Lymphocyte heterogeneity in teleosts and reptiles

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Lymphocyte heterogeneity in teleosts and reptiles
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xii, 162 leaves : ill. ; 29 cm.
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Cuchens, Marvin Agusta, 1948-
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Fishes   ( mesh )
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Immunology and Medical Microbiology Thesis Ph.D   ( mesh )
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Notes

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

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University of Florida
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All applicable rights reserved by the source institution and holding location.
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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
















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.

















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













TABLE OF CONTENTS (continued)

CHAPTER Page

IV Results . . 134
(continued)
Discussion. . . .. 149

LITERATURE CITED....... ................. 153

BIOGRAPHICAL SKETCH . . ... 162
















LIST OF TABLES


Table Page

1 White Cell Differentials of Bluegill Whole Blood and
Hypaque-Ficoll 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 Heterologous
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










Table Page

14 Effect of Sodium Chloride Concentration on Alligator
Peripheral Blood Lymphocytes Cultured with PHA 76

15 Effect of 3H-Thymidine Concentration on Alligator
Peripheral Blood Lymphocytes Cultured with PHA 77

16 Effect of Incubation Time with 3H-Thymidine on PHA
Stimulated Alligator Peripheral Blood Lymphocytes. 78

17 A Comparison of the Mitogen Responses of Alligator
Blood and Splenic Lymphocytes. . ... 95

18 Mixed Lymphocyte Cultures of Alligator Peripheral
Blood Lymphocytes. . . 97

19 Combined Effects of Mitogens on Alligator Peripheral
Blood Lymphocytes. . .. 98

20 Mitogen Responses of Peripheral Blood Lymphocytes from
Alligators Maintained at 160C . .. 102

21 Mitogen Responses of Cell Populations Fractionated on
Glass Wool . . 106

22 The Effects of Cytotoxic Treatment with Rabbit Anti-
Alligator Immunoglobulin on the Mitogen Responsive-
ness of Alligator Peripheral Blood Lymphocytes 112

23 Depletion of LPS and PWM Responsiveness in Alligator
Peripheral Blood Lymphocytes Passed Through an Anti-
Immunoglobulin Immunoadsorbent . .. 114

24 Cytoplasmic Immunofluorescence Studies of Uncultured
and Cultured Alligator Peripheral Blood Lymphocytes. 116

25 Primary In Vitro Immunization withSheep Red Blood Cells
of Alligator Peripheral Blood Lymphocytes. ... 117

26 Quantitation of Surface Immunoglobulin on Bluegill and
Mouse Lymphocytes. . ... 139

27 Effects of Pronase Digestion on Membrane Associated
Immunoglobulins of Bluegill and Mouse Lymphocytes. 147















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

13 Photomicrograph of PHA-stimulated alligator peripheral
blood lymphocytes .................................... 92









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...........

MEM...........

MLC...........

PFC...........

Pi1A...........

PPD ...........

PWM ...........

Ra-BIg........

Ra-GL.........

Ra-M IgM......

RBC...........

RPMI 1640.....

SDS ...........

SRBC..........

TCA ...........


i.


.concanavalin A

.counts per minute

.deoxyribonucleic acid

.alligator mimimum essential medium

.heavy chain

.tritium

.immunoglobulin

.light chain

.lipopolysaccharide

.mimimum essential medium

.mixed lymphocyte culture

.plaque-forming cell

.phytohemagglutinin

.purified protein derivative

.pokeweed mitogen

.rabbit anti-bluegill immunoglobulin

.rabbit anti-grouper light chain

.rabbit anti-mouse IgM

.red blood cell

.Roswell Park Memorial Institute medium 1640

.sodium dodecyl sulfate

.sheep red blood cell

.trichloroacetic acid











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 NaCI concentration in the medium and the age of the

serum donor.










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 320C and 220C, although responsiveness at 220C 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 220C 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








fluctuations in PHA responsiveness, LPS responses dropped significantly

during the winter months or when alligators were housed at 160C.

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 completed

with antisera to fish immunoglobulins. Lactoperoxidase catalyed radio-

iodination, detergent lysis and immunoprecipitation with appropriate

anitsera were 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-cells. 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.















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









mixed lymphocyte reactions (43,53,54,93). B-cells respond to different

mitogens (e.g lipopolysaccharide), 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 inthe control of the 19S to 75 switch (IgM to IgG anti-

bodies) as well as maturation of the humoral response (increase in anti-

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








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 basesfor 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









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

LPS 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.









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

shown 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)








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 birds

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,








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 0.45 v 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 RPMI 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









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 560C and were sterilized by Millipore filtration (0.45 I)


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-Ficoll 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 weighing.

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 Certtrifuge, Boston, Mass.) for 20 min at 1000 RPM. The interface








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 ~) prior to use. One drop was

added to approximately three ml of a cell suspension and the mixture in-

cubated for 30 min at 370C. 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 105 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








were diluted with RPMI 1640 without serum or plasma supplements and

were added to appropriate wells in 20 pl 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 105 cells suspended in 0.1 ml of supple-

mented RPMI 1640 from each cell preparation (total cell concentration

per well was 5 x 105/0.2 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 vCi/10 il/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
2
was routinely measured with a Fryrite CO2 tester (Bacharach Instrument

Company, Pittsburgh, Penn.).

Cells, mitogens and 3H-thymidine 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 serving 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.








White, red, and dead cells were enumerated and 1 x 107 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% C02 95% air humidified

environments as described above.


Assay for 3H-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 3H 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 (Mallinckrodt, 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.;

Model LS-100).








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) 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

Cultured cells, pulsed with 3H-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,








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 with 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

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 et 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








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-iosetting 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 SRBC'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 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 (I 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































C C;


0. X
P0 E-





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uc c
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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

yielded about 5 x 107 and 2 x 107 cells/fish, respectively. Heparinized

blood yielded about 5 x 106 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















Table 1

White Cell Differentials of Bluegill Whole Blood
and Hypaque-Ficoll Isolated Blood Cells


Percent of Totalb
a
Cell Type Blood Hypaque-Ficoll Isolated

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.


















































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









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 pl), Con A (10 pg) or LPS (1 or 10 Vg) at

either 22, 27 or 320C. 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









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 0.15M NaCl. The data obtained with four

bream plasma pools which elicited high background levels prior to

dialysis represented 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 220C and thus resulted in increased stimu-

lation indices with LPS. With three of four pools used with cells

maintained at 320C 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 in 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-















Table 2

Effect of Dialysis of Plasma Supplements on Mitogenic
Stimulation of Bluegill Anterior Kidney Lymphocytes


Stimulation Indexa


PHA (0.1 pl) 32C


Supplement Plasma
Poolb


Undialyzed


(a)
320C
(b)
(c)


Dialyzedc

6.8

4.0

4.0

4.6


LPS (1 pg) 220C


Undialyzed

1

1

5.9

1.5


Dialyzed

1

14.0

16.4

7.4


Triplicate cultures were stimulated with either PHA (0.1 pl) at
or LPS (10 pg) at 220C, pulsed on day 6 and harvested on day 7.
Each pool represented the plasma obtained from at least five bream.
Dialysis was against pyrogen free 0.15 M NaCI.








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















Table 3

Effect of Maintenance Time of Bluegill in
Laboratory Aquaria on Differential White Cell
Counts of Hypaque-Ficoll Isolated Anterior Kidney Cells


Percenta


Maintenance Time


Lymphocyteb


1 day


3 weeks


50-60


Blast-like

0

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.

















































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.















Table 4

Effect of Maintenance Time of Bluegill in Laboratory
Aquaria on the Incorporation of Thymidine by
Unstimulated Anterior Kidney Cell Cultures


CPM/Culturea


Maintenance Timec

1 day

1 day

3 weeks

3 weeks


5 weeks


220C

508

12014

112137

99168

13,197580


(a) Results are expressed as the means of CPM from triplicate
cultures standard deviations.
(b) Cells from individual fish were incubated without mitogens
at 220C or 320C, pulsed with 3H-thymidine on day 2 and harvested
on day 3.
(c) Length of time fish were maintained in laboratory aquaria
before sacrifice.


Fishb


32C

458

446

2645109

1231127


2838181









were cultured for seven days with various doses of PHA at 320C.

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

pl), 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 were

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






























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

















32oC


10 pg o-" 1 \
\


LPS


"0


Ipg o

0.1 pg ,-----


PHA


'0
O.O1pI f.OpZ
3.1lIOr


T I

CON A


/ .-. ,

f/\';


3 5 7 9

DAYS IN


-I


22 C


LPS


0I


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PHA













O *(LI 6*00 *60


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CON A


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CULTURE


20-


10-


200-




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200-





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z





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differences in temperature on maximum stimulation with the various

mitogens. Cells stimulated with PHA (0.1 p-) and Con A (50 ug) re-

sponded well at 32C (p < 0.01) and very poorly, if at all, at 220C

(p > 0.1), whereas LPS (1 pg) responsiveness was higher at 220C

(p < 0.01). There was, however, a significant response (p < 0.05) to

LPS (10 pg) 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 320C were 0.1 pl,

50 ug, and 10 pg for PHA, Con A, and LPS respectively and. 1.0 pg of LPS

at 220C. PHA and Con A responses were greater at 320C than 220C and

LPS responsiveness was greater at 220C than 320C. Optimal culture times

were 5-7 days for all mitogens with the exception of 10 pg of LPS at

320C 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















Table 5

Mitogenic Responses of Bluegill Thymus Lymphocytes


Stimulation indexa


Mitogen


LPS (1 pg)

PHA (0.1 pl)

Con A (10 pg)


320C


220C


4.7


(a) Triplicate cultures were pulsed on day 6 and
harvested on day 7.








(both populations capable of responding) at 220C and 320C. Cultures

were initiated with 0.25 x 106 lymphocytes from each donor fish per

culture well (0.5 x 106 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 320C 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%
















Table 6

Mixed Lymphocyte Responses of Bluegill
Anterior Kidney Lymphocytes


Stimulation Indexa


Mixed
Lymphocyte
Response

Cross 220C 320C


1+2


Mitogen Response


220C

LPSb PHA

62 2.3

17.2 7.2


320C'

LPS PHA


9

8.9


3 22 16 3.4 51
3+4 1 12
4 20 3 2.4 55


3 22 16 3.4 51
3+5 1 9
5 24 2.9 5.3 22


4 20 3 2.4 55
4+5 1 19
5 24 2.9 5.3 22


(a) Triplicate cultures were pulsed on day 6 and harvested on da


Results are expressed as stimulation indicies as defined in Materi
and Methods.
(b) Mitogen concentrations were 1 yg and 10 yg of LPS at 220C and
320C, respectively, and 0.1 pl of PHA at 220C and 320C.
(c) Designates the source of cells used in the mixed lymphocyte
cultures.


7.
als


Fish


y








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 PHA 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 r30% 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 (u 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 former 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.















Table 7

Mitogen Responses of Bluegill Anterior Kidney
Lymphocytes Treated with Anti-Brain Plus Complement
or Rosette Depleted with Rabbit Red Blood Cells


Stimulation Indexa


Expt 1


Treatment


LPSb


Control


Anti-Brain + Complementc


Expt 2


PHA


4.2 9.8

30 1.3


LPS PHA

4 12.4


Expt 3

LPS PHA

8 9.9


26 1.3 ND


Rosette Depletiond


1.8 17


NDe


ND 1 9.0


(a) Triplicate cultures incubated at 320C, pulsed on day 6 and harvested
on day 7.
(b) Mitogen concentrations were 10 pg and 0.1 Vl 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.















Table 8

Rosette Formation of Bluegill Anterior
Kidney Lymphocytes with Red Blood
Cells from Heterologous Species


RBC Source

Bluegill

Human

Ferret

Alligator


Rabbit


Guinea Pig

Horse

Mouse

Sheep


Chicken


% Rosettinga

0

0.65 0.2

0


21 1.5

0.75 0.2

0

0

1.3 0.6

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.









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 prepared

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 was assayed in

a Jerne 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.
















Table 9

Distribution of Antibody Forming Cells in Various Tissues
of Bluegill Immunized with Sheep Erythrocytes


Tissue

Blood (2 ml)

Kidney

Thymus

Spleen

Blood (2 ml)

Kidney

Thymus

Spleen


White
Cells
(xl0-6)

9

100

18

9

10

184

30

24


Number of PFC


Per 106 Cells

4

70

53

52

1

3

4

2


(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).


Fisha

1







2


Total

36

7000

954

468

10

552

120

48








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















Table 10

Immunoglobulin Producing Cells
in the Lymphoid Organs of the Bluegill


Organa % Positiv

Blood 20 5


Spleen

Thymus


Anterior Kidney

Posterior Kidney


45 11

39 15

40 14

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.


eb









organs. All cell suspensions used contained < 30% red blood cells and

" 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 220C

and 320C for various time periods. Two experiments utilizing unimmunized

"normal" bluegill as cell donors are presented in Table 11. In 320C

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 from

immunized than control cultures occurred 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

220C 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 220C.

The magnitude of the response measured on day 7 however was much lower

(control = 0 PFC, "boosted" = 18 PFC/Culture) than that seen at 320C

with 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 (220C, 90% for controls, 285% for boosted;





















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51


320C, 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-

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 factors)

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,








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 320C and very poorly at 220C. 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 320C. The other population of lymphocytes is

LPS-responsive at both 320C and 220C although responsiveness at 220C

was usually greater.

The two subpopulatins were shown to be different by anti-brain

serum cytotoxicity and rosette depletion experiments. The 320C, 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










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 only at 190C 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.









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 320C. The MLC reactive

cells also responded only at 320C 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









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 320C elicited a

very good plaque forming cell response to SRBC's whereas cells main-

tained at 220C 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

220C, though with lower numbers of plaque-forming cells. However 320C

cell cultures were not responsive, contrary to in vivo primary immuniza-

tion studies. This may indicate a secondary antigenic stimulus at 320C

which elicits a tolerant state or suppressive factorss.

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








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 (350C), 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 350C 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 390C rather than 370C.

Armadillos have body temperatures of < 350C, yet the transformation of

lymphocytes stimulated by PHA was increased approximately 2.6 times

when cultured at 370C rather than 33C (91). Perhaps an evaluation of

mitogenic responses of other mammalian lymphocytes cultured in narrower

temperature ranges (370 20C) 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 390C showed the optimal response to both mito-

gens was obtained at 370C.


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









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, 220C cultured

lymphocyte could inhibit capping and membrane events leading to cell

activation, whereas a PHA-responsive, 320C 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

phylogeny of the immune system since phylogenetically they are a

common ancestor of the birds and mammals. However, as pointed out

by Cohen (36), 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.








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 occurring 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








32-370C, whereas at 220C they did not respond at all (80). More

recently, Evans has presented evidence that desert lizards maintained

at 350C responded well to sheep red blood cells, but if maintained at

300C or 40aC, 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 ip

skin allograft rejections, as shown by Borysenko (11). Snapping

turtles accepted allografts when they were maintained at 100C but

were able to reject the allografts at 25C, and more rapid rejections

were seen at 350C.

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








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 NaC1 concentration of

the complete media was increased to 0.157 M by dissolving 2.400 g NaC1

in the medium prior to adjusting the final volume to 1.0 L. The pre-

pared MEM containing extra NaC1 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 If.









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









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 3H-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.








Cytotoxicity Assay

The protocol described in Chapter II was followed.


Rosetting Techniques

The method of Jondal et al. (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 et 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 NaC1. The immunoglobulin

enriched fraction was then dialyzed against 0.15 M NaC1 0.005 M

Na2 407 (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.








Affinity columns were prepared as follows: The coupled Sephadex

G-200 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 drop--

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

NaCI, 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 (320C) 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 320C 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 320C 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

of lymphocytes (1-2 x 107 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 106). 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









seen. Only 2-5 x 107 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)















Table 12

White Cell Differentials of Alligator Whole
Blood and Hypaque-Ficoll Isolated Blood Cells


Percent of Totalb

Cell Typea Blood Hypaque-Ficoll Isolated

Thrombocyte 42 0

Granulocyte 364 55

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.


























0I 26 .
to7


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








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 NaC1), 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 NaC1 concentrations. On the basis of this

finding an experiment in which alligator peripheral blood lymphocytes















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

Alligators AA

BB

CC

DD


Stimulation Indexa

1

36

45

18

18

7

5

19

78

1

90

100


(a) Triplicate cultures were incubated at 320C with or without PHA
(1 pl), 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.









were stimulated with PHA in different MEM preparations containing various

concentrations of NaCI 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 NaC1 concentrations (0.040 M and 0.060 M extra NaC1

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 NaC1 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 3H-thymidine (0.5-1.0 pCi/culture; 24 hr) were applicable for

alligator lymphocyte cultures, the effects of 3H-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

bleedings (4-8 x 108 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 phytohemagglutinin (PHA), concanavalin

A (Con A), lipopolysaccaride (LPS), pokeweed mitogen (PWM), and purified

protein derivative (PPD).
















Table 14

Effect of Sodium Chloride Concentration on Alligator
Peripheral Blood Lymphocytes Cultured with PHA


NaC1 Concentration

0.117 Mc

0.137 M

0.157 M

0.177 M

0.197 M

0.217 M


Stimulation Indexb

47

85

142

145

59

1.0


(a) Final concentrations of NaCl in the culture medium.
(b) Cultures were incubated at 320C with or without PHA (1 pl), pulsed
on day 4 and harvested on day 5.
(c) The concentration of NaC1 in mammalian MEM was calculated from the
GIBCO formulation to be 0.117 M.















Table 15

Effect of 3H-Thymidine Concentration on Alligator
Peripheral Blood Lymphocytes Cultured with PHA


pCi H/Culture

0.05

0.1

0.25

0.5

1.0

2.0


Stimulation Indexa

30

60

66

71

71

67


(a) Cells were incubated at 320C with or without PHA (1 Ip), pulsed on
day 4 and harvested on day 5.















Table 16

Effect of Incubation Time with 3H-Thymidine on PHA
Stimulated Alligator Peripheral Blood Lymphocytes


Length of Time with
3H-Thymidine (Hr)

24

48

72

96


Stimulation Index

108

119

112

110


(a) Cells were cultured at 320C with or without PHA (1 pl). Five-tenths
VCi of 3H-thymidine was added at various intervals prior to the harvest of
cultures. All cultures were terminated on day 5.








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 220, 35, 37, and 400C was significantly lower (p < 0.01)

than in cells cultured at 270, 30, and 320C. Although responses pf

cells incubated at 270, 300, and 320C were not significantly different

from each other (p > 0.1), the length of time required for optimal stim-

ulation of cultures maintained at 320C was shorter (five days) as com-

pared with 270 and 300C 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 320C 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 320C, 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 pg, 10 pl, 1 pi and

20 pg respectively. It should be pointed out that responses of cells

cultured with 20 pg of Con A varied in different experiments and was

attributed to changes in the lot numbers of Con A used, as well as the




























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length of time a mitogen solution was stored at 40C. 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

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 pl) were pre-

dominately in aggregates and looked like lymphoblasts (Figures 13a and

13b). Cells optimally stimulated with LPS (10 pg) 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

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