) to 12.5. Although
the response to PHA was significantly lower (p < 0.05) in one of the
alligators (alligator number 4) than the other seven, there was no
correlation between a low response to PHA and the ability to respond in
MLC.
The Combined Mitogen Effects on Lymphocyte Stimulation
The responses of alligator peripheral blood lymphocytes to various
combinations of the mitogens were assayed to determine if different com¬
binations would give additive, synergistic or antagonistic effects. The
data from one experiment are presented in Table 19. Although in some
cases it was difficult to determine if the combined effects were addi¬
tive, synergistic or antagonistic when compared to the results from
cultures stimulated with only one mitogen, the results from culturing
lymphocytes with combinations of LPS + PHA, PWM + PHA and PWM + Con A

Table 18
Mixed Lymphocyte Cultures of Alligator Peripheral Blood Lymphocytes
Stimulation Indexa
PHA Response
167
143
123
76
157
207
145
172
(a) Cultures were incubated at 32°C, pulsed on day 4 and harvested on day 5.
(b) Cells were stimulated with 1 yl of PHA.
VO

98
Table 19
Combined Effects of Mitogens on Alligator
Peripheral Blood Lymphocytes
Mitogen(s)
Stimulate
LPS (10 pg)
6.7
PWM (10 pi)
3.3
PHA (1
â–  vl)
58
Con A
(20 pg)
25
LPS +
PWMb
17
PHA +
Con A
55
LPS +
PHA
142
LPS +
Con A
31
PHA +
PWM
22
Con A
+ PWM
7
(a) Cultures were incubated at 32°C, pulsed on day 4 and harvested on
day 5.
(b) Optimal concentrations of the mitogens were used in the combined
stimulations.

99
clearly indicated that there were at least two different effects on the
stimulation of lymphocytes by the different combinations of mitogens.
The effect of LPS + PHA was synergistic since the response was ^2.5
times higher (p < 0.01) than the PHA response alone, 'v 20 times (p <
0.001) higher than the LPS response and greater than a twofold increase
of the sum of the LPS and PHA responses. The effect of PWM + PHA or PWM
+ Con A was antagonistic, in that the responses were 'v 0.6-0.7 times
lower (p < o.o5) than the PHA or Con A responses. The results from LPS
+ PWM, PHA + Con A and LPS + Con A stimulations were not sufficiently
conclusive to determine if the effects were additive or synergistic.
Effect of Environmental Temperature on LPS Responsiveness
During the winter months a decrease in LPS responsiveness of alli¬
gator peripheral blood lymphocytes was observed. A subsequent return
of responsiveness to LPS occurred with the arrival of spring weather.
The data compiled from studies of one alligator from Nov. 20, 1975,to
Feb. 27, 1976,are presented in Figure 15. Since the animals were housed
outdoors and the winter was unusually cold, it was hypothesized that
such colder environmental temperatures may have effected the circulating
population of LPS-responsive lymphocytes. To test this hypothesis two
alligators were housed indoors at 16°C for an extended period of time
and the mitogen responsiveness of their peripheral blood lymphocytes
(cultured at 32°C) was monitored periodically. The results presented
in Table 20 indicate that environmental temperature did significantly
effect the LPS-responsive population since a LPS response was not de¬
tected in either alligator after 36 days at 16°C. Although the PHA
response appeared to drop also it was nonetheless present when the

Figure 15. A chronological study during the winter months of alligator
peripheral blood lymphocytes. Beginning and ending dates are indicated
within the parentheses on the abscissa. Cells were cultured at 32°C,
pulsed on day 4 and harvested on day 5. LPS and PHA concentrations were
10 yg and 1 yl, respectively. S.I. = Stimulation Index.

101

102
Table 20
Mitogen Responses of Peripheral Blood Lymphocytes
from Alligators Maintained at 16°C
day 5.
Time at
16°C
Alligator
a
Mitogen
0
7_
16
36
X
LPS (10 yg)
6.5
4.4
1
â– *1
PHA (1 yl)
250
100
30
59
77
LPS (10 yg)
4.9
10
5.8
1
PHA (1 yl)
83
132
75
35
Cultures were
incubated at
32°C,
pulsed on day 4
and
harvested
*

103
LPS response had disappeared. Upon returning alligator X to an outdoor
environment for three months (May, June and July) the LPS responsive
cells were again detectable (S.I.'s for LPS and PHA were 5 and 72 re¬
spectively) .
Evidence for Two Populations of Alligator Lymphocytes
Differences in the magnitude of the stimulation obtained with the
different mitogens as well as variations in the combined effects of dif¬
ferent mitogens suggested that there may be at least two types of lym-
*
phocytes responding in mitogenic assays. The following results are
from experiments designed to demonstrate that at least two different
lymphocyte populations are present in cell suspensions isolated by
Hypaque-Ficoll from the peripheral blood of alligators. To limit the
number of variables tested, PHA and LPS were the primary mitogens used
to follow mitogen responses after various cell manipulations.
Experiments were designed to determine if different populations of
lymphocytes could be separated on the basis of their adherence or non¬
adherence to nylon wool or glass wool. Cells passed through columns
*
filled with nylon wool or glass wool could be separated into two frac¬
tions; nonadherent and adherent fractions. These fractions were then
cultured with various mitogens to determine if they exhibited differ¬
ences in responses to mitogen stimulation. Nylon wool columns proved
ineffective in that there was no difference between the nonadherent and
adherent fractions in response to PHA, LPS, Con A, or PWM stimulation.
However cell fractions obtained fi*om glass wool columns did show differ¬
ent mitogen responses. In three experiments the nonadherent cells re-
producibly exhibited significantly (p < 0.05) higher PHA (and Con A)

104
responses than unfractionated or adherent cells and very low or no LPS
(and PWM) responses. The response of the adherent cells to PHA was not
different from the responses of unfractionated cells (p > 0.1), but
showed a significant (p < 0.01) increase in response to LPS over un¬
fractionated cells.
It was hypothesized that there was an enriched population of cells
responsive to PHA in the nonadherent fraction and an enriched population
of cells responsive to LPS in the adherent fraction. It was further
speculated that the response to PHA in the adherent cell fraction' could
be accounted for by nonspecific trapping of a nonadherent cell popula¬
tion within the glass wool fibers which was eluted along with the cell
population responsive to LPS only after mechanically disrupting the
glass wool. To determine if the cell population responsive to PHA could
be depleted from the LPS-responsive cells in the adherent fraction and
if the PHA-responsive cells in the nonadherent fraction could be further
enriched, the nonadherent and adherent fractions were recycled through
glass wool columns. Figure 16 is a diagram of the procedure used and the
results from oné such experiment are presented in Table 21. A higher
response to PHA was obtained in the cell fraction which was nonadherent
to either column (NA NA) and no response to LPS was detectable. The cell
fraction adherent to both columns (A A) exhibited a higher LPS response
(p < 0.01) than the unfractionated or adherent populations. However the
response to PHA was only slightly lower (p > 0.1) than the original un¬
fractionated population. In fact the response to PHA of the initial ad¬
herent fraction (A) in this particular experiment was significantly
higher (p < 0.05) than the unfractionated population (0).
In additional experiments similar results were obtained. Responses
to LPS were undetectable in the NA NA fractions and the responses to

105
Peripheral Blood
Lymphocytes (0)
1.5 hr
5% C02
Slow Elution
Ncnadherent (NA)
Pressure
Adherent (A)
(NA NA) (NA A)
(A NA) (A A)
1
Figure 16. Diagram of glass wool fractionation procedures. One hundred
million alligator peripheral blood lymphocytes or cells from eluted
fractions were loaded onto the designated columns.

Table 21
Mitogen Responses of Cell Populations
Fractionated on Glass Wool
Stimulation Indexa
0b
NA
A
NA NA
NA A
A NA
A ;
LPS
(10 yg)
1.5
1.4
6.4
1.3
1.0
3.1
40
PHA
(i yg)
87
248
188
463
210
291
72
(a) Triplicate cultures were incubated at 32°C, pulsed on day 4 and
harvested on day 5.
(b) See Figure 16 for an explanation of abbreviations.

107
PHA were significantly higher than the NA fractions or unfractionated
cells. The LPS responsiveness in the A A fractions were significantly
higher than the responses of the A fractions or unfractionated cells.
The NA NA fraction routinely represented 30-50% of the original pupula-
tion and the A A fraction 10-20%. The small percentage of granulocytes
(see Table 12) present in the original cell population isolated from
Hypaque-Ficoll were adherent to the glass wool and were not eluted by
the procedures used in these experiments. The main cell type eluted
(nonadherent or adherent) was a lymphocyte.
The response to PHA in the A A fractions were not reduced to sig¬
nificantly lower levels than the unfractionated cell response, so that
from these experiments it was not possible to determine if the responses
to LPS and PHA in the A A fractions were actually the responses of two
different cell types (both adherent to glass wool or one adherent and one
nonspecifically trapped) or whether there was only one cell type capable
of responding to both mitogens. However the responsiveness of the NA A
fractions only to PHA and not to LPS indicates that indeed there may
be nonspecific trapping in the glass wool of a PHA-responsive cell popu¬
lation. It should be pointed out that the mitogen response of the NA A
fraction could also be explained by an adherent cell population respon¬
sive only to PHA which was present in the NA fraction due to overloading
the glass wool in the initial column fractionation.
The significantly higher responses to PHA in the NA NA fraction and
LPS in the A A fraction could perhaps be explained on the basis of a re¬
moval of suppressor cells (possibly the granulocytes suppressive for a
particular mitogenic response), rather than simply an enrichment of a

108
cell type capable of responding to a particular mitogen. Experiments
were designed to determine if the mitogen responses of isolated fractions
could be depressed by co-culturing, in various ratios of cell numbers,
mixtures of NA NA or A A fractions with either the unfractionated popula¬
tion (0) or each of the fractionated populations. The results from three
separate experiments (not shown) did not lend evidence to the idea that
suppressor cells were removed by passage through the columns. Decreases
in the PHA or LPS responsiveness of the NA NA or A A fractions co-
Y
cultured with various other fractionated or unfractionated cell popula¬
tions could be accounted for by effects of the NA NA or A A populations.
Further evidence which supports the theory that certain cell types
were enriched by passage through a glass wool column was obtained from
experiments in which unfractionated cells were cultured at various cell
concentrations with PHA and LPS. On the basis of the glass wool frac¬
tionation data the unfractionated cell population with an undetectable
LPS response could have contained cells capable of responding to LPS
which were not detectable in the assay due to their low number. The re-
suits presented in Figure 17 indicate that by increasing the number of
cells per culture, a significant increase (p < 0.05) in the response to
LPS was obtained. A similar effect was seen with PHA over a narrower
range of cell concentrations.
To summarize the results from glass wool fractionation studies, the
data indicate that at least two lymphocyte cell populations are present
in the peripheral blood of alligators: 1) a lymphocyte population which
is nonadherent to glass wool and is responsive to PHA and 2) a lymphocyte
population which is adherent to glass wool and is responsive to LPS (and
possibley to PHA).

Figure 17. Effects of increasing the cell density in mitogen stimulated
cultures of alligator peripheral blood lymphocytes. Cultures were incu¬
bated at 32°C, pulsed on day 4 and harvested on day 5. S.I. = Stimula¬
tion Index.

S.I.
LP3
NUMBER OF CELLS PER CULTURE {xlQ6}

Ill
To determine if different populations of lymphocytes could be
isolated on the basis of differences in cell surface antigens, periph¬
eral blood lymphocytes were treated with different antisera plus comple¬
ment and the surviving cells cultured in_ vitro with various mitogens.
Efforts to produce a rabbit anti-alligator brain with specific reactiv¬
ity to a subpopulation of alligator lymphocytes were unsuccessful, in
that 100% of the lymphocytes treated with the immune sera plus guinea
pig complement were killed (as adjudged by trypan blue exclusion and
cell recoveries). Adsorption of the antisera with alligator red blood
cells removed all of the reactivity ('v 100% of the cells were recovered
as viable and had unaltered responses to mitogens). However,similar
cytotoxic experiments utilizing a rabbit anti-alligator immunoglobulin
plus complement were successful. The results of two experiments pre¬
sented in Table 22 show that a population of lymphocytes responsive to
LPS was depleted by anti-immunoglobulin plus complement and the PHA
response remained intact. To determine if any residual responsiveness
to LPS could be detected, the surviving cells in experiment 1 were cul¬
tured with the mitogens at a higher cell density. The rationale for
this approach was based upon the previously mentioned effects of cell
density. An increase in the number of cultured cells was shown to yield
a response to LPS (see Figure 17). Only cells treated with normal rab¬
bit serum plus complement showed an increase in responsiveness to LPS.
As an additional assay to determine if cells responsive to LPS were be¬
ing depleted by the cytotoxic treatment with anti-immunoglobulin plus
complement, the synergistic action of LPS plus PHA (see Table 19) was
measured. As shown in experiment 1, the synergistic effect was ablated
only by the anti-immunoglobulin plus complement treatment. It should be

Table 22
The Effects of Cytotoxic Treatment with Rabbit Anti-Alligator Immunoglobulin
on the Mitogen Responsiveness of Alligator Peripheral Blood Lymphocytes
£
Stiumlation Index
Experiment
Treatment
Q
% Recovered
Cells/Culture
LPS (10 yg)
PHA (1 yg)
LPS+PHA
1
NRS
94
0.5xl06
5
41
84
2xl06
11
34
101
Anti-Ig
71
O.SxlO6
1
45
34
2xl06
1
40
36
2
NRS
97
0.5xl06
9
98
NDd
Anti-Ig
86
0.5x10
2.5
75
ND
(a) Cultures were incubated at 32°C, pulsed on day 4 and harvested on day 5.
(b) Twenty percent rabbit serum (normal rabbit serum [NRS] or rabbit anti-immunoglobulin
[Anti-Ig] plus 10% guinea pig complement was used to treat the cells.
(c) Viability was determined by trypan blue exclusion. The number of recovered viable cells
was expressed as a percent of the original number of viable cells.
(d) ND = not done. v

113
pointed out that the response to I,PS was not always totally depleted,
as shown in experiment 2. Possible reasons for such differences in
anti-immunoglobulin plus complement treatments will be discussed in a
later section.
Ten to 15% of the total number of lymphocytes were killed by anti¬
immunoglobulin plus complement treatment. However < 1% of the total
number of lymphocytes were membrane immunoglobulin positive as measured
by an indirect membrane immunofluorescence technique. Differences in
the results obtained from the two techniques will be discussed in' a
later section.
Results from the cytotoxic experiments described above indicated
that killing the cells with surface immunoglobulin determinants depletes
the response to LPS. Another approach used to selectively remove cells
bearing surface immunoglobulin, without complement mediated killing,
was attempted. Cells were passed through cellular immunoadsorbents
conjugated with either normal rabbit serum or rabbit anti-alligator
immunoglobulin. Results from one experiment are presented in Table 23.
Cells which passed through the anti-immunoglobulin immunoadsorbent
column were stimulated only by PHA and not by LPS (or PWM). Attempts
to elute the retained cells using whole alligator serum (i.e. antigenic
competition) were not successful due to much reduced flow rates.
In conclusion, these results indicated that removal of surface immuno¬
globulin bearing cells depleted the response to LPS and left intact the
population of cells capable of responding to PHA.
To determine if PHA-and LPS-stimulated cultures expressed differ¬
ent cellular characteristics in vitro, the number of cells producing
immunoglobulin (cells containing immunoglobulin in their cytoplasm) were

Table 23
Depletion of LPS and PWM Responsiveness
in Alligator Peripheral Blood Lymphocytes Passed
Through an Anti-Immunoglobulin Immunoadsorbent
Stimulation
Index
cl
Treatment
% Recovered'3
LPS
PHA
PWM
Unfractionated
-
5
74
9.8
NRS Immunoadsorbent
100
5.5
80
12.5
Anti-Ig Immunoadsorbent
67
1
71
1
(a) Immunoadsorbents were prepared from 40% saturated ammonium sulfate
precipitates of rabbit anti-alligator immunoglobulin (Anti-Ig) or normal
rabbit serum (NRS) coupled to Sephadex G-200.
(b) The number of cells recovered is expressed as a percent of the
total number of cells applied to the immunoadsorbent column.
(c) Cultures were incubated at 32°C, pulsed on day 4 and harvested on
day 5. Mitogen concentrations were 10 yg, 1 yl and 10 yl for LPS, PHA,
and PWM respectively.

115
quantitated by indirect immunofluorescence. The results are presented
in Table 24. Very few positive cells were seen in cell preparations
(Hypaque-Ficoll isolates) before culturing. There was an increase in
the number of immunoglobulin containing cells in PHA-stimulated cultures
as compared to cells which were not cultured. However a similar in¬
crease was also seen in unstimulated cultured controls as well, and was
considered to be a nonspecific increase due to culture conditions. On
the other hand LPS-stimulated cultures showed a significant ( p < 0.01)
increase in the number of immunoglobulin positive cells over uncultured
control, cultured control and PHA-stimulated cultures indicating that
LPS stimulates immunoglobulin production in alligator lymphocytes as
it does in mouse lymphocytes.
In Vitro Studies on Antibody Producing Cells
Due to the limited number of alligators available and the necessity
of maintaining "normal" alligators as blood donors for in vitro experi¬
ments, it was not possible to study rn vivo immune responses. However,
one preliminary experiment was performed to determine if peripheral
white blood cells isolated from Hypaque-Ficoll were immunocompetent in
a primary in vitro immunization assay (Mishell-Dutton type cultures).
Control (without SRBC) or immunized (with SRBC) cultures were maintained
at 32°C and assayed for the number of cells producing antibody to SRBC's
after various periods of incubation. The results are presented in Table
25. Only cells from immunized cultures contained plaque-forming cells.
The peak response occured after seven days of culture (two days later
than the optimal time for mitogenic stimulation). It should be pointed
out that a decrease in the number of plaque-forming cells on day 10 may

116
Table 24
Cytoplasmic Immunofluorescence Studies of Uncultured
and Cultured Alligator Peripheral Blood Lymphocytes
% Positive^
Cells3
NRS
Anti-Ig
Uncultured Control
0
0.1-0.3
>
Cultured Control
0
0.5-1.5
Cultured with PHA (1 yl)
0
0.5-1.0
Cultured with LPS (10 yg)
0
7-10
(a) Cultured cells were incubated with or without mitogens at 32°C for
five days. Cytocentrifuged slides were prepared of cells obtained from
cultures or Hypaque-Ficoll isolates of uncultured cells and were stained
for cytoplasmic immunoglobulin.
(b) The number of positive staining cells is expressed as a percent of
the total number of white cells counted. The "sandwich" stain was nor¬
mal rabbit serum (NRS) or rabbit anti-alligator immunoglobulin (Anti-Ig)
and the second stain was fluorescein conjugated goat anti-rabbit IgG.

Table 25
Primary In Vitro Immunization with Sheep Red Blood
Cells of Alligator Peripheral Blood Lymphocytes
Culturea
Days in Culture
PFC/Culture
% Recovered*3
% Viable0
% Rosettes1^
Control
5
0
18
96
0
Immunized
0
34
93
33
Control
7
0
14
80
0
Immunized
908
90
91
29
Control
10
0
16
96
0
Immunized
19
94
54
28
(a)Trilpicate cultures were incubated at 32°C,
(b) The total number of white cells recovered at the end of the culture period is
expressed as a percent of the initial number of white cells.
(c) The number of viable cells is expressed as a percent of the .total number of white
cells recovered. Viability was determined by trypan blue exclusion.
(d)The number of white cells rosetting with SRBC’s is expressed as a percent of the
total number of white cells.
117

118
have been due to a decrease in viability as a result of a nutrient de-
pletion(s) in the culture medium (the pH was lower on day 10). There
was an initial decrease in the number of cultured cells in both control
and immunized cultures followed by an increase in the number of cells
recovered from immunized cultures and an unchanged residual population
in control cultures.
A high percentage ('v 30%) of the cells recovered from immunized
cultures were observed to form rosettes with SRBC. These rosettes per¬
sisted (i.e. not lysed) through the Jerne assay and therefore probably
did not represent antibody-producing cells.
To explore the possibilities that the rosetted cells recovered from
immunized cultures were not antigen induced but rather represented
either a population of cells normally present in the peripheral blood
of alligator capable of rosetting SRBC's or cells which were generated
by the culture conditions, irrespective of the presence of SRBC's, the
following experiment was done. The alligator used as a blood donor was
bled again at the time of culture termination and the blood separated
on Hypaque-Ficoll. Cells recovered from control cultures (no SRBC's
present) and immunized cultures (SRBC's present) as well as Hypaque-
Ficoll isolated, uncultured cells were each tested for their ability
to form rosettes with SRBC's. No rosettes viere observed in cells re¬
covered from control cultures and < 1% of the total number of uncultured
lymphocytes isolated from Hypaque-Ficoll formed rosettes, whereas 31%
of the cells recovered from immunized cultured were rosetted. These
findings indicate that the high number of rosetted cells in immunized
cultures may have resulted from antigenic stimulation.

Discussion
Culture Conditions
As with any initial study involving in_ vitro lymphocyte culture,
it was important to investigate several variables in order to define
optimal culture conditions. The data clearly demonstrated the neces-
*
sity of establishing culture conditions optimal for the alligator rather
than merely employing mammalian criteria of culture media and incubation
temperature. Although mammalian MEM significantly supported stimulation
of cells cultured with PHA, optimal stimulation was approximately three
times higher when extra sodium chloride was added. Similarly, although
cultures incubated at 37°C gave measurable stimulation indices with PHA
stimulation, an assumption that a mammalian temperature optimum was op¬
timal for alligator cultures would have been erroneous in that the tem¬
perature optimum was 5°C lower.
As previously discussed in the bluegill studies, numerous serum
factors conceivably could be influential in the fn vitro culture condi¬
tions and it is only speculation that such factors are responsible for
differences in the supportive capacity observed with different serum
supplements as well as different alligator serum sources. It was in¬
teresting that individual sera from large, mature alligators were rou¬
tinely more supportive as supplements with cells from younger alligators
than pooled sera from younger alligators. This may provide a basis for
future studies on possible influences of serum factors on immune regula¬
tion.
119

120
Temperature Effecct in the Alligator
Longitudinal mitogenic studies of alligator peripheral blood lym¬
phocyte revealed a decreased response to LPS during the cold weather
months. Mitogenic studies of lymphocytes from alligators maintained at
16°C during the summer subsequently showed a similar decrease in LPS
responsiveness and a recovery of responsiveness when returned to a warm
ambient temperature. These data indicate that the peripheral blood cell
which is stimulated by LPS is influenced in some manner by a decrease in
the environmental temperature. Data from glass wool fractionation,stud¬
ies using cells from alligators during the "winter" months suggest that
there were peripheral blood cells capable of responding, but were proba¬
bly present in lower numbers. However, additional experiments are neces¬
sary to determine if the cells capable of responding to LPS are either
sequestered in the lymphoid organs or alternatively are short-lived and
during prolonged periods at low temperatures are not replaced as rapidly.
There is also the possibility of seasonal fluctuations (which may
have been artificially induced in the 16°C experiment) due to hormonal
changes as suggested by Ambrosius' work in the turtle (3), as well as
indirect influences on immune responses due to seasonal changes in the
blood and urine (63). Also it is conceivable that there was a shift in
the temperature optimum for rn vitro incubations of cultures which
coincided with changes in enviromental temperature changes. Further
studies on the influences of environmental temperature on the rn vitro
immune response in alligators are necessary before the significance of
alterations in lymphocyte responsiveness to LPS in vitro with changes
in environmental temperature can be explained adequately.
It should be noted,however,that the effects of environmental
temperature on ijn vitro LPS responses may explain on a cellular basis

121
the in vivo studies by Evans (47) in another reptile, the California
desert lizard. He has shorn that antibody responses in the lizard were
inhibited if the ambient temperature was lowered from 35°C to 25°C,
correlating with a decrease in LPS responsiveness of alligator lympho¬
cytes when the ambient temperature was lowered. Evans has also shown
that even a primed lizard actively making antibody was inhibited from
making additional antibody when shifted to lower temperatures. Such a
"shutdown" of an ongoing antibody response in the lizard when moved to
lower temperatures is in marked contrast to the teleost antibody re¬
sponses in which antibody production continued after a shift to lower
temperatures (7). This sugests that changes in environmental tempera¬
ture may effect reptiles and teleosts differently. Additional experi¬
ments are necessary to determine if the temperature phenomena in the
alligator and the lizard are related (i.e. both are B-cell functions)
and whether monitoring in vitro LPS responses is a valid indicator of
in vivo temperature effects on the immune functions of the reptiles.
Evidence for Two Subpopulations of Lymphocytes
Several lines of evidence have been presented which argue for the
presence of at least two subpopulations in the peripheral blood of alii
gators. Briefly summarized these are 1) differences in the magnitude
of stimulation with the different mitogens, 2) differences in the com¬
bined effects of the mitogens, 3) a significant increase in immunoglobu
lin producing cells in LPS-stimulated cultures, 4) populations of cells
adherent or nonadherent to glass wool with different responses to LPS
and PHA, 5) the depletion of responsiveness to LPS by cytotoxic treat¬
ment with an anti-immunoglobulin plus complement without reducing the

122
response to PHA,and 6) the depletion of the response to LPS by removal
of immunoglobulin-bearing cells. Although inferences can be made from
one experiment to the next, it was not proven that two populations shown
to differ by one set of experimental criteria were also the same sub¬
populations in other fractionation procedures since only one technique
was utilized in any one experiment. For example, on the basis of the pre¬
sent data, it was not proven that the glass wool adherent population re¬
sponsive to LPS was also the same population depleted by cytotoxic anti¬
immunoglobulin treatment. It is certainly conceivable that more than
two subpopulations were present.
Experiments using anti-immunoglobulin plus complement to selectively
remove immunoglobulin bearing cells demonstrated that the surviving
cells had 1) an intact PHA response, 2) a loss of LPS response and 3)
a decreased response to the combined effects of LPS +, PHA. These data
lend some support to the concept that two different cell types are in¬
volved in the events leading to the responses observed when lymphocytes
were stimulated with the combined mitogens. In other experiments antag¬
onistic effects were demonstrated using PHA + PWM, possibly indicating
that different mechanisms, and possibly different cells than those in¬
volved in the synergistic stimulations, were involved.
It should be pointed out that synergistic and antagonistic effects
in combined mitogen stimulations of amphibian lymphocytes have also been
reported (95) and were interpreted as evidence for different subpopu¬
lations in the amphibians. However, until further experiments are done
to elucidate the mechanisms involved in the synergistic and antagonistic
effects seen with the combined mitogenic stimulations, these types of
experiments should be considered only circumstantial evidence for

125
different, subpopulations of lymphocytes in tha alligator and the amphib¬
ians.
Are Peripheral Blood Lymphocyte Subpopulations T- and B-Cell Equivalents?
As already emphasized in the bluegill studies, designations of T-
and B-cell equivalents in any species must await the association of
functional activities with the in vitro markers established in such
studies. However by analogy, T-like and B-like designations seem
appropriate for the lymphocyte subpopulations established in these
studies. The following discussion is presented to compare the cellular
characteristics of alligator lymphocytes established in these experiments
with in vitro characteristics of bird and mammalian T- and B-lymphocytes.
Using indirect fluorescence microscopy, cytotoxic treatment with
an anti-immunoglobulin and anti-immunoglobulin cellular immunoadsorbents
lymphocytes were found with immunoglobulin on their surface, a B-cell
characteristic in mammals. The latter two techniques were used to show
a diminished response to LPS (a B-cell mitogen) by selectively' removing
the surface immunoglobulin-bearing cells. These results are similar to
those obtained with mammalian B-lymphocytes (24). Although it is pre¬
dictable that the surface immunoglobulin-bearing cells are also the
cells stimulated by LPS, such a conclusion is not warranted with the
present data. It was not proven directly that cells removed were the
cells stimulated by LPS nor that the surface immunoglobulin-bearing
cells became the immunoglobulin-producing cells (Table 24) in LPS-stimu-
lated cultures. It is conceivable that another cell type acting in¬
directly was involved. It should be pointed out that an increase in the
number of immunoglobulin-producing cells (B-cells) in LPS-stimulated cul¬
tures is also observed in mammalian lymphocyte cultures (5,70).

124
There was a discrepancy in the quantitation of surface immunoglobu¬
lin bearing cells by fluorescence (< 1%) or cytotoxic treatment (10-15%).
Such discrepancies are also seen in mammalian systems (90) and are prob¬
ably attributable to differences in sensitivities of the assays; cyto¬
toxic treatment is more sensitive since theoretically only two antibody
molecules (anti-immunoglobulins) combining with adjacent cell surface
immunoglobulin determinants should be necessary for complement mediated
lysis. Also, the failure to effectively deplete the LPS response with
anti-immunoglobulin plus complement in each experiment is an inherent
problem with mammalian systems and suggests that the alligator may also
have a subset of B-like cells which escape treatment and which may be
equivalent to a "null" cell in mammalian systems (94,105).
Fractionation of peripheral blood lymphocytes on glass wool columns
demonstrated an adherent lymphocyte population responsive to LPS. This
adherence is consistent with the characteristics of mammalian B-cells
since they are a more adherent cell than T-cells (2,54,110). However,
in order to explain the PHA responses routinely observed in the adherent
fractions, it is necessary to suggest that alligators have a subset of
T-like cells which are adherent to glass wool or alternatively a B-like
cell responsive to both LPS and PHA.
A small subset(s) of the lymphocytes with low stimulation indices
following LPS stimulation, low numbers of surface immunoglobulin bearing
cells, and low numbers of LPS-responsive cells adherent to glass wool
were found in the peripheral blood. Mammalian peripheral blood B-
lymphocytes also represent low percentages of the total number of
lymphocytes present in the circulation.

125
To summarize the comparisons with mammalian B-cells, the data
suggest the alligator has a B-like lymphocyte population which is
1) present in low numbers in the peripheral blood, 2) responsive to
BPS, 3) adherent to glass wool and 4) has immunoglobulin present on its
cell surface.
In mammalian tissue culture studies a positive mixed lymphocyte
reaction is an accepted measure of cell mediated immunity, i.e. a T-cell
function. Again arguing by analogy, the data from mixed lymphocyte
cultures of alligator peripheral blood lymphocytes would indicate,that
the alligator also has a T-like cell involved in cell mediated reactions.
However, since it has not been proven that the B-like cells rather than
T-like cells are the responding cells in the alligator mixed lymphocyte
reaction, such conclusions should be approached with caution.
Recent experiments have demonstrated that both T- and B-cells are
involved in mixed lymphocyte reactions in the human (57). The B-cell
population was shown to be the stimulating population (elicits the re¬
sponse) whereas the T-cells were the effector cells (undergo stimulation).
The magnitude of the T-cell stimulation was dependent on the number of
B-cells present. With this in mind the mixed lymphocyte culture experi¬
ments need to be redone, monitoring the LPS responsiveness as well as
the PHA responsiveness, to determine if the spectrum of low to high re¬
sponses can be correlated with the magnitude of the LPS responses of
the B-like cells.
Further evidence for the presence of a T-like lymphocyte in the
peripheral blood of the alligator was supported by its mammalian T-cell
characteristics (54) of insensitivity to anti-immunoglobulin plus comple¬
ment cytotoxicity, nonadherence to glass wool or nylon wool (with the

126
possible exception of a small subset mentioned above) and responsiveness
to PHA (and Con A). The data also suggest that the T-like lymphocyte
subpopulation in the alligator is the major lymphocyte population present
in the peripheral blood, similar to mammalian T-cells.
In_ vitro studies of antibody production suggest that the necessary
cells for antigen recognition, antigen processing and antibody formation
are present in the peripheral blood of the alligator. An assessment of
the functional activities of the different subpopulations may be pos¬
sible by modifying the population of cells subjected to antigenic, stimu¬
lation In vitro (Mishell-Dutton type assays) with the various fractiona¬
tion procedures described. If both the T-like and B-like populations
are required before antibody production is obtained, functional activi¬
ties of the cell populations could be assigned.
The Alligator as an Experimental Model
The preliminary data presented suggest that the alligator may be
very similar to the chicken in terms of the architecture of the lymphoid
organs, as well as general characteristics of the isolated lymphocytes.
Thymus,, spleen, bursa, and gut associated lymphoid aggregates have been
previously reported in the alligator (36,37). With the exception of the
gut associated lymphoid tissue these results were confirmed in the histo¬
logical studies of the two sacrificed alligators. In addition a lymphoid
aggregate possibly equivalent to the Harder's gland was found in the
orbital sinus. Also, the in vitro characteristics of the lymphocyte sub¬
populations discussed in the previous section very closely resemble the
general characteristics of T- and B-cells in the birds, so that immuno-
logically the alligator may be nothing more than a "cold blooded chicken."
If further functional analysis of the lymphoid organs and cell types

127
support this prediction, the alligator may be a valuable animal model
for immunologic studies. For example, by simply lowering the environ¬
mental temperature it may be possible to achieve the same effect (at
least in the peripheral blood, if not In toto) as bursectomy in the
chicken. The alligator is also oviparous and conceivably embryonic
manipulations that have been successful with the chicken may also be
applicable to the alligator, as well as the possibility of being able
to modify the embryonic response with temperature changes.
In light of the in vitro temperature studies, the alligator is
somewhat of an immunological paradox.. The In vitro data suggest
that the alligator would do best between 27-32°C and would have dif¬
ficulty with environmental temperatures outside of this range. In
nature the alligator prefers the warmer temperatures and yet survives
the cool winters along the northern Gulf Coast. The question of how
tha alligator is able to cope with his environment is yet unanswered and
may offer an interesting insight into the evolution of the immune systems
in ectothermic and endothermic animals.

CHAPTER IV
MEMBRANE IMMUNOGLOBULINS OF BLUEGILL LYMPHOCYTES
Introduction
Mammalian and avian lymphocytes are heterogeneous in terms of
immunological functions and have been divided into two broad cate-
gories, designated T- and B-cells, based upon their embryologic
origins. Since representatives of each cell category have been shown
to possess antigen binding specificity (115) considerable effort has
been devoted to chemical characterization of the antigen receptors
which these cells possess. The available data indicate that the
receptors on B-cell surfaces resemble monomeric immunoglobulins (2
heavy and 2 light chains) of the IgM class, and in some cases the IgD
class (79). The question of T-cell receptors has not been resolved;
some workers have been unable to detect any immunoglobulin on T-cell
surfaces whereas others claim that monomeric IgM is present on such
cells (74,114). It should also be mentioned that a third group of
workers claim the receptor may be a piece (idiotypic regions) of
immunoglobulin (9).
In light of this controversy it was somewhat surprising that
immunofluorescent techniques have demonstrated that immunoglobulin is
present on nearly all the lymphocytes of a wide variety of lower
vertebrates, regardless of the tissue source (22,42,44,46,116). There¬
fore, since bony fish represent the lowest phylogenetic group of
128

129
animals possessing both putative T-like and B-like cells while ap¬
parently having only one class of serum immunoglobulin (discussed in
Chapter II), studies were initiated to quantitate and to characterize
the membrane immunoglobulin of fish lymphocytes. The fish chosen for
these studies was the freshwater bluegill, Lepomis machrocuris.
*

Materials and Methods
Sources of Animals
The source and maintenance of bluegill are described in Chapter
II. Mice used as comparative controls were obtained from an outbred
albino strain and were kindly provided from a colony being maintained
by Dr. George Gifford, University of Florida.
Sources of Antigens and Antisera
Rabbit antisera to grouper (a marine teleost fish) light (L) chains
(Ra-GL chain) were the same as those used previously (29). Rabbit anti¬
sera to bluegill serum Ig were prepared by immunizing rabbits with im¬
mune precipitates of bream serum proteins precipitated at equivalence
with Ra-GL chain. This approach took advantage of the considerable
cross reactivity between L chains of different species of fish (L. W.
%
Clem, unpublished observations). Each of three rabbits received three
biweekly subcutaneous injections of immune precipitate (1 mg/injection)
in complete Freund’s adjuvant. The antisera obtained by bleeding at 7-
9 weeks were pooled and used here. The specificity of the rabbit anti-
bluegill immunoglobulin (Ra-Blg) is discussed under Results. Sheep
antiserum to rabbit IgG was prepared by repeated (weekly for four
weeks) injections of alum precipitated rabbit IgG (purified by DEAE-
cellulose chromatography from Fraction II, Pentex, Kankakee, Ill.). The
antiserum obtained at 5-6 weeks was specific for rabbit IgG and contained
130

131
about 2.5 mg antibody/ml as adjudged by quantitative precipitation.
Rabbit antiserum to mouse IgM (Ra-M IgM) was kindly provided by Mr.
Alan Brown, University of Florida (17) and contained antibodies against
mouse y and k chains. Fluorescein labeled goat antibody against rab¬
bit IgG was purchased from Miles Laboratories.
Bluegill serum immunoglobulins (Ig's) were purified from whole
serum by affinity chomatography, as discussed under Results, using
Ra-BIg coupled to Sepharose 4B by the CNBr technique (39). Elution of
adherent proteins was accomplished with 0.5 M acetic acid, 1.5 M NaCl.
Mouse IgM was kindly provided by Mr. Alan Brown, University of Florida,
after purificantion, as described previously (17), from the serum of
Ascaris infected mice. The extinction coefficient of bluegill and mouse
proteins (E28O * cm) was assume<^ t0 be 13.5.
Preparation of Lymphocytes
The preparation of organ cell suspensions, isolation of leucocytes
from heparinized blood and organ cell suspensions and characterization
of leukocyte isolates have been described in Chapter II.
•?.
Membrane Immunofluorescence Studies
n
Bluegill lymphocytes were suspended at 2 x 10 cells/ml in ice
cold phosphate buffered saline containing 5% fetal calf serum (PBS-
FCS). One tenth ml aliquots were incubated for 30 min on ice with an
equal volume of various dilutions of rabbit sera (normal or Ra-BIg)
and washed three times with ice cold PBS-FCS; in order to inhibit
"capping" the washing solution used contained 0.03 M sodium azide.
The washed cells were then suspended in 0.2 ml of cold fluorescein
conjugated goat anti-rabbit IgG in PBS-FCS (with or without azide

132
depending upon desirability of "capping"), incubated for another 30
min on ice, and washed three more times as above. Cytocentrifuge
(Shandon-Elliott, Inc.) slides were prepared (1-2 x 10^ cells/pellet),
air dried, fixed 1 min with ethanol, air dried and mounted under PBS
buffered glycerol (1:9). Cells (> 1000/slide) were examined using a
Leitz fluorescent microscope (E. Leitz Inc., Rockleigh, N.J.) with a
IJBC 200W mercury bulb (Osrain, Berlin, Germany), a BG^ excitor filter
(Leitz) and a K490 barrier filter (Leitz).
Labeling, Extraction and Immunoprecipitation of Membrane Immunoglobulin
Bluegill lymphocytes (and mouse thymic and splenic lymphocytes
for comparative purposes) were surface radio-labeled (76) and then
lysed by treatment for 3 hr at room temperature with 0.5% Nonidet P40
(Shell Chemicals U. K. Limited, London, England) prepared.in 0.045 M
Tris-HCl, pH 8.5, 0.01 M EDTA. The lysates were centrifuged for 15 min
at 3000 rpm and the supernatent dialysed for 18 hr with stirring at 4°C
against 100 volumes of buffered saline (0.14 M NaCl, 0.01 M Tris-HCl,
pH 7.4). The dialysed lysates were then centrifuged and immediately
subjected to indirect immunoprecipitation as follows. Various volumes
of the lysates (ranging from 250 yl to 1500 yl) were added to 25 yl
rabbit serum (Ra-BIg, Ra-GL chain or normal rabbit serum as a control)
and incubated at 37°C for 30 min. Sheep anti-rabbit IgG at equivalence
(170 yl) was added and incubated at 37°C for 30 min. Immune precipi¬
tates were allowed to form for 16 hr at 4°C, thrice washed and then
counted for radioactivity. The same protocol was used with mouse
lymphocytes except rabbit anti-mouse IgM was used in the "sandwich."
Inhibition experiments involved the same protocol except that various

133
amounts of unlabeled serum immunoglobulin were mixed with the lysate
prior to adding the rabbit antiserum. Washed immune precipitates were
stored at -20°C until assayed by SDS gel electrophoresis in a reduced
(71) or unreduced (87) state. Marker proteins for these gels included
radio-iodinated (78) nurse shark 19S Ig (MW a, 900,000), nurse shark
7S Ig (MW n, 180,000), nurse shark 19S Ig heavy (H) chain (MW 70,000)
and nurse shark 19S Ig L chain (MW n. 22,000). These proteins were
purified as described previously (33). Pronase digestion at 30°()
for one hr of radio-labeled lymphocytes was performed as described
by others (6 7).

Results
Specificity of Rabbit Antisera to Fish Immunoglobulins
The two antisera (Ra-BIg and Ra-GL chain) used in this study
were each examined by immunoelectrophoresis and by immunodiffusion
against whole bluegill serum as a source of antigen(s). Each test
revealed a single precipitation band; the electrophoretic mobility was
similar to that observed with the IgM-like proteins of other fish (29).
The observation that the Ra-GL chain gave a reaction of partial identity
between grouper Ig and bluegill serum whereas the Ra-BIg failed to form
precipitates with grouper Ig indicated that much of the antibody present
in the latter antiserum likely possessed specificity against bluegill H
chains.
A further test of specificity involved immunoprecipitation of
\
radio-iodinated whole bluegill serum by the indirect system using these
antisera. Each antiserum bound considerably more radioactive material
than control precipitations using normal rabbit serum as the middle
reagent. When such specific immune precipitates were dissolved in urea-
SDS and electrophoresed on SDS-agarose-acrylamide gels approximately
60% of the radioactivity was located in gel slices containing 'v 180,000
molecular weight material. The remainder appeared in the n. 700,000
molecular weight regions of the gels. When these immune precipitates
were extensively reduced and then electrophoresed on SDS-acrylamide
gels, radioactivity was found only in the a, 70,000 (H chain) and
134

135
^ 20,000 (L chain) molecular weight regions. Since the gel slice ratio
of L:H radioactivity was 0.5 for both antisera, it seems appropriate to
conclude that each antiserum was precipitating the same radio-labeled
molecules. This suggestion was substantiated by the failure of
additional antiserum to precipitate additional radioactivity from
bluegill serum that had previously been maximally precipitated with
either antiserum. One final approach at characterizing the Ra-BIg
involved preparative affinity chromatography wherein the gamma globulins
from the rabbit antisera were covalently coupled to Sepharose 4B by CNBr
and used to purify Ig from bluegill serum. Gel filtration on Sephadex
G-200 of the 0.5 M HAc, 1.0 M NaCl eluent from such affinity columns
indicated about 70% of the recovered material to be ^ 7S Ig whereas
the remainder was high molecular weight (Sgow = 13S at 2 mg/ml) immuno¬
globulin. Thus it appears as if bluegill serum contains both tetrameric
(*v 700,000 MW) and monomeric ('v 180,000 MW) immunoglobulins and that the
antisera used were specific for these proteins and no others present in
bluegill serum.
Membrane Fluorescence of Bluegill Lymphocyte Immunoglobulin
White cell suspensions from bluegill blood, thymus, anterior
kidney, and spleen were subjected to indirect immunofluorescence in
order to obtain evidence of membrane associated immunoglobulin. The
results from each of three different fish indicated that > 90% of the
white cells exhibited membrane fluorescence with either antiserum
(RA-BIg or Ra-GL chain) but not with normal rabbit serum. Furthermore,
incubation of such cells at room temperature for about 3 hr resulted
in "patching" followed by "capping" on at least 60% of the fluorescing

136
cells. It should also be mentioned that cell suspensions from bluegill
posterior kidney (a nonlymphoid tissue) exhibited only slight membrane
fluorescence, i.e. < 5% of the cells were positive.
Lactoperoxidase Catalyzed Iodination of Bluegill Lymphocytes
Bluegill WBC, anterior kidney and spleen leucocytes and thymocytes
were radio-iodinated by the lactoperoxidase method and lysed by the
detergent NP-40. Each cell population yielded considerable specifically
precipitable radioactivity using Ra-BIg or Ra-G'L chain. Representative
results from one such experiment are presented in Figure 18 and indicate
the specifically precipitated radioactivity to be 3-4 times the non¬
specific levels. Furthermore, based upon the results of four different
experiments, this level of specifically precipitable radioactivity
approximated 1-2% of the TCA precipitable activity present in the
dialysed lysate for each cell population. In addition, whereas the
Ra-GL chain serum appeared to bind only about half as much radioactive
material as did a similar volume of Ra-BIg, reprecipitation of super¬
natants indicated that each antiserum was capable of removing all radio¬
activity that was precipitable with the other antiserum.
A control experiment conducted to assess immunoglobulin degradation,
involved incubating radio-iodinated bluegill serum immunoglobulin (a
mixture of ^ 16S and 7S Ig) with an unlabeled population of bluegill
kidney cells followed by cell lysis with NP-40. After dialysis at 2°C
for 72 hr, the radioactivity was still 94% precipitable with Ra-BIg.
Gel electrophoresis in the presence of SDS of reduced and unreduced
"spiked" lysates indicated no detectable changes as a consequence of
the lysis-dialysis procedure employed.

137
LYSATE ADDED (jul x I0‘2)
Figure 18. Immunoprecipitation of lysates of membrane labeled bluegill
lymphocytes. Open circles = precipitated with anti-bluegill immunoglo¬
bulin. Closed circles = precipitated with normal rabbit serum.

138
Quantitation of Immunoglobulin on the Surface of Bluegill Lymphocytes
The basic protocol employed involved mixing saturating amounts of
radio4abeled cell lysates (such as 500 pi for the lysates depicted in
Figure 18) and varying amounts of unlabeled bluegill serum 7S Ig prior
to adding Ra-BIg. After completion of the indirect precipitation reac¬
tion, the washed precipitates were counted and the 50% inhibition point
was obtained by interpolation. The results of two different experiments
are given in Table 26 and indicated that there was relatively little
difference between the average levels of membrane immunoglobulin >
between cells from blood, spleen, anterior kidney,and thymus. These
-12
values (0.53 - 0.96 x 10 g/cell) were quite similar to the value of
1 x 10~^ g/cell obtained in a control experiment wherein precipitation
of mouse spleen cell lysates by Ra-M IgM was inhibited by mouse 19S IgM.
It shald also be mentioned that this Ra-M IgM failed to detect any
immunoglobulin in lysates of radio-iodinated mouse thymocytes.
Physicochemical Properties of Bluegill Lymphocyte Membrane Immunoglobulins
The polypeptide chain structure of bluegill lymphocyte membrane
•4.
immunoglobulin was assessed by SDS-acrylamide gel electrophoresis of
extensively reduced immune precipitates. As depicted in Figure 19, the
membrane radioactivity from WBC lysates precipitated with normal rabbit
serum was spread throughout the gel with the only significant amount
being localized near the top of the gel. On the other hand WBC lysate
precipitates formed with Ra-BIg or with Ra-GL chain exhibited consider¬
able radioactivity in those gel slices expected to contain peptides with
molecular weights of 70,000 (H chains) and 20,000 (L chains). It should
be pointed out that comparisons of the areas of radioactivity in the H
and L chain regions of the gels depicted in Figure 19 indicated L:H

139
Table 26
Quantitation of Surface Immunoglobulin
on Bluegill and Mouse Lymphocytes
Surface
Immunoglobu1ina
Species
Tissue
Experiment 1
L Experiment 2
Bluegill
Kidney
0.58
0.61
WBC
0.68
0.96
Thymus
0.53
0.65
Spleen
-
0.70
Mouse
Spleen
1.04
-
(a) Results are expressed as 10 ^ g/cell.

140
SLICE NUMBER
Figure 19. Acrylamide gel electrophoresis in sodium dodecyl sulfate
of extensively reduced immune precipitates of bluegill white blood
cell membrane immunoglobulins. Panel 1, open circles = precipitated
with rabbit antiserum to bluegill serum immunoglobulin; closed circles
= precipitated with normal rabbit serum. Panel 2, open circles =
precipitated with rabbit antiserum to grouper light chains, y = posi¬
tion of shark heavy chains. L = position of shark immunoglobulin
light chains.

141
ratios of 0.80 for precipitates formed with Ra-GL chain and 0.79 for
those formed with Ra-BIg. Although the ratio of L to H chain labeling
appeared to vary from one lysate to another (as low as 0.25 in one case)
no differences between the ratios precipitated by the two antisera
were observed. As discussed above for WBC's, immune precipitates of
anterior kidney, spleen, and thymus cell lysates also reproducibly
yielded radioactivity in those gel slices that were expected to contain
H and L chains (Figure 20). A major difference however, was observed
in that these latter cells always yielded considerable (up to 75%'of
that applied in some cases) radioactivity at the top of the gels. For
comparative purposes, immune precipitates of radio-iodinated mouse spleen
cell lysates (prepared as described previously) were also extensively
reduced and subjected to SDS-acrylamide electrophoresis. Although
radioactivity in the L chain region was evident, three major differences
between the mouse and bluegill precipitates were observed, i.e. 1) the
mouse precipitates showed little (< 10% of that applied) radioactivity
at the top of the gels, 2) the mouse major H chain peak was reproducibly
slower than that of bluegill (or shark marker) H chain by about four
gel slices, and 3) the mouse precipitates also contained a component of
intermediate mobility between H and L chains; this component may repre¬
sent the putative mouse 5 chain.
The covalent structure of bluegill cell surface immunoglobulin was
initially studied by gel filtration of lysates using Sephadex G-200
equilibrated with 0.15 M NaCl, 0.01 M Tris-HCl, pH 7.4. Each fraction
eluting from the column was then assayed by the indirect immunoprecip-
itation technique with Ra-BIg. The results obtained (not shown) with
each lymphoid tissue lysate indicated that all immunoprecipitable counts

142
Figure 20. Acrylamide gel electrophoresis in sodium dodecyl sulfate
of extensively reduced precipitates of bluegill spleen and thymus mem¬
brane immunoglobulins. Open circles = precipitated with rabbit anti¬
serum to bluegill serum immunoglobulin. Closed circles = precipitated
with normal rabbit serum. H = position of shark heavy (y) chain. L =
position of shark light chain.

143
were excluded from the column and hence no immunoglobulin appeared
in the volumes expected to contain < 200,000 molecular material.
If on the other hand unreduced specific immune precipitates, as de¬
picted in Figure 21 (left panel) for WBC lysates, are dissolved in
8 M urea, 2% SDS and subjected to SDS-agarose-acrylamide electrophore¬
sis some radioactivity was localized to gel slices expected to contain
180,000 molecular weight material (based upon a simultaneously run
shark 7S Ig marker). Immune precipitates of radio-labeled thymus,
spleen, and kidney lysates also gave demonstrable a. 180,000 molecudar
weight material in such experiments although the proportion of higher
molecular weight material was quite variable. In fact in some cases,
especially with kidney material, > 70% of the applied radioactivity was
of > 180,000 molecular weight. These findings were in contrast to those
with precipitates of mouse lysates wherein 85% of the applied radioac¬
tivity was localized as a single a- 180,000 molecular weight component.
Thus faced with the problem of multiple components (including an
apparently high molecular weight peptide seen on gel profiles of re¬
duced material), specific immune precipitates of radio-labeled blue-
gill cell lysates were dissolved in urea-SDS and subjected to gel fil¬
tration on Biogel 5M equilibrated with 0.5% SDS. As depicted in Figure
21 (right panel) radioactivity originally derived from WBC (and also
with specific precipitates from thymus, spleen, and kidney cell lysates)
was resolved into two major components, i.e. one in a volume expected
to contain a, 180,000 molecular weight proteins (designated pool 2) and
one composed of larger material (designated pool 1). Each of these two
pools were concentrated by pressure dialysis, extensively reduced and
subjected to SDS-acrylamide electrophoresis. As depicted in Figure 22

144
Figure 21. Agarose-acrylamide gel electrophoresis and gel filtration
of unreduced immune precipitates of bluegill white blood cell membrane
immunoglobulins. Left panel: Agarose-acrylamide gel electrophoresis
in the presence ’of sodium dodecyl sulfate of unreduced immune precipi¬
tates of bluegill white blood cell membrane immunoglobulins. Open cir¬
cles = precipitated with rabbit antiserum to bluegill serum immunoglo¬
bulin. Closed circles = precipitated with normal rabbit serum. 19S =
position of shark pentameric IgM. 7S = position of shark monomeric
IgM. Right panel: Gel filtration in the presence of sodium dodecyl
sulfate of unreduced bluegill white blood cell membrane immunoglobulin
precipitated by rabbit antiserum to bluegill serum immunoglobulins.

145
Figure 22. Acrylamide gel electrophoresis in the presence of sodium
dodecyl sulfate of extensively reduced bluegill white blood cell mem¬
brane immunoglobulins fractionated by gel filtration. See Figure 21,
right panel for the fractionation by gel filtration of pools 1 and 2.
Panel 1; electrophoresis of gel filtration pool 1. Panel 2; electro¬
phoresis of gel filtration pool 2. y = position of shark heavy chain
L = position of shark light chain.

146
for WBC material, pool 2 appeared to be composed only of H and L-like
chains whereas pool 1 contained considerable high molecular weight
material in addition to H and L-like chains. Based upon calculations
of recoveries of presumed H and L chain radioactivity from such experi¬
ments, it seems appropriate to conclude that at least 50% of the blue-
gill membrane Ig (WBC, thymus, spleen, and kidney) is a, 180,000 molecu¬
lar weight. The available data do not permit any comment regarding the
structure of the remaining high molecular weight material.
*
Prona.se Digestion of Bluegill Membrane Immunoglobulin
The difficulties encountered above in totally solubilizing bluegill
membrane immunoglobulins suggest that these proteins are tightly bound
to other membrane components and hence may be somewhat "buried. " Thus
pronase digestion of labeled cells prior to detergent lysis was employed
to determine if immunoprecipitable radioactivity could be stripped off
the cell surfaces. The results of two such experiments with bluegill
kidney cells are presented in Table 27 and indicate that such treatment
had no detectable effect on the level of immunoprecipitable radioactivi-
ty. On the other hand similar treatment of labeled mouse splenocytes
appeared to totally remove all surface IgM indicating another difference
between bluegill and mouse membrane immunoglobulins. Of considerable
importance in these experiments was the observation that if immune
precipitates of lysates from pronase-digested bluegill cells were re¬
duced and subjected to SDS-acrylamide electrophoresis (not shown) all
of the radioactivity expected in "H chain" slices was missing and appar¬
ently was in slices expected to contain < 20,000 molecular weight
material. Hence it seems likely that pronase digestion of bluegill
membrane immunoglobulin had occurred but,in the absence of denaturing

147
Table 27
Effects of Pronase Digestion on Membrane Associated
Immunoglobulins of Bluegill and Mouse Lymphocytes
CPM x ID'3 Precipitateda
Experiment
Cells
Treatment
NRS
Ra-Ig
1
Bluegill Kidney
None
14.0
’41.0
Mock Pronase
13.3
38.9
Pronase
14.2
40.5
Bluegill Kidney
None
8.5
39.5
Mock Pronase
7.4
37.0
Pronase
8.5
38.0
5
Mouse Spleen
Mock Pronase
2.0
11.2
Pronase
2.1
2.0
(a)Treated cells were lyzed with Nonidet P-40 and the lysates were pre¬
cipitated with either normal rabbit serum (NRS) or rabbit anti-bluegill
immunoglobulin (Ra-Ig).

148
and reducing conditions, the antigenic integrity of the molecules
was maintained.

Discussion
In any study involving the characterization of membrane proteins
by protocols utilizing lactoperoxidase catalysed iodination, detergent
lysis and immunoprecipitation there are several important factors to
be considered prior to ascribing validity to the results obtained! In
fact it would almost be self-evident that such an approach is no better
than the antisera employed. For this reason considerable effort was
devoted here to ensuring that the antisera were specific for fish im¬
munoglobulins. It would seem as if the argument regarding specificity
was greatly strengthened by the finding of similar results with both
antisera (i.e. anti-bluegill Ig and anti-grouper L chain). Another
factor of considerable importance is the physiological state of the
cells being studied, the major concern being that proteins not on the
cell surfaces are being labeled. While not tested directly here, there
would seem to be several reasons for thinking that bluegill proteins
labeled were on the surfaces of viable cells. The medium employed was
of a tonicity that readily permitted tissue culture studies with blue-
gill cells (see Chapter II). Furthermore, the finding that, while
pronase did not "strip" bluegill membrane immunoglobulin determinants,
it did in fact cleave such proteins must be considered as evidence that
they were exposed to the environment and hence were likely on the
lymphocyte surfaces. It should also be pointed out that the failure to
observe membrane immunofluorescence on posterior kidney cells certainly
149

150
indicates the proteins being detected on fish lymphocytes were not on
all fish cells.
Therefore, assuming adequate specificity of the antisera and the
physiologic state of the labeled cells, the results obtained here
illustrate several important aspects of fish membrane immunoglobulins.
First, as previously described for other fish species (44,45,46,116),
nearly all bluegill blood, spleen, thymus, and anterior kidney lympho¬
cytes possess surface immunoglobulin determinants that appear capable
of "patching" and "capping" when complexed with anti-Ig. Secondly,
quantitation of these antigenic determinants indicated that bluegill
lymphocytes have similar amounts of membrane Ig regardless of the tissue
of origin and , perhaps more importantly, that this amount was quite
similar to that demonstrated for mouse splenocytes (presumed 'v 50%
B-c.ells). It should be mentioned that this level is about 10 times that
reported by others (92) for mouse B cells. In all likelihood this
difference is attributable to the fact that the lysates used contained
undetermined amounts of unlabeled cytoplasmic Ig. Hence the values
reported here for bluegill and mice must be considered as approximations
Thirdly, in terms of the physicochemical properties of bluegill membrane
immunoglobulins, at least half of the immunoprecipitable membrane radio¬
activity was associated with a, 180,000 material and, hence, ressembles the
2H-2L chain covalently linked membrane Ig's found on lymphocytes of
higher animals. It should also be emphasized that, since the H-L
chain rations (in terms of radioactivity) precipitated with the two anti
sera were similar, it becomes highly likely that only one class of H
chains is present on bluegill lymphocytes. The finding of similar H-
like chain molecular weights for material isolated from bluegill

151
thymocytes and other lymphoid tissues does, however, raise a problem in
that similar studies with goldfish membrane Ig's have revealed presumed
size differences in H chains derived from thymocytes and splenocytes
(116). Another somewhat disconcerting aspect of the bluegill results
reported here was the observation that large amounts of extensively
reduced immunoprecipitated radioactivity failed to penetrate the SDS
gels. This raises the question of whether the high molecular weight
material actually had antigenic determinants in common with serum immu¬
noglobulin or alternatively was a ntag-along" portion of incompletely
solubilized membrane. Certainly the finding that gel filtration (in
the absence of SDS) yielded immunoprecipitable material only in >
180,000 molecular weight fractions suggests difficulty in totally
solubilizing the bream membrane Ig with nonionic detergents. It would
therefore seem appropriate here to take a relatively conservative view¬
point in the sense that these studies do not, in fact, prove which of the
components demonstrable on SDS gels actually contained the Ig antigenic
determinants. Future approaches aimed at assessing the antigenicity
of SDS solubilized membrane components may resolve this issue.
Finally, in light of the as yet unresolved issue of membrane recep¬
tors on mammalian T-cells, it would seem appropriate to comment further
on the results with fish thymocytes. As discussed in Chapter II, in
vivo studies indicate that fish have cellular immune functions which,
by analogy, can be called T-like and B-like. Furthermore, in vitro
studies with trout (46) and bluegill (see Chapter II) seem to leave
little doubt that fish have a heterogeneity of lymphocytes much akin to
that seen in mammals. Thus since bluegill thymuses (as well as other
lymphoid tissues) appear to contain T-like cells and since at least 90%

152
of these cells have membrane immunoglobulin determinants, it seems
irrefutable to say that bluegill T-like cells have these determinants
Therefore, the next major question in this areawill be to decide if
these proteins, in fact, are integral membrane components or alterna¬
tively are passively acquired molecules (cytophilic immunoglobulins).

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BIOGRAPHICAL SKETCH
Marvin Agusta Cuchens was born in Fort Walton Beach, Florida, on
June, 7, 1948. He graduated from Tate High School, Pensacola, Florida,
on May 31, 1966. He was awarded a two-year scholarship at Pensacola
Junior College from 1966-1968 and continued his education at the
University of Florida, where he received a Bachelor of Science degree
in chemistry on December 12, 1970. He completed his studies in the
Department of Immunology and Medical Microbiology for the degree of
Doctor of Philosophy in August, 1977.
He will continue his studies in immunology in the Department of
Microbiology and Immunology, University of Oregon Health Science Center,
Portland, Oregon as a Postdoctoral Fellow with Dr. G. A. Leslie.
162

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.
Professor of Immunology and
Medical Microbiology
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of Doctor
of Philosophy.
Kenneth I. Berns, M.D., Ph.D.
Professor and Chairman
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.
jdwúd J? ygk
yff Joseph W. Shands, Jr
Professor and Chief
Infectious Diseases
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.
Í
Richard B. Crandall, Ph.D.
Professor of Immunology and
Medical Microbiology

I certify that I have read this study and that is 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.
Bt^an M. Gebhardt, Ph.D. •'
Associate Professor of
Pathology
I certify that I have read this study and that is 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.
Professor of Immunology and
Medical Microbiology
This dissertation was submitted to the Graduate Faculty of the
College of Medicine and the Graduate Council, and was accepted as
partial fulfillment of the requirements for the degree of Doctor of
Philosophy.
August 1977
f J.J. y
Graduate
Vx

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UNIVERSITY OF FLORIDA
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