AN ABSTRACT OF THE THESIS OF
Ralph A. Tripp for the degree of Doctor of Philosophy in Microbiology presented
on October 3, 1988.
Title: Glucocorticoid Regulation of Salmonid B Lymphocytes
Abstract approved:
Redacted for Privacy
LJI.
tepnen L. iviattan
The in vitro effects of cortisol in regulating salmonid B cell functions were
investigated. B cells at three distinct stages of differentiation were examined for
cortisol sensitivity. B lymphocyte responses were examined 1) during the early stage
of the precursor, 2) during the intermediate stage of differentiation associated with
clonal proliferation, and 3) at the stage of terminal differentiation associated with
antibody secretion. The effect on B cell precursors was assessed by limiting dilution
analysis, clonal proliferation by limiting dilution analysis and mitogenic stimulation,
and antibody secretion by the induction of plaque-forming cells (PFC). Physiological
levels of cortisol inhibited, in a dose-dependent fashion, the proliferative and
antibody-secreting
responses
of
splenic
and
pronephric-derived
Cortisol-mediated cytolysis of the lymphocytes was
inhibition.
lymphocytes.
not responsible for this
Organ-dependent differences in cortisol sensitivity were observed with
pronephric- and splenic-derived lymphocytes. Pronephric lymphocytes were sensitive
to cortisol only at an early stage of differentiation, whereas splenic lymphocytes
were sensitive at all stages. Preincubation of splenic lymphocytes with cortisol
increased their sensitivity to later additions of cortisol, whereas no such increase
occurred with pronephric lymphocytes. Cortisol-induced suppression of pronephric
lymphocytes
was
reversible.
Proliferative
and
antibody-producing
cell
(APC)
responses in cortisol-suppressed cultures were fully restored by
addition of
leukocyte-derived conditioned medium (CM-L). The active material in CM-L was
found to range in molecular weight from 10 to 18 kd. Recombinant bovine and
human interleukins restored the proliferative phases of B cell differentiation formerly
suppressed by cortisol. Studies also revealed that: 1) two cells (B and adherent) are
required for TNP-LPS responses and 2) pronephric lymphocytes can be physically
separated into surface Ig+ and Ig- populations which appear to be functionally
distinct.
Glucocorticoid Regulation of Salmonid B Lymphocytes
by
Ralph A. Tripp
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Doctor of Philosophy
Completed October 3, 1988
Commencement June 1989
APPROVED:
Redacted for Privacy
Associate Professor of Microbiology in charge of major
_
Redacted for Privacy
Chfrman of th
partment of Microbiology
Redacted for Privacy
grItDean of the Gra
to School
Date thesis is presented
October 3, 1988
Typed by
Ralph A. Tripp
ACKNOWLEDGEMENTS
I would like to take this opportunity to acknowledge several people who
have in some manner aided the achievement of this degree.
My sincere gratitude is extended to my major professor and friend Dr.
Stephen L. Kaattari. Through his guidance and genuine interest in my success,
I have been able to advance my knowledge. I would also like to thank him for
allowing me the opportunity to work and pursue my interests in his laboratory.
To my good friend Alec G. Mau le is extended my deepest appreciation.
Many of the experiments comprising this dissertation were directly or indirectly
assisted by him.
For their contribution to this dissertation, I would also like to thank Dr. Carl
Schreck, Dr. Mike "Chi-Town" Irwin, Nancy Frasier, Jennifer Creason, Mary Yui,
Nancy Holland and Sam Bradford HI.
For their timely assistance and support of this dissertation, I would like to
thank Prasad Turaga, the Salmon of Knowledge (Greg Wiens) and Mary
Arkoosh, graduate students in Dr. Kaattari's laboratory.
For the funding of this study, I express my appreciation to Seagrant and the
N.L. Tartar Foundation.
Most importantly, I wish to thank my family, Mr. and Mrs. Ralph A. Tripp
Jr. and sisters Lori and Denise, for their encouragement I received throughout the
pursuit of this degree.
Table of Contents
INTRODUCTION
LITERATURE REVIEW
The Immune System as an Integrative Cellular Network
Functional Characteristics of Interleukins
1
4
4
5
Role of Cell Separation in Cellular Immunology
8
Role of Limiting Dilution Analysis in Cellular
Immunology
9
Determining In Vitro Proliferative Requirements
11
In Vitro Analysis of the Piscine Immune Response
12
The Role of Glucocorticoids in Regulation of
Physiological and Immune Function
14
MATERIALS AND METHODS
Animals
Antigens
Mitogens
Complement
Preparation of Trinitrophenylated Sheep Red Blood
Cells (TSRBC)
In Vitro Tissue Culture Media
Steroid Preparation
Lymphocyte Preparation
Trypan Blue Exclusion Test for Cell Viability
Mitogen Assay
In Vitro Antibody-Forming Cell Assay
Limiting Dilution Analysis
Cellular Partitioning - Adherent Cells
Cellular Partitioning sIg+/sIgColumn Chromatography
Two- and Four-Way Mixed Leukocyte Reactions
Lymphocyte Separation by Ficoll/Dextran
Centrifugation
Protein A-Mouse Immunoglobulin G Purification
Fluorescent Antibody Technique
18
18
18
19
19
20
21
21
22
22
23
24
25
26
26
27
28
29
30
30
RESULTS
Mitogen Dose-Response to Assess Proliferative
Capacity
Dose-Response for the In Vitro Production
of Plaque-Forming Cells
Effects of Cortisol on Lymphocyte Viability
Dose-Response of Cortisol Measured by Lymphocyte
Proliferation
Dose-Response of Cortisol Measured by Antibody
Cell Production
The Effects of Cortisol Preincubation on PFC
Generation
The Kinetics of Cortisol-Induced Suppression
Effects of Cortisol Pulsing on the Generation of
Plaque-Forming Cells
Identification of the Cellular Target of
Suppression by Limiting Dilution Analysis (LDA)Analysis of Mixed Leukocyte Reactions
Development of Cell Partitioning for Use in the
Limiting Dilution Assay
Effect of Cortisol on Precursor Frequency and
Clone Size - Limiting Dilution Analysis
Restoration of Cortisol-Mediated Suppression by
Conditioned Medium
Effect of Conditioned Medium on the Precursor
Frequency and Clone Size of Suppressed Lymphocytes
Isolation and Partial Characterization of Factor(s)
in Conditioned Media (CM-L)
Ability of Monokine(s) (CM-M) to Restore CortisolMediated Suppression
Analysis of Recombinant Interleukin on
Restorative Capacity
The Relationship of B Cell Proliferation and the
Generation of PFC In Vitro
Separation and Characterization of the Cellular
Constituents of Hematopoietic Tissues
32
32
32
35
42
42
47
47
52
52
55
60
65
70
70
77
82
89
97
DISCUSSION
114
SUMMARY AND CONCLUSIONS
139
BIBLIOGRAPHY
141
APPENDIX
154
List of Figures
Figure
1
2
3
4
5
6
7
8
Page
In Vitro Dose-Responses for Concanavalin A, Pytohemagglutinin,
and E coli Lipopolysaccharide on Pronephric and Splenic
Lymphocytes.
33
In Vitro Dose-Response of TNP-LPS on Pronephric and Splenic
Lymphocytes.
36
Lymphocyte Viability After Cortisol Preincubation.
38
Kinetics of the In Vitro Addition of Cortisol on Lymphocyte
Viability.
40
Effect of Cortisol and Aldosterone on the Response to E. coli
LPS.
43
Effects of Cortisol and Aldosterone on the Plaque-Forming
Response to TNP-LPS.
45
Effect of Cortisol Preincubation on the Plaque-Forming Response
to TNP-LPS.
48
Kinetics of Steroid Suppression of the Plaque-Forming Cell
Assay.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
Effects of Cortisol Pulsing on the Generation of PlaqueForming Cells.
Effects of Two-Way and Four-Way Mixed Leukocyte Reactions
on the Mitogenic Response to E. coli LPS.
Effects of Two-Way and Four-Way Mixed Leukocyte Reactions
on the Plaque-Forming Response to TNP-LPS.
Limiting Dilution Analysis of the Precursor Frequency
Resulting From Saturating Concentrations of Adherent,
Nonadherent and Whole Pronephric Leukocytes.
Limiting Dilution Analysis of the Precursor Frequency
Resulting From Cortisol-Induced Suppression.
Effect of Conditioned Medium (CM-L) on Cortisol-Mediated
Suppression of the Plaque-Forming Response.
Titration of Conditioned Medium (CM-L) for the Ability to
Reverse Cortisol-Mediated Suppression.
Limiting Dilution Analysis of the Precursor Frequency
Resulting From the Reversal of Suppression by
Conditioned Medium (CM-L).
Clone Size Distribution for Treated Pronephric Cultures.
The Elution Profile for the Activity of Chromatographed
Conditioned Medium (CM-L).
Addition of Macrophage-Derived Conditioned Medium (CM-M)
to Cortisol-Suppressed Pronephric Cultures.
In Vitro Induction of Nonspecific Proliferation by the
Addition of CM-M.
In Vitro Induction of Nonspecific Antibody by the Addition
of CM-M.
Effect of Recombinant Human Interleukins One and Two on
50
53
56
58
61
63
66
68
71
73
75
78
80
83
23
24
25
26
27
28
29
30
31
32
33
Cortisol- Suppressed Proliferation.
Restorative Capacity of Recombinant Bovine and Human
Interleukin One on Cortisol-Suppressed Proliferation.
In Vitro Dose-Response of Recombinant Human Interleukin
One on Restoration of the Antibody-Producing Cell
Response.
Hydroxyurea Inhibition of E coli LPS-Induced Proliferation.
Late Inhibition of Proliferation by Hydroxyurea.
The Role of Ongoing Proliferation in the Expression of
Plaque-Forming Cells in Pronephric Cultures.
The Role of Ongoing Proliferation in the Expression of
Plaque-Forming Cells in Splenic Cultures.
Ficoll/Dextran Density Gradient Separation of Lymphocytes.
Mitogenic Analysis of Ficoll/Dextran Separated Lymphocytes.
Fluorescent Antibody Analysis of Panned Lymphocytes.
Mitogenic Profiles of Panned Lymphocytes.
Antigenic Analysis of Panned Lymphocytes.
85
88
90
93
95
98
100
102
104
107
109
112
Glucocorticoid Regulation of Salmonid B Lymphocytes
INTRODUCTION
Physiological systems, such as the immune and endocrine systems, have evolved to
a high level of interdependency. This interdependency allows for sophisticated regulation
which results in the maintenance of a relatively stable internal environment for the
organism. The maintenance of a stable environment is referred to as homeostasis. When
homeostasis fails, dramatic events usually ensue. Such is the case for the diabetic who
is unable to breakdown and use sugars properly, due to an insulin deficiency. These
sugars (glucose) accumulate in the blood and eventually are excreted into the urine;
however, over a period of time, if a sufficient quantity of glucose accumulates, it can
damage the eyes, kidneys, nerves and other parts of the body (84). Thus, one can readily
observe the integration of the endocrine system (responsible for the production and
release of insulin) with metabolism. However, only recently has such unification been
documented for the immune and endocrine systems (10,16,17,45). The immune system
is a highly versatile, interacting network of specialized cells and cell products capable
of acting at sites distal from their point of origin. This system is essential in the overall
maintenance of homeostasis and health of an organism. This integrative network is
involved in the biological response to the invasion by infectious agents and must be
highly regulated. The cellular, and molecular components of the immune system are
combined in an exquisitely complex communications network whose regulatory control
of these defense mechanisms is essential for a stable or homeostatic environment. The
2
endocrine system, which is comprised of a large number of adrenal, pituitary and sex
hormones and prohormones, which include corticosteroids, mineralocorticoids, and
gonadotropins, is also highly regulated. Regulation is primarliy accomplished by a series
of feedback loops which either stimulate or down-regulate the production of precursor
molecules necessary for the generation of specific hormone(s). Recently, bidirectional
communication between endocrine and immune systems has been demonstrated in both
higher and lower vertebrates (9,10,11,13,16,17,24,53,79, 81). Of the endocrine hormones
examined, the glucocorticoids appear to have the most dramatic effects on the immune
system (45). These hormones are able to down-regulate immune responses both in vivo
and in vitro (79,80,83,91). Such integration allows for increased regulation of the immune
response, as well as a high degree of control in the monitoring and maintenance of
homeostasis within the organism. The goals of this study were to determine the role of
glucocorticoids in regulating salmonid B cell responses. This was addressed by
determining the effects of cortisol on the B cell precursor level, on clonal proliferation,
and on the terminal differentiation stage associated with antibody secretion. The role of
lymphokines in restoring cortisol-suppressed immune responses was also examined. In
addition, the cellular requirements for in vitro antibody responses to a specific antigen
were determined. These findings were essential to determine the precise cellular target
of cortisol suppression. This study represents the first delineation of the mechanism of
glucocorticoid-induced immunosuppression of the antibody response in fish. In addition,
this study pioneers the use of limiting dilution techniques for the examination of B cell
precursors in the salmon. Also, physical techniques to separate the salmonid lymphocyte
compartment into surface immunoglobulin positive (B cells) and surface immunoglobulin
3
negative (T cells) populations was devised. Notwithstanding, this study also demonstrates
the existence and activity of lymphokine(s) and monokine(s) recovered from stimulated
salmonid
leukocytes.
These
agents
were
able
glucocorticoid-suppressed B cells to secrete antibodies.
to
restore
the
ability
of
4
LITERATURE REVIEW
The Immune System as an Integrative Cellular Network
B cells, their mature plasma cell progeny, and their immunoglobulin products have
been found in every vertebrate examined including both cartilagenous and bony fish,
reptiles, amphibians, and mammals (85). B cell activation
is
induced by the
cross-linkage of their Ig receptors by multivalent ligands such as antigen or anti-Ig
antibodies (86,97). B cell activation results in clonal expansion. Clonal expansion is
accomplished by cellular proliferation of precursor lymphocytes. The role of proliferation
is to expand the number of cells reactive to a specific antigen. Due to the initial low
frequency of B cells specific for any antigen (41), cellular proliferation is essential to
maintain immunological proficiency. Macrophages, T cells and their products have a
paramount role in expansion of
activated B cell pools (63). Antigen is trapped,
concentrated and processed by the macrophage (86,152). These macrophages can then
interact with two classes of mature T cells, helper (in the case of T-dependent antigens)
and amplifier T cells which can induce and augment B cell proliferation (63,86). T cell
proliferation, which is essential for the initial
development of the T cell immune
response to T-dependent antigens (e.g. foreign proteins), is augmented by interleukin
one (86,97,154). This interleukin is produced
T-independent antigens
by the activated macrophage (63).
(e.g. polysaccharides) may also require the elaboration of
several B cell growth and differentiation factors
(63,65,67,86,97,103,117,135,144).
5
Specifically activated helper T cells boost B cell clonal expansion by the release of
soluble B cell growth factors (interleukin 4 and 5, 97,154). In addition, they specifically
interact with the
activated B cells in the presence of antigen and major
histocompatibility (MHC) gene products, located on the surface of the B cells (97,154).
This interaction eventually leads to the differentiation of the antigen activated B cells
into plasma cells (86). During
differentiation, daughter B cells express specific
receptors for B cell differentiation factor (interleukin 6,
which allows for plasma cell
154), another T cell product
differentiation (97). Thus, cellular interaction and
interleukin elaboration are essential to the development of an antibody response.
Functional Characteristics of Interleukins
The advent of advanced molecular and cellular approaches to lymphokine isolation
and characterization has led to considerable progress in the study of these factors (97).
A wide variety of known nonspecific, soluble mediators exist that regulate both the
afferent and efferent limbs of the immune response (16,51,79,81,97,154). These include
interleukins one (I1-1) through six (11 -6), each having distinct but pleiotropic regulatory
functions (154). The more well-characterized interleukins, one and two, which serve
as endogenous second signals in conjunction with antigens, rapidly amplify both local
and systemic host defense mechanisms involving B and T lymphocytes (reviewed in
86).
6
Interleukin one (11-1) was first detected in cultures
of human peripheral blood
adherent cells (reviewed in 86). The biological properties originally ascribed to this
molecule included the synergistic enhancement of the proliferative responses of murine
splenocytes and thymocytes to lectin stimulants such as concanavalin A (Con A) and
phytohemagglutinin (PHA), as well as the enhancement of antibody production by T
cell-depleted murine splenocytes (reviewed in 97). Presently, 11-1 is known to affect B
and T lymphocyte proliferation and function (86). 11-1 has been demonstrated to increase
the expression of Ig
receptors (97), and also to promote the expression of
differentiation antigens on T lymphocyte membranes (63).
I1-1
also affects
nonlymphocytic cells, such as natural killer, neutrophilic, hypothalmic and connective
tissue
cells
(reviewed in 97).
Perhaps more
importantly, I1-1
antigen-stimulated helper T cells to produce interleukin two (11 -2).
promotes
The elaboration
of 11-2 from mature helper T cells requires at least three signals which result from: 1)
the interaction of antigen or lectin with T cell membranes, 2) MHC antigen interaction
and 3) EA (86). The primary function of 11-2 is to expand and maintain continuous T
cell growth (97), its elicitation, therefore, is highly regulated. T lymphocyte acquisition
of 11-2 responsiveness is the direct result of the appearance of 11-2-specific receptors
found on activated T cells which have received the three signals described above (86).
Interleukins three through six (11-3,-4,-5,-6) are functionally less characterized than
I1-1 or -2 in both murine and human systems. Their effects on B cells and other cell
types are often pleiotropic supporting the growth and maturation of both B, T and
progenitor cells and cell lines (154). For example, 11-3, -4, -5, and -6 support the
growth of pre-B and B cell lines, and 11-3 and -4 also support the growth of mast cells
7
(154), whereas
11-5 and -6 do not (154). Due to their recent isolation,
complete
functional characterization has yet to be accomplished (154).
Conventional isolation of lymphokines is a slow and tedious process, due to the
number of biochemical similarities found between the lymphokines. Employment of gel
filtration chromatography is a primary method of
isolating distinct lymphokines.
Monitoring the purification of the proliferative interleukins, such as I1-1 and 11-2, has
traditionally been accomplished by examining the mitogenic potential of processed
material on
sensitive cell lines or types. For example, I1-1 is often assayed by its
mitogenic effect on murine thymocytes (99).
induce 11-1
As bacterial LPS is commonly used to
production, the LPS-unresponsive C3H/HeJ mouse is usually used as the
thymocyte source (99). Thymocytes are selected for the target cells, principally because
thymocyte
suspensions contain less than 0.1% adherent cells and they rapidly
proliferate in response to I1-1 (86). Other assays for I1-1 activity have been described
using cloned neoplastic T cell lines (100,101). However, to define interleukins which
have the capacity to activate B cells and mediate their growth and maturation to
antibody-secreting cells, the induction of Ig secretion by B cells or B cell lines is often
used (102,103). One such line is the murine B cell line, WEHI 279, which synthesizes
and displays membrane IgM (97). Lymphokine activity can then be indirectly assessed
by its ability to nonspecifically induce IgM production and secretion into the culture
supernatants of these cells (97,102,103).
8
Role of Cell Separation in Cellular Immunology
The cellular requirements for an integrated network
lymphocytes, macrophages and their
development of
consisting of B and T
products were not fully recognized until the
cell separation techniques. The cellular requireinents for in vitro
antibody responses were first elucidated in
experiments designed to ascertain the
mechanisms of T cell help for the generation of antibody-forming cells (104). This
helper function was felt to be mediated by a soluble
factor. This contention was
supported by experimental evidence which demonstrated that supernatants derived from
lectin or alloantigen-activated T cell cultures could
However, a major
replace T cells in vitro (105).
difficulty which confounded the execution of these initial
experimental designs was the inability to generate a homogeneous B cell population.
This was essential for demonstrating that lymphokines are responsible for the activation
of many B cell responses. Cell separation
techniques were thus developed. These
techniques not only provided the defined cellular populations needed for the in vitro
experiments, but also allowed for further identification and characterization of cells as
new antibody specificities were developed. The development of these techniques which
used antibodies to probe the surface membrane antigens of cells and thus elucidate
lymphoid cell
heterogeneity (106-108), provided the basis for modern
cellular
immunology (69-71). Two of these techniques that have been exceptionally useful are
the direct labeling and separation of lymphoid cells by the use of fluorescently tagged
antibodies
(ie.
fluorescence
activated cell
sorting,
FACS),
and
the
use
of
antibody-mediated cellular adherence (ie. panning). FACS, although presently efficient
9
and accurate in quantitating the proportion of cells bearing specific marker antigens,
requires very sophisticated instrumentation
.
Alternatively, panning techniques do not
require sophisticated instrumentation while still allowing for the separation of large
numbers of cells on the basis of the presence of a marker/receptor for which antibodies
are available (110). Consequently, many of the earliest
cellular identification and
receptor studies were conducted by the use of panning procedures (69,70,71,111). Most
panning experiments were designed to determine the effects of deletion or addition of
cell subsets on various aspects of the immune response. This ultimately was used to
delineate the cellular requirements for the in vitro antibody response. However, pooling
of phenotypically similar cellular subsets, either by panning or FACS methods, cannot
supply information as to the number of these cells which are functionally competent.
Subsequent
assay systems utlizing these separated cells can only
provide a
non-quantitative assessment of the functionality of the lymphocyte pool. Often, in order
to obtain a quantitative estimate as to the number of functional cells, or precursors,
limiting dilution analysis (LDA) is utilized.
Role of Limiting Dilution Analysis in Cellular Immunology
The in vitro form of this analysis employs microculture techniques which allow for
the development of a functional population of progeny lymphocytes from one precursor.
Quantitation of precursor frequencies is accomplished by assessing the responses of
replicated (>96) cultures at various initial lymphocyte concentrations (59). A positive
10
B cell response could be the production of antibody, whereas when enumerating T cell
precursors the elaboration of lymphokines could be used (41). The estimation of the
precursor frequency is performed by determining the fraction of negative replicates out
of the total number of replicates at each dilution. The statistical procedure used for the
estimation of the unknown frequency is based upon the assumption that the number of
specific precursors distributed over all the replicated cultures are binominally distributed,
that is, having a Poissonian distribution (66). If there is a Poissonian distribution and
if the cultures are only limiting for the precursor under analysis then a yield of 37%
negative cultures represents the concentration of lymphocytes possessing one precursor
(41,66). It is necessary, from the outset, to assume one of four possible models for the
generation of a positive response: 1) only one cell of one cell type is necessary for a
positive
response (single-hit kinetics), 2) at least two cells of one cell type are
necessary (multi-hit kinetics), 3) only one cell of a given cell number (>2) are necessary
(multi-target kinetics), and 4) a combination of multi-hit and multi-target models. LDA
is commonly employed in immunology for cloning cells (59), as well as in such studies
as the analysis of the developmental changes in T cell populations including those
associated with aging (112), self-tolerance (113), recovery from irradiation (114), and
immunosuppressive drugs (115). In addition, LDA has been used to determine the role
of T cell-derived lymphokines in early activation, proliferation and antibody production
of B lymphocytes isolated from both murine (60,67) and human (61,62) sources.
However in lower vertebrates, LDA has not been utilized until the advent of the studies
reported in this thesis. Thus, essential phases
in specific immune responses which
presently can only be determined through the use of LDA have not been studied in
11
lower vertebrates.
Determining In Vitro Proliferative Requirements
The role of B cell proliferation in the generation of antibody-producing cells (86)
has yet to be determined in both higher and lower vertebrates (116,118). It has been
demonstrated that small resting murine B cells, separated from larger cells by velocity
sedimentation, were capable of maturing to IgM-secreting (19S) cells in the absence of
Similarly, it was shown that
DNA synthesis and cellular proliferation (116,117).
hydrocortisone could stimulate immunoglobulin production without B cell proliferation
in cultures of human peripheral blood lymphocytes (119). Contrary to these studies, and
in support of the currently held paradigm of a proliferation prerequisite for B cell
differentiation, are the results of studies that utilized
the cell cycle-specific drug
hydroxyurea (HYU) to inhibit DNA synthesis. The inability of activated murine B cells
to generate antibodies in the presence of HYU indicated that B cell division is required
for the generation of antibody-producing cells (77). Contraindicatory data such as this
emphasizes the fact that the cellular requirements
responses have yet to be
for in vitro antibody immune
totally resolved. Through future use of HYU these
proliferative requirements can be addressed in both higher and lower animal systems.
12
In Vitro Analysis of the Piscine Immune Response
In the piscine system, there is circumstantial evidence suggesting, by analogy with
higher vertebrates,
that B and T cells exist in fish (1,3,4,8,15,38,42,68,72, 74,78),
although direct, formal proof is still lacking. The discovery by Nowell in 1960 (89) of
the mitogenic activity
of PHA, a plant lectin isolated from the red kidney bean,
Phaseolus vulgaris, and the subsequent elucidation of roughly twenty other mitogenic
plant lectins (88) with immunologic activity, has led to the identification of lymphocyte
specific mitogens in mammals (88). Of the various mitogens, some stimulate only B
cells (e.g. polysaccharides), some T cells (e.g. phytohemagglutinin) and others, B and
T cells (e.g. pokeweed mitogen, reviewed in 88). It is, therefore, possible to use specific
mitogens to characterize and quantify lymphocyte populations. However, only recently
has such mitogenic characterization of lymphocyte populations been attempted in lower
vertebrates such as fish. This is due primarily to the lack of a physical distinction
between lymphocyte populations, that is, B and T-like cells. In the rainbow
Salmo gairdneri, thymic cells were demonstrated to
trout,
respond only to an optimal
concentration of Con A but not lipopolysaccharide (LPS). In contrast, splenic-derived
lymphocytes were found to respond to all mitogens tested, and pronephric lymphocytes
could only respond to LPS (31). These distinct patterns of mitogenic responses from the
cells of each immune organ demonstrated that lymphoid heterogeneity is coupled with
unique tissue distribution in
the teleost. A more definitive study utilizing fine
specificity analysis of anti-trinitrophenyl (anti-TNP) antibodies generated in vitro by
rainbow trout lymphocytes has expanded on these findings by demonstrating B cell
13
heterogeneity in teleost lymphoid organs (8). Physical cell separation of lymphocytes
derived from the bluegill,
Lepomis macrokinus, also demonstrated differences in
mitogenic responses can be used to divide lymphocytes into two distinct subpopulations
(4). One population was found to be responsive to PHA and Con A but not LPS (T-like
cells) and the other was responsive only to LPS but not PHA or Con A (B-like cells).
Similar patterns of lymphocyte heterogeneity were observed in both carp, Cyprinus
carpio (73), and catfish Ictalurus punctatus (2).
The development of in vitro techniques which permit the study of normal immune
reactions such as antibody production (92) allow one to remove lymphocytes from
variable external sources of influence, such as cell trafficking and hormonal fluxes. Such
environment is essential to the analysis of the processes
a controlled
of cellular
interaction in the generation of immune responses. A widely used culture system that
allows for
such analysis, originally developed by Mishell and Dutton
for murine
lymphocytes (92), has been modified for use in the catfish (3) and more recently for the
salmonid (12). Subsequent development and utilization of this microsystem to detect
antibody-secreting cells has allowed for the
delineation of the functional roles of
different lymphocyte subpopulations in the catfish (3). The value of this culture system
is particularly apparent when used in conjunction with hapten-conjugated antigens and
appropriate cell separation techniques (1,3). Such studies have helped to develop in
vitro correlates of mammalian immune
generating in
responses. The cellular requirements for
vitro immune responses has also been approached in other piscine
systems. The cellular response of goldfish,
erythrocytes
Carassinus auratus to heterologous
demonstrated the requirement for cell-cell interactions as well as for
14
antigenic recognition (15). Recognition of major histocompatibility antigens was also
documented in the carp (120), and the catfish (6), through the use of one- and two-way
mixed leukocyte reactions. T cell activity, measured by the migration inhibition factor
(MIF) assay, to sheep red blood cells has been measured in the tilapia (121) and trout
(72). In addition, adherent cells
(macrophages) were shown to be required for
TNP-LPS-induced in vitro immune responses in the catfish (3). Beyond this, direct in
vitro piscine correlates with mammalian systems have yet to be demonstrated.
The Role of Glucocorticoids in Regulation of Physiological and Immune Function
In mammals, induction of glucocorticoids involves
neural stimulation of the
hypothalamus, resulting in the release of corticotropin-releasing factor (CRF). CRF
induces the production and release of adrenocorticotrophin (ACTH) from the pituitary,
and the subsequent release of glucocorticoids from the adrenals (81). Independent of
other actions, glucocorticoids are involved in the control
of their own plasma
concentrations through a negative feedback loop involving CRF, ACTH and as more
recently demonstrated the lymphokines (79). Such negative feedback is not limited to
CRF, ACTH and lymphokines, because other messengers are linked to the secretion of
the ACTH precursor molecule pro-opiomelanocortin (POMC) such as beta-endorphin
and melanocyte-stimulating hormone (51). The two major glucocorticoids are cortisol
and corticosterone
(79). Rats, mice, rabbits, birds
and snakes make mostly
corticosterone. Humans, monkeys, hamsters, guinea pigs and fish secrete primarily
15
cortisol. Cortisol is, by several criteria, the more potent hormone (79).
Glucocorticoids and their production mediate the glycogenolytic, gluconeogenic and
ketogenic actions of glucagon (16,51,80). They influence carbohydrate, protein and lipid
metabolism, as well as having generalized effects (such as anti-inflammatory; 24,75,79)
on the immune system.
around
the
release
An intriguing aspect of glucocorticoid regulation revolves
of
the
hormone
during
periods
of
stress
(16,17,30,32,33,34,45,49,51,54,58). In general, the activities of the glucocorticoids on
the immune system are
down-regulatory (reviewed in 51), thus, this leads one to
question why an organism would produce a substance that decreases its ability to
produce immunity during stress. It has been postulated that the function of
glucocorticoids released during stressful events is not to protect the organism from the
source of stress itself but rather to protect it from possible abnormal defense reactions
that may be activated by stress (80), ultimately preventing a hyperimmune immune
response. The indirect effects of glucocorticoids on immune cells was realized early in
the 1940's during the treatment of rheumatoid arthritis patients with pharmacological
doses
of cortisone and ACTH (reviewed in 80). Administration of pharmacological
doses of cortisone or ACTH dramatically reduced the swelling and inflammation of
afflicted tissues. It is now realized that the actions of the glucocorticoids cover a vast
range of phenomena, from general suppression of immune inflammatory reactions in the
whole organism
(24), to the subtle modulation of interactions among various cell
populations (reviewed in 79-81). Glucocorticoids appear to exert their actions through
a
receptor-mediated mechanism requiring RNA and protein synthesis (46,51). The
ability of glucocorticoids to delay the primary immune response against skin allografts
16
in
non-immunized animals was noted by Medawar (93). It was demonstrated that
cortisone could prolong survival of skin allografts by a factor of three-to-four fold, over
control animals immunized against the donor. Similar observations were made for
human renal allografts (94). Glucocorticoids were shown to be less effective in allograft
patients who received them only after grafting had begun rather than prior to grafting.
Thus, glucocorticoids could suppress the generation of an immune response, but had
little effect
on an ongoing immune response. On the basis of these and
similar
observations obtained from experiments utilizing in vitro culture systems (18,19,51), it
was postulated that
nonimmunized lymphocytes are glucocorticoid-sensitive, but
immunized lymphocytes are relatively resistant. However, several reports have shown
that lymphocytes stimulated by
mitogen (95) or antigen (96) are as sensitive to
glucocorticoids as nonstimulated lymphocytes from the same donor and contain larger
numbers of glucocorticoid receptors (95,96). Thus, it appears unlikely that the failure
of glucocorticoids to suppress an established
immune response could be solely
attributable to a decreased sensitivity of activated, immune lymphocytes. Instead, it has
been proposed that glucocorticoids inhibit the early proliferative phases of lymphocyte
differentiation
as
a
consequence
of
inhibiting
lymphokine
production
(10,11,19-23,39,44,47,48,52). Glucocorticoids have been shown to inhibit mammalian
I1-1 (20,44), 11-2 (19,21,23,52) and 11-3 (22) production. The actions of glucocorticoids
on murine macrophages has been demonstrated to decrease MHC gene product (Ia)
expression (20), as well as suppress its overall immune function (39,44). Restoration of
macrophage Ia expression was achieved upon the addition of 11-1 (44). In addition,
glucocorticoid-induced immunosuppression of murine lymphocyte cultures could be
17
reversed by addition of 11-2 (21,23,52). Such restorative capacity of the interleukins has
not been reported for 11-3. Glucocorticoids have been demonstrated to down-regulate
several immune functions in lower vertebrates. In fish, the administration of cortisol to
brown trout, Salmo trutta, resulted in increased susceptibility to saprolegnia infection
and furunculosis (25). In addition, cortisol administration induced lymphocytopenia in
brown trout (26), which could play a role in the increased susceptibilty to the fish
pathogens responsible for saprolegnia and furunculosis (25). More specific effects of
glucocorticoid down-regulation of the
demonstrated. The in
salmonid immune response have been
vivo generated antibody-producing cell responses to a fish-
pathogen-polysaccharide antigen, the 0-antigen extract from Vibrio anguillarum, has
been demonstrated to be inhibited by in vivo cortisol administration (9). Overall, a
paucity of data exist concerning the interrelationships of immune-endocrine regulation
in the piscine system.
18
MATERIALS AND METHODS
Animals
Yearling (75-175g) coho salmon (Oncorhynchus kisutch) were obtained from the
Oregon Department of Fish and Wildlife Fall Creek Hatchery. These coho salmon were
used in all assays, except for the cell partitioning assays, where 2+ year old (250+ g)
coho salmon were used. All fish were raised in fish-pathogen-free well water (12°C in
880 L circular tanks) at the Oregon State University Fish Disease Laboratory. Fish were
fed a diet of Oregon Moist Pellet at a ration of 1-2% body weight/day.
Antigens
Trinitrophenylated-lipopolysaccharide (TNP-LPS) was prepared by the method of
Jacobs and Morrison (14). Lipopolysaccharide W (161 mg) from E. coli (055:B5, Difco,
Detroit, ML) was suspended in 8.05 ml cacodylate buffer (see appendix) with constant
stirring. The pH of the solution was adjusted to 11.5 using 10 N sodium hydroxide. The
tube was foil-wrapped and 8 ml of a picyrlsulfonic acid solution was added dropwise
with constant stirring. The picrylsulfonic acid solution contained 96 mg picrylsulfonic
acid dissolved in 8 ml of cacodylate buffer. The solution was stirred for 2 hours at
ambient temperature, afterwhich it was dialyzed against four, one liter changes of
19
phosphate buffered saline (PBS; see appendix) and finally against one liter of RPMI
1640. TNP-LPS was pasteurized for 45 minutes in a 70°C water bath and stored at 4°C.
All dilutions of stock TNP-LPS were prepared in tissue culture medium (TCM).
Trinitrophenylated-keyhole limpet hemocyanin (TNP -KLH) was purchased as a 1
mg/ml solution (Sigma, St. Louis, MO.) and stored at 4°C. Dilutions of stock TNP -KLH
were made in TCM.
Mitogens
E. coli. (055:B5) lipopolysaccharide W (Difco, Detroit, MI) (10 mg/ml in TCM)
stock was pasteurized for 45 minutes in a 70°C water bath and stored at 4°C for 6
months. All dilutions were made in TCM prior to use. Both PHA and concanavalin A
(Con A; Sigma, St. Louis, MO) were prepared to a concentration of 1 mg/ml by the
addition of TCM to the sterile, lyophilized poNVder, and stored at 4°C. All dilutions were
made in TCM prior to use.
Complement
Spawned adult steelhead trout (Salmo gairdneri) were bled by severing the caudal
peduncle, their blood collected in 50 ml centrifuge tubes (Corning Glass, Corning, NY),
and the sera removed after being allowed to clot overnight at 17°C. The sera were
20
centrifuged in a Beckman microcentrifuge (Beckman, Palo Alto, CA) for 4 minutes to
remove residual red blood cells. The cleared sera were pooled, then divided into 0.5 ml
aliquots, and stored at at -70°C until use. The sera were periodically titrated by dilution
in modified barbital buffer (see appendix) for optimal activity in the plaque-forming cell
assay.
Preparation of Trinitrophenylated Sheep Red Blood Cells (TSRBC)
Sheep red blood cells (SRBC) were collected and stored in Alsever's medium (see
appendix) at 10°C. Prior to haptenation, a 2 ml aliquot was washed two times in 1 x
modified barbital buffer and centrifuged at
1400
g
(4°C).
The final supernatant was
removed and the SRBC container foil-wrapped. A 3.5 ml aliquot of a picrylsulfonic acid
solution, containing 10 mg picrylsulfonic acid dissolved in 3.5 ml of cacodylate buffer
(see appendix), was added dropwise to the wet-packed SRBC. The tube was capped and
slowly tumbled on a rotating tumbler (Model 151, Scientific Indust., Bohemia, NY) for
15 minutes at ambient temperature. The TNP-coated SRBC (TSRBC) were centrifuged
at 1400 g, for 5 minutes at 4°C. The supernatant was removed and the TSRBC pellet
resuspended in a glycylglycine solution containing 3.7 mg glycylglycine dissolved in 5.83
ml of a lx modified barbital buffer. The TSRBC were again pelleted by centrifugation
at 1400 g for 5 minutes at 4°C and resuspended in lx MBB (10% v/v) for use.
21
In Vitro Tissue Culture Medium (TCM)
The tissue culture medium (TCM), used in all assays involving cell culture, was
modified from that originally described by Mishell and Dutton (92). TCM consists of the
following sterile-filtered reagents: RPMI 1640 (Gibco, Grand Island, MI) supplemented
with sodium bicarbonate (2 g/L); 10% hybridoma screened fetal bovine sera (M.A.
Bioproducts, Wallcersville, MD, or Hyclone, Logan, UT); 0.1% gentamicin sulfate (50
mg/ml, M.A. Bioproducts); 50 uM 2-mercaptoethanol; 2 uM L-glutamine and nucleosides
(10 ug/L) adenosine, cytosine, guanosine and uracil (Sigma, St. Louis, MO).
Steroid Preparation
Cortisol (hydrocortisone, Sigma, St. Louis, Mo) and aldosterone (Sigma) dilutions
used in tissue culture were prepared from a stock solution (8000 ng/ml steroid in RPMI
1640). The stock solution was prepared by dissolving an appropriate quantity of cortisol
or aldosterone in 99% ethanol, making appropriate dilutions, and allowing the ethanol
to completely evaporate. The residual steroids were subsequently resuspended in TCM
and the solutions filter-sterilized.
22
Lymphocyte Preparation
Fish were euthanized by anesthetic overdose in 200 mg/ml tricaine methane
sulfonate, a concentration known not to cause an endogenous cortisol response in
salmonids (51). The pronephric and splenic tissues were then dissociated in TCM by
repeated aspiration and expulsion of tissues through a 1 ml syringe. The resulting cell
suspensions were held for 3 minutes on ice to allow larger organ fragments to settle. The
supernatant, a single cell suspension, was washed once in TCM by centrifugation at 500
g for 10 minutes at 4°C. The cell pellets were resuspended in TCM to 2x107 viable
lymphocytes/ml, as determined by trypan blue exclusion.
Trypan Blue Exclusion Test for Cell Viability
The trypan blue exclusion test is a method for differentiating viable from nonviable
lymphocytes. Trypan blue stain is prepared by suspending trypan blue dye (0.04 g/L;
Sigma, St. Louis, MO) in saline, and adjusting the pH to 7.4 with 1N HC1. An aliquot
of trypan blue is added to an appropriate lymphocyte dilution, and the number of viable
(clear) and nonviable (blue) lymphocytes counted using a hemocytometer under 100x
magnification.
23
Mitogen Assay
The number of coho salmon used in all mitogen assays ranged from two to four
animals
(n=2-4). Lymphocytes were pooled from the appropriate organs for
experimentation. All sampling for experimentation occurred during the months of
September through March. Fish were selected irrespective of their sex. Each mitogen
assay was replicated a minimum of four times.
Fifty microliters of the lymphocytes suspensions were added to individual wells of
a 96-well, flat-bottom microculture plate (Corning Glass, Corning, NY). To these
suspensions, 50 ul of the mitogen, with or without cortisol, were added. The cultures
were incubated in an airtight incubator (Model 624, C.B.S. Scientific, Del Mar, CA) at
17°C under a blood-gas environment. The cells were fed 10 ul of a feeding cocktail (see
appendix) every other day for the duration of the culture period. On day four, all cultures
were pulsed with 1 uCi of tritiated thymidine (NEN, Wilmington, DL) in 10 ul of TCM.
Cells were harvested on day five with distilled water onto glass fiber filters (Skatron,
Sterling, VA) with a Skatron cell harvester. The filters were allowed to air dry and then
placed in scintillation vials with cocktail (6 g 2,5-diphenyloxazole (PPO), 5 mg
p- bis(2- (5- phenyloxazolyl) benzene (POPOP) Amersham, Arlington Heights, IL) in one
liter of toluene, and counted on a Beckman liquid scintillation counter (Model EL 3800,
Fullerton, CA).
24
In Vitro Antibody-Forming Cell Assay
The number of coho salmon used for all assays, except the cell partitioning assays,
ranged from two to four animals (n=2-4). For cell partitioning assays the number of coho
salmon used ranged from four to eight animals (n=4-8). Lymphocytes were pooled from
the appropriate organs for experimentation. Fish were sampled for experimentation during
the months of September through March. Fish were selected irrespective of their sex.
For all assays, except the cell partitioning assays, 200 ul of the lymphocyte
suspension were added to 24-well, flatbottom microculture plates (Corning Glass Works,
Corning, NY). To these suspensions, 200 ul of the appropriate dilutions of antigen, with
or without cortisol or hydroxyurea, were added. The cultures were incubated in an
airtight chamber at 17°C in a blood-gas environment. The cells were fed 50 ul of feeding
cocktail (see appendix) every other day for the duration of the culture period. After nine
days of incubation, cell cultures from individual wells were removed via gentle aspiration
using a Pasteur pipette and deposited into separate plastic radioimmunoas say tubes
(VWR, Seattle, WA) and held on ice. The tubes were centrifuged at 500 g for 10
minutes at 4°C. The supernatants were removed and the cell pellets resuspended in RPMI
1640 to an appropriate lymphocyte concentration for plaque-forming cell (PFC)
enumeration. Cells secreting anti-TNP antibody were then detected by a modification
of the Cunningham plaque assay (190). One hundred microliters of the lymphocyte
suspension, 25 ul of an appropriate dilution of steelhead serum (see appendix), and 25
ul of TNP-coated sheep red blood cells (see appendix) were mixed in individual wells
of a 96-well, roundbottom microtiter plate (Falcon, Oxnard, CA). This mixture was
25
deposited in a Cunningham slide chamber, sealed with paraffin, and incubated for 1.5
hours at 17°C. Plaques were enumerated using a low power dissecting microscope.
Lymphocyte numbers were determined using a Coulter counter (Model ZM, Coulter,
Hialeah, FL) with PBS (see appendix) as a diluent. Plaque-forming responses were either
expressed as PFC/106 lymphocytes or PFC/culture.
Limiting Dilution Analysis
To assess the frequency of TNP-specific PFC precursor cells, limiting dilution
cultures were seeded into all 96 wells of flat-bottom microculture plates (Corning) using
cell concentrations comprised of 1, 2, 4 or 6x104 lymphocytes/ml with a complementary
balance of mitomycin C-treated lymphocytes, to achieve the final concentration of 1 x106
lymphocytes per culture well. A 50 ul aliquot of pronephric cells at a concentration of
2x107 lymphocytes/nil was then applied to fibronectin-coated wells and allowed to
incubate for 2 hours at 17°C in a blood-gas environment. This incubation was followed
by a wash in TCM to remove the nonadherent cells. Lymphocyte-derived conditioned
medium (CM-L) was prepared by the incubation of pronephric lymphocytes with 0.5
ug/ml TNP-LPS for four days. The supernatant was removed, centrifuged at 500 g for
10 minutes at 4°C to remove residual cells, and cortisol added to achieve a concentration
of 100 ng/ml.
26
Cellular Partitioning- Adherent Cella
Adherent cells (macrophages) were highly enriched by isolation on fibronectin-coated
microculture wells. Briefly, 24-well, flat-bottom microculture plates (Coming) were
coated with 1 ug/ml fibronectin (Sigma) for 2 hours at 17°C. Excess fibronectin was
removed by inverting the plate and shaking loose the free liquid. A 100 ul aliquot of
2x107 pronephric leukocytes/nil were applied to the fibronectin-coated wells and allowed
to incubate for 2 hours at 17°C in a blood-gas environment. This incubation was followed
by two gentle washes in TCM to remove nonadherent cells.
Macrophage-derived
conditioned medium (CM-M) was generated by stimulating the fibronectin-enriched
adherent cells and collecting the cell-free supernatants four days later. Briefly, 150 ul of
0.5 ug/rnl TNP-LPS was added to all adherent cell-coated wells and allowed to incubate
in a blood-gas environment for 4 days at 17°C. The supernatants were then collected,
pooled, and centrifuged at 500 g for 10 minutes at 10°C to remove residual cells. All
CM-M was stored at 4°C for up to 2 weeks.
Cellular Partitioning - sIg+/sIg-
Polystyrene (100x15 mm) bacteriological petri dishes (Falcon) were used in all
experiments. Protein A-purified mouse anti-fish immunoglobulin monoclonal antibodies
(from hybridoma 1-14), to be bound to the plates, were diluted in 0.05 M Tris-HC1, pH
9.5, and 10 ml was incubated immediately in the plates. The solution was swirled on
27
each plate until the bottom surface was evenly covered. After 40 minutes incubation at
ambient temperature, the buffer was decanted and the plates were washed two times with
PBS (see appendix) and once with 7 ml of 1% fetal bovine sera (FBS) in PBS (1%
FBS/PBS). A single cell suspension of 2x107 lymphocytes/ml was diluted in 3 ml 5%
FBS in PBS (5% FBS/PBS) and incubated on each antibody-coated plate, with care taken
to avoid the generation of bubbles. The plates were incubated on a level surface at 17°C
for 60 minutes. After 30 minutes, unattached cells were redistributed by gently tilting and
swirling the plate(s). After incubation, the nonadherent cells were removed again by
swirling the dish and decanting the supernatant. Following this procedure, the plate(s)
was tilted and washed gently with an additional 1% FBS/PBS and the resulting
supernatant collected. If only the nonadherent population was to be used, two additional
1% FBS/PBS washes were sufficient. To recover the bound cells, the plate was filled
with 15 ml of 1% FBS/PBS and the entire surface of the plate flushed by using a Pasteur
pipette.
Column Chromatography
Molecular sieve chromatography was used to isolate the active fractions of
conditioned medium (CM-L). The inert carrier, Sephadex G-100 (Pharmacia, Uppsula,
Sweden), was prepared by allowing 3 g of Sephadex G-100 dry beads to swell overnight
in 300 ml of distilled water at room temperature. The slurry was transferred to a vacuum
flask and subjected to 20 inches of mercury vacuum for 20 minutes with occasional
28
swirling to remove trapped air. The slurry was allowed to settle for 10 minutes, negative
pressure was removed, and the "fines" were carefully removed from the surface by
repeated aspiration and discharge with a 10 ml pipette. The slurry was then poured into
a glass chromatography tube (9x280 mm Scientific Glass Co., Bloomfield, NJ) with a
standard taper (10/18) sintered glass disc bottom. The column was allowed to pack by
repeated 50 ml flushes with PBS (see appendix). The column was moved to a 10°C
environment and calibrated using five distinct molecular weight marker proteins: bovine
albumin (MW = 66kd), carbonic anhydrase (MW = 29kd), alpha- lactalbumin (MW =
14.2kd), cytochrome c (MW = 12.4kd), and aprotinin (MW = 6.5kd). Upon calibration,
3 ml of concentrated CM-L was applied and fractions were collected in PBS as the
elution buffer.
Two- and Four-Way Mixed Lymphocyte Reactions (MLR)
The pronephros was removed from four individual animals and individually prepared
to a concentration of 1x106 lymphocytes/ml in TCM. For two-way MLR, 5x105
lymphocytes/ml from one fish were combined with 5x105 lymphocytes/ml from another
and 50 ul of all possible two-way MLR combinations were aliquoted into individual
wells of a 96-well, flat-bottom microculture plate (Corning). Four-way MLR was
accomplished by pooling 2.5x105
lymphocyte/ml from each of the four fish and
aliquoting 50 ul of the cells into individual wells of 96-well flat-bottom microculture
plate (Corning). All lymphocyte dilutions were done in TCM. Individual and mixed
29
lymphocyte combinations were incubated for 5 days in an incubator (CBS Scientific)
in a blood-gas environment at 17°C. On day 4, 1.0 uCi of tritiated thymidine (ICN,
Cleveland, OH) in 10 ml of TCM was added to all cultures and the cells were harvested
24 hours later onto glass fiber filters (Skatron). Counts per minute (CPM) were assessed
using a Beckman liquid scintillation counter (Model EL 3800).
Lymphocyte Separation by Ficoll/Dextran Centrifugation
Pronephric lymphocytes were separated using the technique of velocity sedimentation
through an alternating ficoll (Sigma) / dextran (Sigma) density gradient. Fifteen milliliter
polypropylene centifuge tubes (Corning) were layered with 2 ml each of (w/v in TCM)
18% ficoll, 18.5% dextran, 19% ficoll, and 19.5% dextran. Two milliliters of 2x107
pronephric lymphocytes/m1 in TCM were applied over the 18% ficoll layer and the
gradient tube centrifuged at 500 g for 40 minutes at 17°C. An 18 gauge needle and 3 ml
syringe were used to individually extract the separated cells. Extracted cells were washed
once in TCM by adding 3x the extracted volume of TCM to each layer and centrifuging
at 500 g for 10 minutes at 10°C.
30
Protein A-Mouse Immunoglobulin G Purification
A lx10 cm Econo-column (Biorad, Richmond, CA) was packed with 3 ml of
prepared Affigel Protein A (Biorad) agarose and equilibrated to pH 9.0 with five bed
volumes of binding buffer (see appendix). Four milliliters of ascites generated from a
female BALB/c mouse intraperitoneally carrying a 1-14 hybridoma was diluted 1:1 in
binding buffer and applied to the column at approximately 5 mg immunoglobulin/ml.
Unbound material was allowed to pass through the column. The column was then washed
with 15 bed volumes of binding buffer. The protein A-bound IgG was eluted by initially
passing five bed volumes of elution buffer (see appendix) through the column, followed
by an additional 10 bed volumes of elution buffer to ensure total removal of IgG. The
eluate containing IgG was immediately neutralized in 3.5 tnls of 1 M Tris-HC1 (Sigma),
pH 9.0. The column was subsequently regenerated for further use by passing five bed
volumes of regeneration buffer (Sigma) through the column. The column was stored at
4°C.
Fluorescent Antibody Techniques (FAD
Indirect immunofluorescence was performed by co-incubating 2x107 pronephric
lymphocytes/ml in PBS (see appendix) with 10 ug/mi protein A-purified mouse anti-fish
immunoglobulin monoclonal antibody for 20 minutes at 17°C. After incubation,the cells
were centrifuged at 600 g for 7 minutes at 4°C and the supernatant decanted. The cells
31
were gently resuspended in a 1/20 dilution of goat anti-mouse fluorescein-labeled
polyclonal antibody (Hyclone, Logan, UT) and allowed to incubate for 20 minutes at
17°C. The labeled cells were centrifuged at 500 g for 5 minutes at 4°C and resuspended
in PBS (see appendix) for viewing under 100x magnification with a UV microscope
(Zeiss, West Germany).
32
RESULTS
Mitogen Dose-Response to Assess Proliferative Capacity
The direct effects of cortisol on both proliferative and antibody-producing
stages of B lymphocyte differentiation were analyzed using an in vitro cell
culture system. Several mitogens were analyzed for their ability to induce
maximal proliferation of lymphocytes
derived from the pronephros (A) and
spleen (B). A concentration range of 25-100 ug/tnl of concanavalin A (Con A)
produced maximal stimulus, based upon stimulation indices, for both pronephros
and spleen-derived lymphocytes, while 10 ug/tril and 100 ug/ml were found to
be optimal for phytohemagglutin (PHA) and E. coli lipopolysaccharide (LPS),
respectively (Figure 1).
Dose-Response for the In Vitro Production of Plaque-Forming Cells
The ability of B cells to generate specific antibody in response to antigen
was assessed by determining the number of TNP-specific plaque-forming cells
(PFCs) induced
by
in vitro
exposure
to
trinitrophenylated-lipopolysaccharide (TNP-LPS).
several concentrations of
The
optimal concentration
which induced the maximum number of TNP-specific PFCs was determined by
33
Figure 1. In Vitro dose-response of concanavalin A,
and
E.
coli
lipopolysaccharide
on
pronephric
(A)
phytohemagglutinin,
and
splenic
(B)
lymphocytes. The stimulation indicies (SI) were generated by determining the
mean incorporation of tritiated thymidine in triplicate cell cultures divided by
the mean response of triplicate non-stimulated cultures.
Vertical bars
represent the standard errors of the mean of the stimulation index. Responses
were assessed five days after culture initiation.
The background counts per
minute (CPM) for pronephric (A) and splenic (B) cultures were less than
14,280 and 10,220 CPM, respectively.
34
PRONEPHR1C RESPONSE (A)
o in
O N
IP*
CON A
C 4C1
CN
0
1-
o o -o
Oo
LPS
131-1A
SPLENIC RESPONSE (B)
N;
O
O
41 0
v-
p.
O
O
1-0
CON A
PHA
O
O O
O O
0
-
LPS
Figure 1. In Vitro dose-response for concanavalin A, phytohemagglutinin and E. coli Iipopolysaccharide on pronephric and splenic
lymphocytes.
35
a dose-response experiment such as that depicted in Figure 2. Maximum PFCs
generation for pronephric 2192+22 PFC per 106 lymphocytes)
and splenic
(1028+2 PFC per 106 lymphocytes) cultures was achieved using 0.4 ug/ml
TNP-LPS (Figure 2).
Effects of Cortisol on Lymphocyte Viability
The possible cytocidal activity of the highest concentration of cortisol used
in vitro (100 ng/ml) was determined by the assessment of the percent viable
cells
after cortisol preincubation of splenic and pronephric
lymphocytes.
Preincubation for 24, 48 and 72 h. did not significantly affect the viability of
pronephric
lymphocytes, but did decrease the viability of splenic cultures
preincubated for 48 hours (27%), as well as 72 h. (9%), as determined by trypan
blue exclusion (Figure 3). The kinetics of the decrease in viability for both
splenic and pronephric lymphocytes cultured with 0.4 ug/ml TNP-LPS and 100
ng/ml cortisol was assessed over a 15 day incubation period. No decrease in
viability was observed through day 11 for either cell source, however viability
steadily declined thereafter, reaching 70% on day 15
(Figure 4). These data
demonstrate that addition of 100 ng/ml cortisol at the initiation of culture does
not completely cause suppression by decreasing lymphocyte viability.
36
Figure 2. In Vitro dose-response of TNP-LPS on pronephric and splenic
lymphocytes. The number of PFC per 106 lymphocytes induced by: 1) 0.04
ug/ml, 2) 0.4 ug/ml, 3) 4.0 ug/ml TNP-LPS and 4) TCM (0 ug/ml) are
plotted. Vertical lines represent the standard errors of the mean response of
three culture wells. Optimal responses were assessed nine days after culture
intiation.
37
PRONEPHR1C
SPLEN1C
3000
40
0
2000
C.)
C-
1000
-
0
0
er
0
a
TNP - LPS
(ug/ ml)
Figure 2. In Vitro dose-response of TNP-LPS on pronephric
and splenic lymphocytes.
38
Figure 3. The percentage of viable lymphocytes remaining after cortisol
preincubation. Splenic 6 and pronephric () lymphocytes were preincubated
with cortisol for 24, 48 or 72 hours. The percent viability as determined by
trypan blue exclusion, is plotted. The dashed horizontal lines represent the
maximum standard errors about the mean percent viability for untreated
pronephric and splenic lymphocytes. Vertical lines represent the mean
standard errors about the mean of the percent viability for treated cultures.
39
//
/
24 24
,
0
//
48 48
72 72
HOURS OF FREINCUSATICN
Figure 3. Lymphocyte viability after cortisol preincubation.
40
Figure 4. Kinetics of the In Vitro addition of cortisol on
lymphocyte
viability of splenic (o---o) and pronephric (o---e) lymphocytes. The percent
viability on the
indicated day is shown. The vertical lines represent
standard error of the mean
.
Viability, determined by trypan blue exclusion,
was assessed for 15 days after the
culture.
addition of cortisol at the intiation of
41
100
90
80
70
60
50
40
30
20
10
intzr,ezoo .
4
Qr
C%1
Q.
4
4
N 4",
cl"
to
DAYS OF INCUBATION
Figure 4. Kinetics of the In Vitro addition of cortisol on
lymphocyte viability.
42
Dose-Response of Cortisol Measured by Lymphocyte Proliferation
The effects of cortisol on the initial phases of B cell activation were
examined by co-culturing splenic or pronephric lymphocyte preparations with 1,
10 or 100 ng/ml cortisol with 100 ug/tnl E. coli LPS. A dose-dependent
inhibitory effect on proliferation was observed with both pronephric (Figure 5A)
and splenic (Figure 5B) cultures. Aldosterone, a steroid with no known immune
function in salmonids, produced no inhibition with either lymphocyte source
(Figures 5A,B), indicating the specificity of the steroid.
Dose-Response of Cortisol Measured by Antibody Cell Production
The possible suppressive effects of cortisol on the
production of
antibody-secreting B cells were assessed by examining the in vitro PFC response
to 0.4 ug/ml TNP-LPS. The generation of TNP-specific PFCs was shown to be
inhibited, in a dose-dependent fashion, by the addition of either 1, 10 or 100
ng/ml cortisol. The specificity of the PFCs was assured by the inability of the
lymphocytes to produce plaques when plated with unhaptenated sheep red blood
cells (data not shown). These immunosuppressive effects elicited by cortisol
occurred in lymphocytes derived from both the pronephros (Figure 6A) and the
spleen (Figure 6B). The control steroid, aldosterone, had no apparent effect on
the PFC response (Figures 6A,B).
43
Figure 5. Effect of cortisol and aldosterone on the mitogenic response to E.
coli lipopolysaccharide (LPS).
incubated with LPS
Cortisol () or aldosterone (m) were co-
and pronephric cells (A) or splenic cells (B). The
plotted values represent the mean of triplicate cultures expressed as counts
per minute (CPM) x 10' +1 SE. Background CPM were less than 18,900
for A, and 600 CPM for B.
PRONEPHROS (A)
44
180
160
140
120
100
80
SPLEEN (B)
4
I...............
.... ..... .
1
STEROID
(ng/m1)
Figure 5. Effect of cortisol and aidosterone on the response to
E. coil LPS.
45
Figure 6. Effect of cortisol and aldosterone on the plaque-forming response
to trinitrophenylated-lipopolysaccharide (TNP-LPS). Pronephric (A) cells or
splenic (B)
cells were co-incubated with TNP-LPS and cortisol (N) or
aldosterone (. The plotted values represent the mean of triplicate cultures
expressed as PFC/106 lymphocytes ±1 SE. Background PFCs were less than
150/106 lymphocytes for A and 170/106 lymphocytes for B.
46
PRONEPHROS (A)
4000
: ..........
3000
...
i
II
2000
1000
0.
-k
0
.11
"PO
0
0
STEROID
(ng/ml)
SPLEEN (8)
2000
}
-.
a
Ow*
1
aai
....
STEROID
(ng/rnI)
Figure 6. Effects of cortisol and aldosterone on the plaqueforming response to TNP-LPS.
47
The Effects of Cortisol Preincubation on PFC Generation
Preincubation of 100 ng/m1 cortisol with either lymphocyte source, for 0 -
72 h. prior to the addition of 0.4 ug/m1 TNP-LPS, modestly affected the
pronephric PFC response, but dramatically affected the splenic PFC response
(Figure 7). Splenic lymphocytes, preincubated
with 100 ng/ml cortisol,
demonstrated enhanced suppression upon the re-addition of 100 ng/m1 cortisol
plus 0.4 ug/ml TNP-LPS, while this was not observed for pronephric cultures.
These data are in agreement with earlier findings that cortisol preincubation
mainly affects the viability of splenic lymphocytes.
The Kinetics of Cortisol-Induced Suppression
The kinetics of cortisol-induced suppression was analyzed to determine if
early or late events in the antibody response were affected. Cortisol was capable
of inhibiting 50% of the PFC response of pronephric lymphocytes (Figure 8A)
when added at any time from the initiation of the culture period through day 4.
However, after 4 days, the addition of cortisol had progressively less effect. In
contrast, splenic lymphocytes (Figure 8B) were inhibited to the same degree at
all days of cortisol addition. Addition of an equivalent amount of the control
steroid, aldosterone, produced no significant inhibitory effects (Figures 8A,B).
48
Figure 7. Effects of cortisol preincubation on the plaque-forming response to
TNP-LPS. Cortisol was preincubated with 1x106 pronephric (A) and splenic
(B) lymphocytes per milliliter for 24, 48 or 72 hours then removed. Fresh
antigen () or antigen plus cortisol *) were added to cultures initially
receiving TCM or cortisol,
respectively. The number of PFC/culture
generated after 9 days of culture
is
plotted. Vertical lines represent one
standard error about the mean response of three culture wells.
24
72
72
0 48
Cm 48
z
rn
xim
0
24
-n
cn
0
c
0
!it r.17.*.xi:
ir.):';:!8!.,:: ',..:.4},*.,,i Z. ei:S;
I.
I
1
0
10
is3
00
4.1
Xi ii, 1
a
PFC/CULTURE
z
Cu)
z
rn
72
72
48
48
0
24
-n
13 24
c
0
k$'0!..1.
\\\
sr._
0
0
41h
\ \ \ \ \\ \
0
0
CO
I 11_1 _a -.1
0
0
Cn
MISSMMM1
0
0
0
kl
PFC/CULTURE
0
0
0
-4
0
0
t.5
-4
50
Figure 8. Kinetics of steroid suppression of the plaque-forming cell assay.
Cortisol
and aldosterone (0) were added to yield a concentration of 100
ng/ml on different days to both pronephric (A) and splenic-derived (B)
cultures. All cultures were harvested day 9 after the initiation of culture.
Data points indicate the mean + 1 SE of triplicate assays. The upper
horizontal set of dashed lines represents ± 1 SE about the mean value of
cultures stimulated with TNP-LPS only. The lower set of horizontal lines
represents the range of standard errors about the mean value of cultures
without TNP-LPS or cortisol.
PRONEPHROS (A)
51
120
t,....
Sif....
.....
ONI110..
I...
80
60
40
4
0
6
8
10
DAY OF ADDITION OF STEROID
SPLEEN (8)
DAY OF AOOITION OF STEROID
Figure 8. Kinetics of steroid suppression of the plaque-
forming cell assay.
52
Effects of Cortisol Pulsing on the Generation of Plaque-Forming Cells
To determine the period in the generation of the antibody response most
affected by cortisol, pulsing assays were performed from day 0 to day 8 and the
number of PFCs assessed. Splenic cultures were inhibited to a similar degree
(approximately 83%) at all days of cortisol (100 ng/ml) pulsing. Conversely,
pronephric cultures became cortisol insensitive by day 3 of culture (Figure 9).
The
early sensitivity observed, coupled with previously
observed data
concerning the periods of cortisol sensitivity (Figures 4,7,8), suggest that early
B cell differentiation stages may be the primary target of cortisol-induced
immunosuppression, at least in the pronephros.
Identification of the Cellular Target of Suppression by Limiting Dilution Analysis
(LDA). Analysis of Mixed Leukocyte Reactions.
The analysis of the effect of cortisol on the frequency of responding
precursors and their resultant clone sizes was addressed by the use of limiting
dilution analysis (LDA). Because of the large number of lymphocytes required
for this assay, pooling of pronephric lymphocytes from more than one fish was
necessary. Before such analysis could be performed, the possible confounding
effects due to mixed lymphocyte reaction (MLR) had to be evaluated. Four
individual coho salmon pronephric lymphocyte preparations, and all possible
53
Figure 9. Effects of cortisol pulsing on the generation of plaque-forming cells
induced by TNP-LPS. Both splenic and pronephric cultures were pulsed at
the days indicated, for 24 hours with cortisol plus TNP-LPS. After pulsing
medium from treated cultures was replaced with TNP-LPS in TCM (spun).
The number of PFC per 106 lymphocytes is plotted. Vertical bars represent
the standard error about the mean response of triplicate wells. Responses
were assessed nine days after culture initiation.
54
PRONEPHRIC
RESPONSE
SPLEN1C
RESPONSE
3000
co
is
0
u-
2000
1000
CS
3578
4 SPUN
03
4.J
s-
CI
C3
<
u0
1
3578
SPUN
Figure 9. Effects of cortisol pulsing on the generation of
plaque-forming cells.
55
combinations thereof, were tested. Cellular proliferation was assessed by tritiated
thymidine uptake. A positive MLR (indicated by increased tritiated thymidine
uptake in lymphocyte combinations over autologous cell culture responses)
occured for all combinations employed; however, MLR had little effect on 100
ug/ml E
soli, LPS-induced proliferation (Figure 10). Using an identical protocol
of lymphocyte combinations, the effects of MLR on the PFC response to 0.4
ug/ml TNP-LPS was assessed. No lymphocyte combinations tested had an effect
on the pronephric PFC response to TNP-LPS (Figure 11). In addition, the MLR
did not induce the formation of
lymphocytes; Figure 11),
TNP-specific PFCs (<50 PFC per 106
thus the pooling of pronephric lymphocytes from
various fish was deemed appropriate for LDA.
Development of Cell Partitioning for Use in the Limiting Dilution Assay
An initial LDA was performed using whole pronephric
lymphocyte
preparations ranging in concentration from 1x104 to 8x104 lymphocytes per
milliliter. These cultures were
supplemented with mitomycin C-inactivated
lymphocytes from the same pronephric pool, to reconstitute all cultures to a cell
concentration of 1x106 per milliliter. Plotting of the
data revealed multi-hit
kinetics, indicating that more than one cell type was required for the generation
of the
antibody response. Previous studies using Ictalurus
punctatus had
demonstrated the requirement for adherent cells (macrophages) to produce an
56
Figure 10. Effects of two-way mixed lymphocyte reaction (MLR) on the
mitogenic response to lipopolysaccharide (LPS). All possible mixtures of
pronephric lymphocytes consisting of 5x105 cells/culture from each of two
fish, and 2.5x105 cells/culture from each of four fish were co-cultured with
(j-) and without (N) LPS. The data are expressed as the mean counts per
minute (CPM)x10-3 +1 SE for triplicate cultures pulsed with tritiated
thymidine 24 hours prior to harvesting.
57
PRONEPHRIC RESPONSE
CZ
17.
.0
CZ
0
Li
Figure 10. Effects of two-way and four-way mixed
leukocyte reactions on the mitogenic response to
E. coli LPS.
58
Figure
11.
Effects
plaque-forming
of mixed lymphocyte reaction
(MLR)
on
the
response to trinitrophenylated-lipopolysaccharide (TNP-LPS).
All possible mixtures
of pronephric lymphocytes consisting of 5x105 cells/
culture from each of two fish, and 2.5x105 cells/culture from four fish were
co-cultured with TNP-LPS. Vertical
bars represent the mean response of
three culture wells + 1 SE. Responses were assessed nine days after culture
initation for magnitude of MLR.
59
PRONEPHRIC RESPCNSE
co
Figure 11. Effects of two-way and four-way mixed
leukocyte reactions on the plaque-forming response
to TNP-LPS.
60
antibody response to TNP-LPS (1). Adherent cells were therefore added to
cultures in saturating concentrations by adherence to fibronectin-coated tissue
culture plate wells. Lymphocytes were then cultured with either adherent or
nonadherent cell monolayers in the presence of antigen, and LDA performed.
The results demonstrated that single hit kinetics was
restored in adherent
cell-saturated cultures (Figure 12C), but not in nonadherent cell cultures (Figure
12A). Consequently, further LDA were performed in the presence of saturating
levels of adherent cells.
Effect of Cortisol on Precursor Frequency and Clone Size- Limiting Dilution
Analysis
Armed with a functional LDA, studies could now be conducted on the
effect of cortisol on B cell precursor frequencies and clone size. Precursor
frequency analysis
revealed that incubation of pronephric lymphocytes with
cortisol resulted in the reduction of the precursor frequency from 24 precursors
per 106 lymphocytes to 10 precursors per 106 lymphocytes (Figure 13). Thus, a
primary cellular target associated with cortisol-induced immunosuppression is the
B cell precursor.
61
Figure 12. Limiting dilution analysis of the precursor frequency performed
with saturating concentrations of adherent (C) or nonadherent (A) cells, with
the unfractionated (B) cell control added in situ to pronephric lymphocyte
titrations. Each point represents the fraction of negative-response (Fo) cultures
in a population of 96 individual cultures stimulated with trinitrophenylatedlipopolysaccharide (TNP-LPS).
62
LYMPHOCYTES / WELL x 1041
Figure 12. Limiting dilution analysis of the precursor
frequency resulting from saturating concentrations of
adherent, nonadherent and whole pronephric lyphocytes.
63
Figure 13. Limiting dilution analysis of the precursor frequency performed
with pronephric lymphocytes cultured in the presence () or absence
(o)
of
cortisol, with conditioned medium (CM-L) added to all cultures at day four
of culture; each point represents the fraction of
cultures in
a population
of 96
negative-response (F0)
individual cultures
trinitrophenylated- lipopolysaccharide (TNP-LPS).
stimulated with
64
1
2
LYMPHOCYTES/ml (x !Ch)
4
6
3
10
Figure 13. Limiting dilution analysis of the precursor
frequency resulting from cortisol-induced suppression.
65
Restoration of Cortisol-Mediated Suppression by Conditioned Medium
Conditioned medium, a putative source of lymphokines, and recombinant
mammalian interleukins were assayed for
their ability to overcome
cortisol-mediated suppression. Conditioned medium (CM-L) was generated by
incubating pronephric cells with 0.4 ug/ml TNP-LPS for a period of 4 days, at
which time the cells were removed and the remaining supernatant, or CM-L,
collected and utilized. Addition of 100 ng/ml cortisol to TNP-LPS-stimulated
pronephric cell cultures resulted in approximately 50% inhibition of the PFC
response (Figure 14). However, addition of CM-L on day 4 of culture, to the
cortisol-suppressed cultures completely restored the PFC response (Figure 14).
Such restoration suggested the presence of factors in the CM-L which were
required for the
restoration of the PFC response. Subsequently, additional
pronephric-derived CM-L was generated and titered for restoration activity of
PFC production in the presence of cortisol. Two-fold titrations were made to 1:8
in TCM containing 0.4 ug/ml TNP-LPS plus 100 ng/ml cortisol. Dilutions were
added day 4 of culture to cortisol-suppressed (100 ng/ml) splenic and pronephric
cell cultures. CM-L concentrations of 1:1 (100%) and 1:2 (50%) were able to
completely restore the PFC response, while concentrations of 1:4 (25%) and 1:8
(12.5%) only partially restored PFC ability (Figure 15). CM-L, at any dilutions
did not restore the cortisol-suppressed (100 ng/ml) splenic PFC response and
undiluted had little reconstitutive capacity (Figure 15).
66
Figure
14.
Effect of conditioned media (CM-L) on cortisol-mediated
suppression of the plaque-forming response. Pronephric cell cultures were
cultured with TNP-LPS alone, TNP-LPS plus cortisol, and TNP-LPS plus
cortisol in the presence of CM-L. The number of PFC per 106 lymphocytes
is plotted. Each bar represents one standard error about each mean.
67
PRONEPHRIC RESPONSE
2000
tO
o
I
1000 '"
s'
C.)
LL
.
s
; ,
,.
s ."-.3:--
s
s
s
'
sa--;.
0
..........
Ag
..
Ag+C
Ag+C+CM-L
Figure 14. Effect of conditioned medium (CM-L) on cortisolmediated suppression of the plaque-forming response.
68
Figure
15.
Titration of conditioned medium (CM-L) for
cortisol-mediated (100 ng/ml) suppression. The
lymphocytes resulting from day 4
made in TNP-LPS plus
splenic
reversal of
PFC response per
106
addition of 10% (v/v) CM-L titrations,
cortisol, to cortisol-suppressed pronephric (0) and
() lymphocyte cultures is plotted. Vertical bars
represent one
standard error about each mean of triplicate cultures. The upper and lower
dashed horizontal lines
of
represents one standard error about the mean value
pronephric and splenic cultures stimulated with only
respectively. Responses were assessed nine days
culture.
TNP-LPS,
after the initiation of
69
CONDITIONED MEDIA (CM-L.)
,::,7000 ,.
000
.,-,;,?.?,
..
......
...
,.. .. er,
,
,
I
i
cz
G,
C:11
v..
CV
r
C)
Cr
'Cr
In
co
t CZ
CI
(::,
v
1....
PERCENT CM L.
Figure 15. Titration of conditioned medium (CM-
ab
to reverse cortisol- mediated
mediated suppression.
for the
70
Effect of Conditioned Medium on the Precursor Frequency and Clone Size of
Suppressed Lymphocytes
Addition of 10% (v/v) pronephric-derived CM-L to the cultures restored the
precursor frequency of cortisol-treated lymphocytes to that observed with
untreated lymphocytes (Figure 16). Addition of CM-L had no
effect on the
precursor frequency of untreated lymphocytes. The average number of PFCs
originating from a single B cell precursor (i.e. clone size) was calculated for
each treatment (Figure 17). The average clone size for both cultures with or
without CM-L, in the presence of 100 ng/ml
cortisol (Figure 17B,D) was
approximately 17% less (p < 0.05, one-tailed test) than in cultures without
cortisol (Figure 17A,C).
Isolation and Partial Characterization of Factor(s) in
Conditioned Medium
(CM-L)
Conditioned medium, derived from pronephric leukocytes
(CM-L), was
concentrated by Amikon filtration to molecular weight species ranging between
10 - 30 kd. This material was applied to a Sephadex G-100 column. The
resulting elution profile is demonstrated in Figure 18. The resulting eluates
were tested for their ability to re-establish PFC generation in cortisol-suppressed
pronephric cell cultures. Pronephric cultures could be significantly restored to
71
Figure 16. Limiting dilution analysis of the precursor frequency performed
with pronephric lymphocytes cultured in
the presence (s) or absence (o) of
cortisol. Conditioned medium (CM-L) was added to all cultures at day four
of
culture. Each point represents the fraction of
cultures
in a
population
of 96
negative-response (F0)
individual cultures
trinitrophenylated- lipopolysaccharide (TNP-LPS).
stimulated with
72
LYMPHOCYTES / WELL! x
2
s
5
8
104
10
LLB
Figure 16. Limiting dilution analysis of the precursor
frequency resulting from the reversal Of suppression
by conditioned medium (CM-L).
73
Figure 17. Clone size distribution for pronephric lymphocyte concentrations
yielding primarily a single precursor (Fo>0.37) in cultures with antigen only
(A), antigen with cortisol (B), antigen with conditioned medium (CM-L;C),
and antigen with cortisol and CM-L (D). u = average number of precursors
per well, c= average clone size expressed as PFC per precursor.
74
C
0.316
u:10.302
60
C:17.0
:1Z5
v-
50
40
.11111,
30
20
nn
n
10
u :0.302
n
'
70
0.3C2
: 13.0
60
50
40
111.10111,
30
20
r-i
10
2 - 3 4 -7 5-15 16-31 32-63 64+
2 - 3 4 -7 846 16-31 32-63 64+
PFC/WELL
Figure 17. Clone size distribution for treated pronephric
cultures.
75
Figure
18.
The elution profile for the activity of
conditioned medium (CM-L). Solid peaks
chromatographed
represent the elution profiles of
protein molecular weight markers: A= bovine albumin (MW=66 kd), 13=
carbonic
anhydrase (MW= 29 kd), C= alpha-lactalbumin (MW= 14.2 kd),
D= cytochrome C (MW= 12.4 kd), and E= aprotinin (MW= 6.5 kd). The
dashed peak represents the MW at which CM-L-like activity was found.
A11Al2.0V 3A11..01S323
01
O
Lii
d 1VW1XvW INEOEct
0
.....................
33111181:10S8V
...........................
OSZ
-O
CO
Figure 18. The elution profile for the activity of chromatographed conditoned medium (CM-L).
7f7
77
approximately 80% of the response observed for non-suppressed cultures. The
apparent molecular weight of the active fraction ranged between 14-17 kd.
Ability of Monokines (CM-M) to Restore Cortisol-Mediated Suppression
Having demonstrated the existence of PFC restorative
activity in
antigen-stimulated culture supernatants, it was of interest to determine if specific
cell types could
generate such factors. Macrophages, highly enriched by
recovery on fibronectin-coated petri plates, were stimulated with 100 ug/ml E.
coli LPS for 5 days, then the cell-free supernatants were collected (CM-M) and
assayed for their ability to restore the antibody-producing cell
cortisol-suppressed pronephric lymphocyte
response in
cultures (Figure 19). CM-M was
supplemented with 100 ng/ml cortisol and added to suppressed pronephric
cultures. CM-M additions failed to restore the antibody-producing cell response.
CM-M was subsequently tested for mitogenic activity as determined by the
enhancement of the
proliferative responses of both splenic and pronephric
lymphocytes with and without the addition of 100 ug/ml E. coli LPS (Figure
20). The addition of the putative monokines (CM-M) enhanced the LPS response
as well as inducing proliferation without mitogenesis in splenic cell cultures
(Figure 20A). The augmentation of mitogenic responses was also observed for
pronephric cell cultures (Figure 20B). A unique activity was demonstrated when
78
Figure 19. The addition of macrophage-derived conditioned medium (CM-M)
to cortisol-suppressed pronephric cultures. Pronephric lymphocytes were
co-cultured with either antigen alone or antigen plus cortisol. CM-M was
added day four to cortisol-suppressed cultures (TLPS+C+CM-M). The number
of PFC/106 lymphocytes were detected nine days after the initiation of
culture. Vertical bars represent + 1 SE about the mean response of triplicate
cultures.
79
PRONEPHRIC RESPONSE
3000
2500
2000 -
(13
>C
Um.
a.
1500
'
1000
500 -
...44.;.:".,
7
4-:.;:--
0
TLPS
TLPS+C TLPS+C+CM-M
Figure 19. Addition of macrophage-derived conditioned
medium (CM-M) to cortisol-suppressed pronephric
cultures.
80
Figure 20. The effects of in vitro addition of
conditioned medium (CM-M) to induce
macrophage-derived
nonspecific proliferation in splenic
(A) and pronephric (B) cultures, with and without mitogenic stimulation by
100
and 25 ug/ml Con A
ug/ml E. coli LPS
Proliferation was
assessed five days after initiation of culture by determining the counts per
minute (CPM) per 106
error
lymphocytes. Vertical bars represent one standard
about the mean response of three culture wells. Horizontal dashed
lines represent one standard
triplicate cell cultures.
error about the
mean response of untreated
SPLENIC (A)
12 -
81
10Cl
8
CD
wo
6
eT
4
(i
2
m000smimpodile
0
0
50
50
100
PERCENT CM- M
PRONEPHRIC (8)
0
50
0
50
100
PERCENT CM- M
Figure 20. In Vitro induction of nonspecific proliferation
by the addition of CM-M.
82
CM-M was assayed for its ability to nonspecifically induce
TNP-specific
antibody (Figure 21). Addition of CM-M to pronephric cultures nonspecifically
induced a 40% increase in TNP-specific antibody over that generated by E. coli
LPS stimulation (Figure 21). Moreover, this activity of CM-M appears exclusive
from that of CM-L, since CM-L is able only to restore the PFC response during
cortisol-mediated suppression (Figure 14).
Analysis of Recombinant Interleukins on Restorative Capacity
Homogenous recombinant human and bovine interleukins one and two, were
assessed for their ability to re-establish
cortisol-suppressed
the proliferative response in
pronephric cell cultures. Initially, recombinant human
interleukin (rHuIl) one and two were tested individually and together at five
concentrations: 0.0001, 0.001, 0.01, 0.1 and 1.0 U /ml. None of the recombinant
interleukin concentrations restored E. coli LPS-induced, cortisol-suppressed
proliferation (Figure 22).
Subsequently, recombinant bovine interleukin one
(rBoll) was cultured in parallel with rHuIl at five concentrations (10 ug/ml, 1
ug/ml, 10Ong/tnl, lOng/ml, lng/m1), and assayed for their ability to re-establish
proliferation. At these concentrations, both rHuIl-1 and rBoll-1 restored the
proliferative response, in addition, the 10 ug/ml rBoIl-1 concentration augmented
proliferation 58% more than the rHuI1-1 concentration (1 ug/ml) producing the
greatest restorative capacity (Figure 23A). Using identical recombinant
83
Figure 21. The ability of macrophage-derived conditioned media (CM-M) to
nonspecifically induce TNP-specific antibody in pronephric cell cultures.
Pronephric cell cultures were included with TNP-LPS alone, TNP-LPS plus
CM-M, TCM alone, and CM-M alone. The number of PFC per
106
lymphocytes is plotted. Each bar represents one standard error about each
mean. The lower horizontal dashed line to the abscissa represents the
standard error about the mean response of pronephric cells treated with 100
ug/ml E. coli LPS only. All wells were harvested 9 days after culture
initiation.
84
1000
PRONE?HRIC RESPONSE
800
00
a
()
U.
CL
600
400
200
"'''''" 7777
4
col
a.
Figure 21. In Vitro induction of nonspecific antibody by
the addition of CM-M.
85
Figure 22. Effect of recombinant human interleukins (rHuIl) one and two, on
cortisol-mediated suppression of the proliferative response of pronephric
lymphocytes. The day 3 of culture addition of rHuIl-1, -2, and a
1:1
combination thereof to 100 ug/ml E. coli LPS-stimulated, cortisol-suppressed
pronephric cell cultures was assessed. The plotted values represent the mean
of triplicate cultures expressed as counts per minute (CPM) ±1 SE per 106
lymphocytes. Nonstimulated background CPM were less than 4,200 CPM.
86
PRONEPHRIC RESPONSE
60
50
40
X
30
C.
20 --10
0
000
Op
0 0. 0
000 0 Op
0000
0 CO
0 .0
0
o
0
0 0
0 0
-
CLJ
0
0
CC
+
cr)
rHuIL 1
rHuIL 2
rHuiL 1 + 2
C.
_1
UNITS/n-11
Figure 22. Effect of recombinant human interleukins
one and two on cortisol-suppressed proliferation.
87
Figure 23. The restorative capacity of recombinant bovine interleukin one
(rBolL-1)
proliferative
and
recombinant
responses
of
human
cortisol-
interleukin
one
suppressed
(rHuIl -1)
pronephric
on
the
cultures
mitogenically-stimulated with 100 ug/ml E. coli LPS (A) or 25 ug/ml Con A
(B). The effect of day 3 additions of rBoIl -1 and rHuIL -1 were assessed five
days after the initiation of culture. Vertical lines represent the standard error
about the mean response of three culture wells.
=
.-.
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F.
-4
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.
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100 liinfirialtilleeittrA10 I I
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1
1,3
i
89
interleukins and a concentration of 25 ug/ml Con A and 100 ng/ml cortisol,
pronephric cultures were analyzed for restorative capacity upon the addition of
the
interleuldns. All rBoI1 and most rHuIl concentrations
established the proliferative response,
completely re-
however, only the 10 ug/ml rHull-1
concentration restored proliferation by approximately 50%, which was probably
supraoptimal for this concentration of interleukin (Figure 23B). Reversal of 100
ng/ml cortisol-induced suppression of the PFC response by rHuIl-1 and rHull-2
was also determined. Maximal restoration was approximately 52% for both
rHuIL-1 and -2 at 50 U/ml, followed by 45% at 5 U/ml and 42% at 0.5 U/ml
(Figure 24). Lack of complete restoration of the PFC response by both rHuIl-1
and -2, suggests that either the interleukins modulate early B cell activational
events, or they are sufficiently phylogenetically distant from salmonid- produced
molecules that they bind with poor affinity to interleukin receptors.
The Relationship of B cell Proliferation and the Generation of PFC In Vitro
Hydroxyurea (HYU), a cycle specific drug capable of killing proliferating
cells by preventing the reduction of ribonucleotides to deoxyribonucleotides
(152), was used to study the relationship between B cell proliferation and the
generation of PFCs in vitro. Two hundred micromolar HYU completely inhibited
the capacity of 0.5 ug/ml TNP-LPS to generate PFCs (data not shown). This
inhibition resulted primarily from suppression of B cell proliferation. This
90
Figure 24. In vitro dose-response of recombinant human interleukins -1 and
-2 on cortisol-suppressed pronephric lymphocytes stimulated with TNP-LPS.
The number of plaque-forming cells (PFC) per culture restored by: 1) 0.5U/ml,
2) 5.0 U/ml, and 3) 50.0 U/ml of the interleukins are plotted. Vertical bars
represent one standard error aboutthe mean response of three culture wells.
Responses were assessed nine days after culture initation.
91
200 -
PRONEPHRIC RESPONSE
100 .;
.41
rHuIL-1
rHuIL- 2
U/ml
Figure 24. In Vitro dose-response of recombinant human
interleukin one on restoration of the antibody-producing
cell response.
92
suppression of proliferation was directly observed in LPS-stimulated pronephric
cultures (Figure 25). Experiments were conducted to ascertain the length of time
that B cells
remained sensitive to the actions of HYU. A representative
experiment is depicted in Figure 26. Both splenic and pronephric lymphocyte
cultures were stimulated with 0.5 ug/ml TNP-LPS and the number of PFC were
assessed 24 h. after the addition of 200 uM HYU. As expected, the earliest
events, equivalent to the period of active proliferation (days 2 and 3), were the
most sensitive to HYU inhibition (Figure 26). However, addition of HYU as
late as day 6 resulted in a dramatic decrease in the number of PFC observed for
both splenic (25%) and pronephric (39%) cultures. This would suggest that
during the time period between days 6 and 7, very active cellular proliferation
occurs, this may be due to one or more of the following: 1) active cell division
due to initial clonal commitment in response to antigen; and 2) the activation
of a previously antigen insensitive pool. In an effort to understand the nature
of this inhibition in greater detail, the PFC response was assayed on a daily
basis in both the presence and the absence of HYU. PFC were detected in
control cultures after 3 days of incubation with 0.5 ug/ml TNP-LPS (24 PFC/
culture), they reached maximum numbers day 7 (137 PFC/culture) and were
maintained approximately at this level until day 9 (135 PFC/ culture)(Figure 26).
The
addition of HYU at the initiation of culture completely blocked the
development of PFCs throughout the length of the incubation. When HYU was
added on days 3 and 4 of incubation, an increase of PFCs was detected,
suggesting some maturation had occured days 2 and 3. The addition of HYU
93
Figure 25. Complete inhibition of LPS-induced proliferation by 200 uM
hydroxyurea. The proliferative response of pronephric lymphocytes was
assessed for the co-incubation with LPS and TCM, and for the additions of
hydroxyurea
or TCM. Counts per minute (CPM) were determined by
measuring tritiated thymidine incorporation. All responses were assessed 5
days after the initiation of culture as CPM x 10-3 +1 SE.
94
PRCNEPHRfC RESPONSE
20 -
15
10i
50
(15
C.
Figure 25. Hydroxyurea inhibition of E. con LPS-induced
proliferation.
95
Figure
26.
Late addition
of hydroxyurea inhibits the generation
of
plaque-forming cells (PFCs). Pronephric (s) and splenic (o) lymphocytes were
cultured with 0.4 ug/ml
TNP-LPS and hydroxyurea was added at various
times during the incubation as indicated. The number of PFC per culture for
all treatments was determined at 9 days. Vertical bars represent one standard
error about the mean of three culture wells.
96
400 -
300 200 -
100
0
1
0
.
2
4
6
.
i
8
10
DAY OF ADDITION OF HYU
1200 uM I
Figure 26. Late inhibition of proliferation by hydroxyurea.
97
on days 5 through 8, at a time when PFCs are already present, resulted in a
marked reduction of detectable PFCs within 24 hours. No HYU-resistant PFCs
appeared in pronephric cultures after day 5 (Figure 27). These data argue for
a continuously proliferating lymphocyte pool, which contributes mature PFC to
the in vitro system during the entire period of incubation. A similar pattern was
observed for splenic lymphocyte preparations, albeit HYU-resistant PFC were
present from days 4 through 7, thus suggesting a shifted kinetics (Figure 28).
Separation and Characterization of the Cellular Constituents of Hematopoietic
Tissues
Cellular separation studies were conducted to explore
various cell types in the pronephros.
characteristics of
Initial studies employed velocity
sedimentation techniques involving alternating concentrations (w/v) of 18.0%
ficoll,
18.5 dextran, 19.0% ficoll and 19.5% dextran to separate
subpopulations of pronephric lymphocytes (Figure
possible
29). The mitogenic
responsiveness of each population was tested using 25 ug/ml PHA and 100
ug/ml LPS. Population
B, which sedimented to the 19/19.5% interface,
responded approximately 6-fold greater to either mitogen than did population
A (18% interface) or population C (19.5% layer)(Figure 30). When population
B was co-cultured
with A, only PHA-responsiveness was restored, but if
co-cultured with C, only LPS-responsiveness was restored (Figure 30). Thus,
98
Figure 27. Role of ongoing B cell proliferation in the continued expression
of 0.4 ug/ml TNP-LPS-stimulated plaque-forming cells (PFC). Pronephric
lymphocytes were stimulated with TNP-LPS and assayed for the number of
PFC per culture found on days
1
through 9 of culture. Hydroxyurea was
added at the initiation of culture or on consecutive days thereafter (T). For
each set of cultures, the number of PFCs was then assayed daily. Control
cultures consisted of pronephric lymphocytes receiving TCM at the same
time the experimental cultures received hydroxyurea. The addition of TCM
had no significant effect on the number of PFC generated (data not shown).
Vertical bars represent the standard errors about the mean of three culture
wells.
99
PRONEPHRIC RESPONSE
200
100 Y
..
-.
....
T....
.
. ..
..... ..,
.0 _
;
.
.
......... ..t.
.. : .............
la
0
0
2
4
6
8
10
LENGTH OF INCUBATION
DAYS I
Figure 27. The role of ongoing proliferation in the
expression of plaque-forming cells in pronephric
cultures.
100
Figure 28. Role of ongoing B cell proliferation in the continued expression
of TNP-LPS-stimulated plaque-forming
were stimulated with
culture
cells (PFC). Splenic lymphocytes
TNP-LPS, and assayed for the number of PFC per
found on days 1 through 9 of culture. Hydroxyurea was
the initiation of culture or on consecutive days
added at
thereafter W. For each set
of cultures, the number of PFC was then assayed daily. Control cultures
consisted of
splenic lymphocytes receiving TCM at the same time the
experimental cultures received hydroxyurea. The addition
of TCM had no
significant effect on the number of PFC generated (data not shown). Vertical
bars represent the standard errors about the mean of three culture wells.
101
SPLENIC RESPONSE
o
4
6
8
10
LENGTH OF INCUBATION
1DAYS I
Figure 28. The role of ongoing proliferation in the expression
of plaque-forming cells in splenic cultures.
102
Figure 29. Effects of the concentration ranges of ficoll and dextran for
density gradient centrifugation on the separation of pronephric and splenic
lymphocytes. After centrifugation of whole leukocytes on the density
gradients, three cellular populations of leukocytes and erythrocytes were
resolved: the first banded at the 18% (w/v) ficoll interface (A), the second at
the 19/19.5% (w/v) ficoll-dextran interface (B), and the last in the 19.5%
(w/v) dextran gradient.
103
DENSITY GRADIENT CENTRIFUGATION
CELLS >,
18.0%
1 HR, 174C
.lc A
500 xg
18.5% --
19.0% ).
19.5>
--ic 8
--KC
Figure 29. Ficoll/Dextran density gradient separation
of lymphocytes.
104
Figure 30. Effects of mitogenic stimulation on the density gradient-separated
leukocyte
populations.
lipopolysaccharide (LPS,
Phytohemagglutinin
10Oug/m1) and TCM
(PHA,
10
ug/ml),
were co-incubated with
individual and combined leukocyte populations A (18% ficoll interface) B
(19/19.5% ficoll-dextran interface) and C (19.5% dextran gradient). The
proliferative responses were determined by the degree of tritiated thymidine
incorporation, measured by counts per minute (CPM) x 10' +1 SE. All
responses were assessed at 5 days after culture initiation.
105
PRONEPHRIC RESPONSE
50
40
30 >c
c_.
20 10. -1
0
11-11=-:
<
4t C CI CI
4 vs
O.
2V 4= r 2V itS
a.
s...
a.
I.-
C.)
is
e
vs
...a
Figure 30. Mitogenic analysis of Ficoll/Dextran separated
lymphocytes.
106
there appeared to be at least two
subpopulations of lymphocytes in the
pronephros; one PHA-responsive and the other LPS-responsive. Subsequently,
panning experiments were conducted to separate the lymphocytes by absorption
of sIg+ lymphocytes. Using purified mouse anti-fish immunoglobulin monoclonal
antibody (MaFIg)-coated petri plates, pronephric lymphocyte preparations were
panned into two distinct subpopulations based on the presence (sIg+) or absence
(sIg-) of surface
immunoglobulin. These subpopulations were analyzed by
fluorescent antibody techniques (FAT) to determine the purity of separation.
Unpanned pronephric lymphocytes, sIg+, and sIg- cells were incubated with an
appropriate
concentration of MaFIg. After incubation, excess MaFIg was
removed and an appropriate concentration of polyclonal goat
anti-mouse
immunoglobulin conjugated to fluorescein (GaMIg -Fl) applied. Controls included
non-MaFIg retreated
panned cells and whole pronephric cells treated with
GaMIg-Fl. All cell preparations were viewed by ultraviolet microscopy. Cells
panned one time for slg+ characteristics were found to be 80% pure, while sIg-
panned cells were contaminated by only 7% fluorescing lymphocytes. If the
same subpopulations (sIg+/-) were re-panned, slg+ panned cell recoveries were
increased to 99% homogeneity and contamination of the slg- fraction decreased
to
approximately 2% (Figure 31). Whole cell preparations were
contain 32% slg+ cells by FAT.
found to
Functional characterization of the Spanned
cells was conducted by determining their mitogenic profile (Figure 32) to 100
ug/ml LPS, 10 ug/ml PHA or 25 ug/ml Con A. Surface Ig+ cells dramatically
responded to stimulation by LPS, but only minimally to PHA and Con A. The
107
Figure 31. Fluorescent antibody analysis of panned lymphocytes. Pronephric
lymphocytes were separated on the basis of being phenotypically surface
immunoglobulin positive (sIg+) or negative (sIg-), by panning, using protein
A-purified mouse anti-fish Ig. The percentages of each cell phenotype were
determined by observing fluorescein-conjugated goat anti-mouse staining
using UV microscopy. The percent sIg+/- cells found in the unimmunized
pronephros were determined after both single (1X) and multiple (2X) panning
for sIg+ lymphocytes.
108
LYMPHOCYTES
F.A.T. ANALYSIS OF PANNED PRONEPHRIC
2X
UNPANNED LYMPHOCYTES
32%
32%
SURFACE 1g + CELLS
80%
99%
- CELLS
7%
2%
SURFACE
1g
Figure 31. Fluorescent antibody analysis of panned lymphocytes.
109
Figure 32. The mitogenic profiles of panned lymphocytes. Lipopolysaccharide
(LPS, 100 ug/ml), phytohemagglutinin (PHA, 10 ug/ml), and concanavalin A
(Con A, 25 ug/ml) were included in unpanned, surface immunoglobulin
positive, surface immunoglobulin negative, and 1:1 cell ratios of surface
immunoglobulin positive/negative cell cultures. The stimulation indices (SI)
were determined by assessing the mean response of triplicate mitogen
cultures over the mean response of triplicate control cultures. The vertical
bars represent the mean standard errors of stimulated cultures, over the mean
SE of nonstimulated cultures. Responses were assessed 5 days after culture
initiation by determining the levels of vitiated thymidine incorporation.
110
PRONEPHRIC RESPONSE
+
C/
masa
(0%
,
LPS
I
41%
I
+
C
C
CS
C
N
C.
us
C
CJ
C
C
LI
CI
Uf
is
CO
C-
C
PHA
CON A
Figure 32. Mitogenic profiles of panned lymphocytes.
111
opposite
occurred for sIg- cells; optimal stimulation by PHA and Con A,
minimal by LPS. Co-culturing sIg+ with sIg- cells
resulted in complete
restoration of all mitogenic responses when compared to whole cell indices.
Panned sIg+, sIg- and 1:1, 1:4 and 1:8 sIg+/sIg- cell combinations were also
assayed for their ability to produce TNP-specific antibody
stimulation with 0.5 ug/ml TNP-LPS. Panning
generated by
enriches for lymphocytes,
therefore all cells were added to wells with adherent cell monolayers. The PFC
response by sIg+ cultures was approximately 70% (210 PFC/culture) that of
unpanned whole cells (300 PFC/culture), while sIg- cells failed to produce a
significant PFC response above nonstimulated (TCM) controls. The 1:1 sIg+/sIg-
cell ratio cultures did respond (120 PFC/culture), but only to approximately
40% that of unpanned controls (Figure 33).
112
Figure 33. Plaque-forming cell (PFC) response of unpanned and
cells
to
0.4
ug/ml TNP-LPS.
panned
Surface immunoglobulin positive and
surface immunoglobulin negative lymphocytes were co-incubated alone and in
combination (1:1, 1:4,
1:8),
with TNP-LPS. Unpanned cells were also
incubated with TCM. All lymphocytes were added in situ to adherent cell
monolayers, and the number of PFC per culture assessed nine days after the
initiation of culture. Vertical bars represent ± 1 SE about the mean response
of three culture wells.
113
PRONEPHRIC RESPONSE
+
0
1
Figure 33. Antigenic analysis of panned lymphocytes.
114
DISCUSSION
An integrative view of immunoregulation involving several closely linked
biological systems, such as the immune and endocrine systems, is currently
held for all
warm-blooded species examined (16-18,24,45,46,50,51,79,
81-83). Such integration permits for a high degree of
response to antigen. This current view
versatility in the
of immunoregulation provides a
broader framework in contrast to the traditional concept of a self-contained,
self-monitored immune system. It was formerly held that immunoregulation
involved solely autoregulatory mechanisms
such as idiotypic-anti-idiotypic
networks, helper/ suppressor cells and regulatory lymphokines and monokines.
One area of research in which this integrative view has
produced much
interest is that of the effects of stress and stress-induced glucocorticoids on
the immune response.
The importance of glucocorticoid regulation in the
salmonid is demonstrated by its dramatic increase in plasma associated with
stress (13,29,30,32-34,58) and smoltification (30,49,53,55-57). This response
is so characteristic that blood corticosteroid levels have been used to assess
both the magnitude of stress and progress of smoltification (30,49,53,55-57).
Smoltification (the
parr-smolt transformation) of juvenile, anadromous
salmonids involves a morphological, behavioral and the
metamorphosis of the fish from a
saltwater-adapted form
physiological
fresh-water-adapted form to the
(30). The functional significance of this increase of
corticosteroids during smoltification remains unclear,
however, it has been
115
postulated that corticosteroids released during smoltification may function to
prevent the generation of autoimmunity to metamorphically changing tissues
(9). A similar postulate was made for the surge of glucocorticoids associated
with stress in mammals (123). A
stressful situation in mammals, such as
injury to tissues, can result in the exposure of antigens normally privy to the
immune system. Thus, the surge of glucocorticoids is postulated to protect
against an autoimmune response
against
those antigens by inducing
generalized immunosuppression.
The consequences of the glucocorticoid surge are many,
tissues bear glucocorticoid receptors, such as
since many
adipose, bladder, bone, brain,
fibroblast, lung, and lymphoid tissues (51). The effects on lymphoid tissues
are as diverse. Glucocorticoids have been demonstrated to
affect each stage
of the mammalian immunological response. These include antigen processing
and the penetration of basement membranes by antigen-antibody complexes
(123);
actions on macrophage function and phagocytosis (20);
helper cell
functions (19); inflammatory reactions (24); production of interleukins (19-23)
and down-regulation of
including
humoral and cellular immunological responses,
direct cytolysis of lymphocytes (reviewed in 51). However,
the
immunological effects of the surges of glucocorticoids associated with stress
and smoltification have not been thoroughly investigated. Thus, the dynamics
of cortisol
interactions in B cell immunoregulation were addressed
coho salmon (0. kisutch).
using
116
To investigate the role of glucocorticoids on modulating B cell regulation
in the salmonid system, all cellular analysis was performed in vitro. This
afforded
the use of a closed, tightly controlled system, free from external
influences which might interfere with the direct effects of glucocorticoids on
B
cell
regulation.
glucocorticoid-mediated
attention in
Since
the
molecular
mechanisms
of
immunosuppression have received only limited
the field of fish immunology (9-11,25-27), all maturational
phases of B cell development were examined. Before such analyses could be
performed, proliferative and
optimized.
antibody-producing cell assays needed to be
Since species sensitivity differences to mitogens have been
reported (88), assessment of the proliferative phases of B
were determined using graded concentrations
cell maturation
of several mitogens. A
dose-response was performed using the mitogens Con A, PHA, and LPS. No
significant organ-dependent differences in the optimal mitogen concentrations
were observed for either pronephric or
splenic responses were
splenic responses; however, the
generally one-half that
of those observed for
pronephric-derived lymphocytes (Figure 1). This is in direct contrast to the
studies reported in rainbow trout in which the organ distribution of mitogenic
responses to Con A, LPS, and purified protein derivative of tuberculin (PPD)
were examined (31). The inability of rainbow trout pronephric lymphocytes to
respond to Con A or PPD may have been the result of suboptimal culture
conditions or inappropriate temperatures. Similar results as those observed in
the rainbow trout were revealed to be temperature-mediated in the catfish (5).
117
LPS-induced
responses were shown to be temperature-independent, while
Con A responses were differentially suppressed at lower temperatures.
Trinitrophenylated-lipopolysaccharide (TNP-LPS) was used as the antigen
for the induction of TNP-specific B cells (Figure 2). The results of kinetic
studies of the peak response indicated that day 9 of culture was optimal for
the generation of TNP-specific antibody-producing cells
vitro assessment of glucocorticoid
(8,12). Thus, the in
modulation on all phases of B cell
differentiation in the salmonid could be determined.
The cytocidal effects of cortisol on splenic and pronephric lymphocytes
were investigated. The effects of
and 72 hours with
100 ng/ml cortisol co-cultured for 24, 48
lymphocytes from either source was determined.
Pronephric lymphocytes were demonstrated to be resistant to any
cytocidal
effects attributed to cortisol as measured by the percent lymphocyte viability;
however, after 48 hours of preincubation there was approximately a 25%
decrease of viable splenic lymphocytes (Figure 3). This dramatic decrease in
viability was not observed for splenic cultures preincubated for 72 hours in
results were likely to be due to increased
cortisol; consequently, these
proliferation
loss
between 48-72 hours. The kinetics of the in vitro
of viability
upon
the
addition
TNP-LPS-stimulated pronephric and splenic
determined (Figure 4). The
of
100
ng/ml
lymphocyte
cortisol
to
lymphocyte cultures was also
resistance of salmonid lymphocytes to the
cytocidal actions of cortisol was evidenced by the fact that the viability for
both lymphocyte cultures was not altered by cortisol
during the 11 days of
118
possible that TNP-LPS-induced proliferation
co-incubation. However, it is
could mask the possible cytocidal effects of cortisol by adding more viable
cells to the lymphocyte pool. These cytocidal effects would had to have been
extremely minimal, because
(Figure 3).
they were not detected in preincubation studies
If they occured, they would have happened after 72 hours of
co-culturing.
The effects of cortisol on B cell maturation were examined using various
equimolar concentrations of cortisol and aldosterone (Figure 5). Both splenic
and pronephric
lymphocyte proliferation was inhibited, in a dose-dependent
fashion, upon exposure to 10 or 100 ng/ml cortisol. There was no significant
enhancement or suppression upon the addition of 1 ng/ml cortisol nor at any
concentrations of
aldosterone. These findings are important because this
study utilized the same physiological levels of cortisol (1, 10, 100 ng/ml) that
are associated with the lowered
immune responses observed in vivo during
smoltification (9) and during stress (13). The specificity of this suppression
was confirmed by the use of the control steroid, aldosterone, not known to be
produced or have
function in salmon
(11).
In
addition, a similar
dose-dependent suppression of the antibody-producing cell
response was
observed upon the addition of 1, 10 or 100 ng/ml cortisol to either splenic or
pronephric lymphocytes
co-cultured with TNP-LPS (Figure 6). Addition of
the control steroid, aldosterone, had no effect on the response elicited with
cells from either source. Thus, cortisol was not only able to down-regulate
cellular proliferation but also TNP-specific antibody production.
119
The possible periods of cortisol sensitivity that
might influence
proliferation or antibody production were examined. Splenic and pronephric
lymphocytes were preincubated with and without 100 ng/ml cortisol for 24,
48
or 72 hours after which antigen or antigen plus cortisol was added and
the cells cultured (Figure 7). This experiment would determine if splenic or
pronephric resting
lymphocytes (non-activated) were sensitive to cortisol
modulation and if organ-dependent sensitivity to
occurs. Preincubation of splenic
suppression of the
glucocorticoid suppression
lymphocytes with cortisol increased the
anti-TNP-LPS response with cortisol. This was not
observed with pronephric lymphocytes treated in an identical fashion. Thus,
organ-dependent sensitivity to cortisol
appeared to exist, the spleen being
more sensitive to cortisol even when the cells are in an unactivated state.
Similar organ-dependent sensitivity to the glucocorticoid
immunoglobulin secretion has been observed
experiments in the mouse
suppression of
in the mouse (37). These
demonstrated that the in vivo administration of
dexamethasone resulted in an immediate decrease of the
production of
immunoglobulin by B cells in the spleen but not by bone marrow B cells.
This difference could not be
ascribed solely to cortisol effects on the
trafficking of lymphocytes, because the total number of antibody-producing
cells as well as the serum immunoglobulin levels decreased as a result of the
exposure. These findings are of particular relevance to the present studies in
that, morphologically and functionally, the salmonid pronephros is analogous
to the mammalian bone marrow (8,35,36). This
apparent organ-dependent
120
sensitivity to cortisol was verified through kinetic analysis. The kinetics study
of the cortisol-induced suppression of the antibody-producing cell response
was conducted for both splenic and pronephric-derived lymphocytes (Figure
8). These studies
demonstrated that there is a distinct, cortisol-sensitive
phase during the development of the antibody response. The data revealed
that lymphocytes from the pronephros
possessed a different pattern of
sensitivity to cortisol compared to lymphocytes from the spleen (Figure 8).
Pronephric lymphocytes demonstrated a marked sensitivity to cortisol during
the first four days of culture. Early differentiation events in this population of
lymphocytes
were
therefore
likely
to
be
the
primary
immunosuppression. The observed decreased sensitivity
of
targets
of
pronephric
lymphocytes to cortisol over time may reflect the fact that the pronephros
possesses interrenal cells which are responsible for the production of cortisol
in
fish (28,29). Such a close association of these cells may
decreased sensitivity of pronephric lymphocytes
lead to a
to the actions of cortisol.
This would likely be a receptor-mediated phenomenon, because there is little
deterioration of culture conditions which could lead to
alterations of cell
membranes. It is possible that splenic lymphocytes possess cell membranes
different from those of
(spleen)
/
pronephric lymphocytes, in light of the mature
immature (pronephros) theory of lymphoid cell heterogeneity
proposed for salmonids (8).
Through the use of cortisol pulsing experiments, the periods of cortisol
sensitivity occurring during the
generation of the antibody response to
121
TNP-LPS were assessed. Splenic cultures could be inhibited by at least 80%
at all time points throughout the generation of the antibody response by the
addition
of
cortisol.
pronephric
Conversely,
cultures
became
cortisol-insensitive by day 3 of culture (Figure 9). The demonstration of
cortisol
sensitivity of pronephric cells by day 3 of culture correlated well
with the kinetic analysis (Figure 8). The
cell-induced insensitivity
However, the
hypothesis concerning interrenal
of the pronephros can be applied here
also.
uniform sensitivity of splenic cultures to cortisol-induced
immunosuppression may also reflect a differential cortisol
sensitivity of
well documented in mice that
lymphocytes from different organs.
It
lymphoid cells from the thymus
and bone marrow display a similar
is
differential steroid sensitivity (reviewed in 18). In the thymus, cells bearing
the TL (thymus-leukemia) antigen are exquisitely sensitive to the suppressive
actions of glucocorticoids, while cells from the same tissue which bear H-2
isoantigens are not (124). Lymphocytes in the mouse bone marrow have been
shown to be resistant to steroids, while the B lymphocytes in the spleen are
sensitive (125). These results demonstrate that in mammals, lymphoid cells
may be steroid-sensitive or -resistant, depending on their location or stage of
maturation. Thus, the persistent
cortisol sensitivity observed in the splenic
lymphocytes of salmon (Figure 9) may best be explained by this differential
steroid sensitivity, rather than some affect
of the interaction of resident
interrenal cells. This is reasonable to assume since both the bone marrow of
mice and the pronephros of salmon (i.e. the physical and functional analog
122
and yet mouse bone
of the bone marrow; 8,35,36) lack this sensitivity
elaborate
marrow does not possess cells which
glucocorticoids
(i.e.
adrenal-like function) as does the pronephros.
Glucocorticoids have been shown to suppress mammalian
responses by inhibiting the production of
possibility
that
such
immunosuppression
examined.
a
mechanism
immune
interleukins (19-23,44, 52). The
played
a
role
in
early
the
observed in pronephric cells (Figures 5,8,9)
The addition of antigen-stimulated culture supernatants
was
to
cortisol-suppressed (100 ng/ml) cultures (Figure 14) demonstrated that: 1) the
suppression is reversible, and 2) B cells are relieved from active suppression
by a soluble factor in the supernatant from non-suppressed cultures. It is
unlikely that cortisol directly suppressed the
pronephric B cells, since
restoration of B cell responses was acheived using antigen-stimulated culture
supernatants
supplemented with cortisol. Instead, it appears that
cortisol
inhibits the elaboration of a requisite factor(s) that may be necessary for B
cell differentation. However, another possibility could be that the reversal of
suppression by the supernatant may have been due to
antigen-insensitive B cell precursors from
recruitment of
the lymphocyte pool into an
antigen-sensitive pool. This possibility was examined using limiting dilution
analyses (LDA).
Limiting dilution assays were used to precisely determine the effect of
cortisol on the number of
subsequent
antigen-sensitive B cell precursors and the
generation of antigen-specific clonal progeny. The
presence of
123
PFC in cultures, initially receiving dilutions
of lymphocytes prior to the
introduction of antigen, were determined. At low dilutions of lymphocytes the
cultures
became limiting for B cells specific for TNP-LPS, and thus
an
estimate of the frequency of precursors could be made. Potential obstacles in
performing such analyses are both the number of lymphocytes required to do
LDA and the possibility of confounding results due to a mixed lymphocyte
reaction (MLR). The generation of a MLR was
order to obtain sufficient cell
proliferative and
felt to be unavoidable in
numbers. The effects of the MLR on both
antibody-producing phases of the immune response were
investigated. The lymphocytes from four coho salmon were
mixed in all
possible combinations to induce proliferation via an MLR (Figure 10). The
data revealed that the MLR did occur for certain lymphocyte combinations,
especially when the lymphocytes from four fish (A-D) were pooled. However,
if the same lymphocyte combinations which produced an MLR
mitogenically stimulated with LPS, no significant
LPS-proliferation occurred. (Figure
observed in the
were
increase or decrease in
10). Also, no effect of the MLR was
generation of PFC for the same lymphocyte combinations
stimulated with TNP-LPS (Figure 11). Thus, the MLR does
ongoing immune response of stimulated
appropriate to use multiple
not alter the
lymphocytes. Therefore, it was
and potentially histoincompatible lymphocyte
sources in the execution of LDA with salmonid cells.
Initial LDA were preformed using dilutions of
cultured with TNP-LPS only (Figure
pronephric leukocytes
12B). The characteristic multi-hit curve
124
that was generated indicated that the system was limiting for, or required,
more than just the antibody-producing B lymphocyte (Figure 12B). Previous
data in the catfish, had demonstrated a strict requirement for adherent cells
(macrophages) in TNP-LPS responses (38). This observation
the phenomenon of multi-hit kinetics observed
would explain
in the salmonid responses.
Thus, pronephric lymphocytes were separated into adherent and nonadherent
cell
populations by adherence to fibronectin-coated microculture
wells.
Identical lymphocyte dilutions were made and co-cultured in situ with either
adherent (Figure 12C) or
Lymphocytes
nonadherent (Figure 12A) cell monolayers.
co-cultured with nonadherent cells responded in multi-hit
kinetic fashion, while single-hit kinetics resulted for lymphocytes co-cultured
with adherent cells. Thus, the
necessity for adherent cells in response to
TNP-LPS was confirmed for the salmonid.
The effect of cortisol on TNP-reactive B cell precursors and the cellular
mechanisms responsible for the
CM-L restoration of the antibody-producing
cell response were investigated. Results demonstrated that cells
cultured in
the presence of cortisol undergo a shift in the precursor frequency from 24
precursors per 106 lymphocytes (for cells cultured in the absence of cortisol)
to 10 precursors per 106 lymphocytes (Figure 13), thus indicating that the
reduction in PFC is likely due to suppression
primarily of the B cell
precursor prior to the proliferative phase of differentiation. More importantly,
the original precursor frequency can be fully restored to 24 precursors per 106
lymphocytes, upon the addition of
CM-L to cortisol-suppressed cultures
125
(Figure 16).
Furthermore, cells cultured in the absence of cortisol do not
produce an increase in the precursor frequency when CM-L is added (Figure
16). Thus, the inability of CM-L to
cultures without
increase the precursor frequency in
cortisol indicates that the increased response in cortisol
cultures does not occur by the activation of another
precursor pool. In
average clone size for cultures with or
addition, cortisol decreased the
without CM-L by approximately 17% (p < 0.05) (Figure 17B,D). Suppression
of pronephric B cell responses by cortisol occurs at the precursor level. Data
demonstrating the lowered clone size
(Figure 17B,D), are
observed in factor-restored cultures
then explained by the fact that later addition of the
factor to suppressed cultures would result in less time for clonal expansion,
thus
a
lowered
clone
size.
The
of cortisol-induced
vitro antibody-producing cell response to
immunosuppression of the in
TNP-LPS may
mechanisms
involve the direct inhibition of lymphokine production
thereby indirectly leading to inhibition of B cell
precursor differentiation.
This inhibition of the B cell precursors by cortisol would then be responsible
for the immunosuppression also seen at later stages of B cell maturation in
the salmon as has also been demonstrated to occur in the murine system.
Dexamethasone, a synthetic
suppressive
glucocorticoid, was shown to manifest its
effects on the immune response to TNP-LPS at the B cell
precursor level (155). The suppressive effects of dexamethasone were shown
to be the result of inhibition of
pathway. Such
the phosphoinositide signal transduction
similar glucocorticoid-induced immunosuppression observed
126
between salmonids and mice suggest that a similar suppression of signaling
events may occur for the salmonids, however this has yet to be determined.
Irrefutably, the adherent cell is required for the
activation of B cell
precursors in the response to antigen. In the piscine system, this mechanism
of action is unknown but probably is similar to the mechanisms decribed for
mammalian systems (193). This may occur through the necessity for antigen
processing and presentation, coupled
with nonspecific cellular activation by
lymphokine secretion. Paradoxically, the ability of the B cell to retain
an
antigen-activated state during glucocorticoid-induced immunosuppression, may
reflect
a
potential
protective
mechanism
elicited
by
cortisol
on
antigen-reactive B cells. Mammalian studies conducted in vitro on the effects
of glucocorticoids on lysosomes, a class of cytoplasmic organelles containing
hydrolases and
other bioactive enzymes (127), have shown that cortisone,
disruptive agents
cortisol and their analogs protect biomembranes against
(126). This protection may occur through the stabilization of the movement
of phospholipid acyl chains in the bilayers of leukocytes (128). Thus, it can
be envisioned that cortisol may indirectly stabilize the
antigen receptor of
salmonid lymphocytes, allowing for future lymphocyte activation by antigen.
Theoretically, this membrane stabilization could be different for pronephric
and splenic lymphocytes, assuming that maturational differences exist, as has
been postulated (9). However, it is more likely that the effects of cortisol on
lymphocyte membranes, when they occur, are deleterious to
immunological
responsiveness. This would more likely be the case for cortisol suppression of
127
the antibody-producing cell response (Figure 8). For suppression to occur in
a rapid fashion (i.e. even 12 hours prior to harvest), cortisol would
such
likely have direct affects on the B cell membrane leading to the prevention of
anti-TNP antibody secretion.
The existence of putative lymphokines in
antigen-activated leukocyte
supernatants was investigated. Dilutions of conditioned medium derived from
pronephric
leukocytes stimulated with TNP-LPS (CM-L) were added to
cortisol-suppressed (100 ng/ml) pronephric and splenic cultures (Figure 15).
The
highest
CM-L
antibody-producing cell
CM-L
concentration
(100%)
completely
restored
the
response for suppressed pronephric cultures, while
concentrations of 50%, 25% and 12.5% were able to restore
the
response to approximately 83%, 56% and 45% of the control, respectively.
The suppressed splenic responses were never fully restored by the addition of
any CM-L concentrations. Maximal splenic restoration (approximately 72%)
occurred using the 100% CM-L concentration. In an attempt to further define
the properties of the putative
activity was
lymphokine(s), the material exibiting this
isolated and characterized. Antigen-stimulated pronephric
supernatants were concentrated and fractionated using
cell
a Sephadex G-100
column. The fractions were then tested for antibody-producing cell restorative
capacities in cortisol-suppressed pronephric cultures. All restorative capacity
was eluted in the narrow molecular weight (MW) range of 10-18 kd (Figure
18). These data implicated the existence of a lymphokine-like factor(s) that
can be found
in pronephric cell culture supernatants. By analogy with
128
mammalian systems, the apparent MW range (10-18 kd) of the
soluble factor, most closely resembles that of
piscine
mammalian B cell growth
factor (11-4) whose MW has been reported to be 16-18 kd (129). In the lower
examined to date, the MW of the salmonid lymphokine -like
vertebrates
factor is closest to chicken (15-17 kd) and Xenopus laevis
(11-16 kd)
interleukin two (reviewed in 153). The actual source of mammalian 11-4 has
not been definitivly proven,
(129),
cells
although the fact that murine thymic leukemia
human T cell hybridomas
(130), and cloned interleukin
2-dependent human T cells (131) produce 11-4 argues
strongly that T cells
produce the 11-4. As such, even less is known about the sources of chicken,
amphibian and piscine lymphokines. Future considerations for characterizing
the piscine soluble factor would include delineation of the cell source(s) and
identification of the
factor
by
native
and
sodium dodecyl
sulfate
polyacrylamide gel electrophoresis. Since other cell types exist in pronephric
cultures,
such
as
fibroblasts,
erythrocytes,
macrophages, it was of interest to
granulocytic
cells,
and
determine if isolated cell types could
generate lymphokine -like factors.
Macrophages, highly enriched by fibronectin panning,
were stimulated
with E. coli LPS to generate lymphokine-like factors (CM-M). These factors
were
analyzed for their ability to restore the
antibody-producing cell
response in cortisol-suppressed pronephric cultures (Figure 19). Additions of
CM-M
to
suppressed
pronephric
cultures
failed
antibody-producing cell response. Therefore these cells
to
restore
the
appear to lack the
129
restorative capacity observed for CM-L
(Figure 14). Subsequently, CM-M
was assayed for its ability to induce nonspecific lymphocyte proliferation in
both
splenic and pronephric cultures (Figure 20).
The 50% CM-M
concentration employed enhanced the proliferative responses induced by both
LPS and Con A for splenic cultures and LPS-stimulated pronephric cultures;
however, enhancement of Con A-stimulated pronephric cultures did not occur.
For
each cell source, a 100% CM-M concentration induced
nonspecific
proliferation without prior mitogenic stimulation (Figure 20), while the 50%
concentration did
not (data not shown). These results correlate well with
earlier elucidation of macrophage-derived cytokine (I1-1)
properties in the
murine system (86). However, 11-1 is traditionally assayed by its effect on
LPS-unresponsive
used
murine thymocytes, because bacterial LPS is commonly
to induce 11-1 production (86). Although salmonids contain
two
somewhat distinct thymi (64), the availability of thymocyte numbers and the
T cell homogeneity of the
salmonid thymus is questionable (132). This is
different from most mammals, excluding the rabbit (133), whose thymi are
mostly free of mature antibody-producing B cells.
assayed for its ability to induce
Therefore, CM-M was
nonspecific proliferation in lymphocyte
cultures derived from both the pronephros and spleen. The enhancement of
mitogen-induced responses observed after the addition of 50% CM-M (Figure
20) also fits well with the emerging I1-1 model in mammalian systems. It was
recently demonstrated
responses
that macrophages were required for lectin-induced
after rigorous depletion of these cells revealed that H-1
was
130
essential for the generation of a response (134). The current view now is that
macrophages serve as accessory cells to the T cell by providing: 1) antigen
presentation in association with self histocompatibility antigen (class II), and
2) the elaboration of I1-1 (86). These functions have not been demonstrated to
exist in the salmonid but the elucidation of the functional properties of this
monokine(s) is an essential first step in the resolution of
Interestingly, addition of CM-M to pronephric
generation of TNP-specific
only 40% of
this problem.
cell cultures induced the
antibody (Figure 21); however, approximately
the nonspecifically-induced antibody could be attributed to
induction by CM-M over that seen with the LPS control. The co-incubation
of CM-M with TNP-LPS failed to enhance the number of antibody-producing
cells observed and appeared to inhibit their generation (Figure 21). A possible
mechanism by which this apparent inhibition of the antibody-producing cell
response by CM-M addition may be elicited could be through suppression by
supraoptimal doses of LPS, since the optimal TNP-LPS dose is approximately
1000-fold lower than
that of LPS (Figure 2 and 1, respectively). Another
putative mechanism of suppression may involve the
disruption of a required
mitogenic signal by the LPS on TNP-LPS due to saturation with LPS found
in
CM-M. A
required
mitogenic
signal for the
generation of an
antibody-producing cell response to T-independent antigens, such as produced
by LPS, has been documented in the murine system (135).
In order to study the role of purified interleukins in
cortisol-mediated immunosuppression, recombinant
reversing
mammalian interleukins
131.
were tested for restoration of the
lymphocytes inhibited
by cortisol
proliferative responses of pronephric
(Figures
22 and
23). Mammalian
recombinant interleukins were used because there is currently no available
sources of purified or recombinant salmonid interleukins. Recombinant human
interleukin 1 and 2 and a 1:1 combination of rHuIl -1 and -2 failed to restore
cortisol-suppressed, LPS-induced proliferative responses at
concentrations tested (Figure 22). Subsequently,
bovine interleukin 1 were analyzed
than those used
rHull -1 and recombinant
at proportionally higher concentrations
previously. Both rHuI1-1 and rBoll -1
re-establish the proliferative response in identically
addition, a 10 ug/ml rBoI1-1
approximately 55%
producing
were able to
suppressed cultures. In
concentration augmented proliferation by
more than did the rHuI1-1 concentration (1 ug/ml)
the greatest restorative capacity (Figure 23,A). The
response was also restored
pronephric cultures
any of the
in
proliferative
cortisol-suppressed, Con A-stimulated
by identical rBoI1-1 concentrations and most rHuI1-1
concentrations (Figure 23,B). The 10 ug/ml rHuI1-1
proliferation by only 50%, which was
concentration. The fact that
concentration restored
probably supraoptimal at this
rBoll -1, in general,
elicited the greatest
restorative capacity at identical concentrations as those for rHuIl -1,
suggests
a possible phylogenetic similarity of interleukin receptors between the teleost
and the bovine systems with increasing disparity between human and teleost
systems.
132
Recombinant human 11 -1 and -2 were tested for their
capacity to
response in cortisol-suppressed
re-establish the antibody-producing cell
pronephric cultures (Figure 24). As expected, neither rHuIl -1 nor -2 were able
to
completely restore the antibody-producing cell response to
TNP-LPS.
Approximately 50% restoration was achieved at all concentrations tested, for
both interleukins. Taken
CM-L
together, antibody-producing cell restoration by
(Figure 14), rBoll -1 (Figure 23) and rHull -1 (Figure 22) in
cortisol-suppressed cultures,
implicates
the
production (such as 11-1) by cortisol, as a
inhibition
of
lymphokine
primary mechanism of cortisol
immunosuppression in the salmonid.
The kinetics of glucocorticoid inhibition of cellular
proliferation were
initially thought to be somewhat similar to those mechanisms ascribed for the
inhibition of
proliferation by hydroxyurea in the murine system (77);
therefore, analysis of hydroxyurea studies in salmon were
conducted.
Hydroxyurea, a cell cycle-specific drug, prevents DNA synthesis during the S
phase of the cell cycle (76). The kinetics of HYU sensitivity was used to
identify the crucial periods of proliferation during the response to TNP-LPS
(Figure 26). In mammalian systems, proliferation
prerequisite to antibody-producing
is a believed to be a
cell development (86), although this has
not been definitivly proven. It is generally accepted that naive B cells must
first be activated by a stimulus such as provided by antigen or mitogen. The
activated B cells then undergo proliferation and differentiation, two distinct
phases,
before
the
production
of antibodies
occurs.
The
resultant
133
antibody-producing cells are considered to be stable, terminally-differentiated
Ig-secreting plasma cells
mononuclear cells,
Conversely, human
(86).
peripheral blood
stimulated with pokeweed mitogen (a polyclonal B cell
activator), were shown to be actively cycling during Ig secretion and were
not terminally differentiated as past
was required for Ig
theories propose, because proliferation
(77). The addition of 200 uM HYU to
secretion
LPS-stimulated pronephric lymphocytes completely abolished the proliferation
of those cells (Figure 25), because HYU kills only those cells involved in
active DNA synthesis (76). Thus, the relationship of B cell proliferation and
the generation of immunoglobulin-secreting cells could be directly examined.
In addition, HYU utilization would allow
periods lymphocyte
for the precise analysis of the
proliferation which might be sensitive to cortisol. The
kinetics of HYU sensitivity for both salmonid splenic and
lymphocytes
indicated
that
antibody-producing
cells
were
pronephric
probably
continually proliferating during the secretion phase (Figure 26). Earlier data
which indicated that maximal mitogen-induced proliferation occurred during
days one through four (Figure 1) correlated well with the period observed for
maximal HYU sensitivity (days
sensitivity during the period
antibody-producing
1
through
4). The expected loss of HYU
of suspected terminal differentiation to
cells (days 6-8, based on previous kinetic studies (11),
did not occur for either organ culture examined, thus indicating active DNA
synthesis during this period. The apparent reason for the late HYU sensitivity
is the DNA synthesis due to cellular proliferation.
134
HYU pulsing assays were performed to examine more closely this late
HYU sensitivity. Hydroxyurea was added
cultures of pronephric (Figure
every 24 hours to individual
27) and splenic (Figure 28) lymphocytes,
throughout the development of the in vitro PFC response to TNP-LPS. The
inhibition of the PFC response demonstrated that proliferation was required
for the development of these cells. In addition, the results of the kinetics of
HYU sensitivity (Figure 26) and HYU pulsing (Figure 27 and 28) assays,
argue for a continually proliferating pool of
lymphocytes due to the
sensitivity to HYU at early and late time points. These proliferating cells are
thus capable of
adding to the anti-TNP-secreting antibody-producing cell
pool in vitro. A possible argument that HYU does not effect proliferation, but
rather antibody secretion of the
Ig molecule is unlikely for a number of
reasons. First, later in cultures, HYU-resistant antibody-producing cells were
found that indicated these cells were capable of synthesizing Ig despite the
presence of HYU (Figure 26).
Second, the number of HYU-resistant
antibody-secreting cells increased as the culture was prolonged even further.
Moreover, the addition of HYU just before the cells were
effect on the number of antibody-secreting
harvested had no
cells detected, confirming that
HYU was not directly inhibiting Ig secretion. Studies in the murine system
using synchronized splenocytes responding to a sheep red blood cell stimulus
(37) suggest the possibility that antibody-secreting cells in the salmonid are
not terminally differentiated cells but capable of continued proliferation. The
kinetic analysis of Ig secretion in the mouse demonstrated that antibody is
135
secreted throughout the
cell cycle with maximal secretion in the early S
phase of the cell cycle. It was also demonstrated that continued proliferation
was necessary to maintain optimal antibody
secretion, as indicated by a
reduction in the number of antibody-producing cells as a result of treatment
with colchicine. It is possible that subpopulations of lymphocytes exist in the
hematopoietic tissues of the
salmon, which vary in sensitivity to HYU
because of possible organ-dependent maturational differences. Differences in
maturity between the lymphocytes of the spleen and pronephros could explain
the shift in kinetics of HYU sensitivity observed (Figure 26). Furthermore,
the sensitivity. of HYU at later B cell differentiative phases, indicates that at
least two important stages of proliferation. These phases of proliferation may
be due to an autocrine mode of growth. The proposed autocrine hypothesis
in which salmonid B cells produce their own
growth factors to aid in
expansion of antigen-reactive clones fits well with the observed HYU results
(Figures
27 and 28). Because late HYU sensitivity is indicative of late
lymphocyte proliferation, such proliferation may be due to the elaboration of
autocrine factors. Subsequent expansion of the B cell clones would allow for
an amplified response to the antigen, thus this could account for the dramatic
increase in PFC observed for cultures derived from either organ between days
seven and eight (Figure 26). In the
virus-transformed B cell line
human system an Epstein-Barr
(RPMI 1788) was demonstrated to produce
autocrines which were required to sustain the proliferation of transformed B
cells (156). The human autocrines were shown to have a molecular weight of
136
17 kd, 24 kd and 35 kd. The major autocrine activity was determined to
reside in 11-la (interleukin -one alpha) which was found to correlate with the
three molecular forms of natural 11-1 (156). These results are similar to the
data observed in the salmonid. Both recombinant human and bovine IL-1 and
whole
supernatants
cell
could
(CM-L)
cortisol-suppressed cultures (Figures 23 and 14,
proliferation
restore
in
respectively), although
adherent cell supernatants (CM-M) could not (Figure 19). This may suggest
that at least two forms of cytokines are required in the salmonid response;
one, a lymphokine form which acts as an autocrine to expand antigen-reactive
B
cell
clones,
and
the
other
a
monokine
possibly
required
in
TNP-LPS-induced in vitro immune responses. The major restorative salmonid
factor was shown to have a molecular weight range of 14-18 kd (Figure 18),
which is quite similar to the molecular weight of the human autocrine 11-1 a
(17 kd). Thus, it is reasonable to
postulate a role for autocrine-induced
proliferation in the salmonid.
Cellular separation studies were conducted to explore the characteristics
and possible functions of cell subsets found in the pronephros. Future work in
this field will
aid in the dissociation of the effects of glucocorticoids on
other cell types, such as putative B and T cell subsets.
Initial studies
incorporated density gradient separation of lymphocytes based on differential
densities in
ficoll/dextran gradients (Figure 29). Data revealed that
distinct subpopulations of leukocytes existed which
mitogen-responsive and -unresponsive to
two
were, respectively,
LPS and PHA (Figure
30).
137
Subpopulation A,
which
banded at
the
ficoll
18%
interface,
was
mitogen-unresponsive when cultured alone with either LPS or PHA, as was
subpopulation
C (19.5% dextran layer). Conversely, subpopulation B was
mitogen-responsive for both LPS and PHA. Co-culturing
subpopulation B
with either subpopulation A or C, differentially suppressed the proliferative
responses of subpopulation B to LPS or PHA. Thus, subpopulations A and C
are distinctly different from subpopulation B in response
to polyclonal
activators. It is possible that the lack of mitogenic responsiveness observed
for subpopulations A and C result from density gradient-mediated seperation
of a required cell type. This cell type is presumed to be the macrophage,
since previous results using LDA (Figure 12)
requirement for macrophages in
demonstrated the strict
TNP -LPS responses. In addition, in
mammalian and piscine systems, the macrophage has been demonstrated to be
required for lectin-induced responses (2,38,63).
Since velocity sedimentation is a method of isolating cells based upon
density but not surface membrane
were conducted,
then
verified
phenotype, cellular panning experiments
by
determining
the
percent
surface
immunoglobulin positive (slg +) or -negative (slg -) lymphocytes found in the
unimmunized pronephros (Figure
lymphocytes analysis revealed
surface Ig+ with
31).
After
one
panning
for slg+
the panned cells to be approximately 80%
only 7% contamination by slg- cells. Re-panning of the
slg+ population resulted in an increased degree of homogeneity with only 2%
contamination by slg- cells.
138
Previous evidence from catfish studies demonstrated a functional analogy
between the sIg+ and sIg- cells of fish to mammalian B and T cells (1,2).
Consequently, the functionality of salmonid sIg+ and sIg- cells was explored
by determining their mitogenic profiles to LPS, PHA and Con A (Figure 32).
LPS-induced proliferation
of the control
responses from
unpanned
lymphocytes was only adequate in comparison to the proliferative responses
observed for unpanned lymphocytes stimulated with either PHA or Con A.
However, when comparing the mitogen-induced responses of sIg+ versus sIg-
cells there is a dramatic augmentation of the proliferative response to LPS, a
mammalian B cell mitogen, by the slg+ cells while the responses to PHA and
Con A, mammalian T cell mitogens,
are marginal. Conversely,
sIg-
lymphocytes responded well to both PHA and Con A but only marginally to
LPS. When both phenotypes were assayed
antibody-producing cells in
lymphocytes
for their ability to generate
response to TNP-LPS (Figure 33), the slg+
responded with approximately 70% (205+90 PFC per culture)
of the responses seen with the unpanned control (300 ±30 PFC per culture),
while the sIg- lymphocyte response was statistically insignificant (10+2 PFC
per culture). This
suggested that the sIg+ population was comprised of B
cells, and the sIg- population contained T cells and possibly other cells which
do not bear antigen receptors,
cells and granulocytes.
such as the mammalian equivalent of pre-B
139
SUMMARY AND CONCLUSIONS
Interactions between the immune and endocrine system
demonstrated to exist in the salmonid. Cortisol,
salmonids, suppressed B cell
have been
a primary glucocorticoid in
proliferation and antibody secretion. More
specifically, these suppressive effects were found at physiological concentrations
of cortisol.
Cortisol-induced immunosuppression was reversible.
Leukocyte-derived
conditioned medium (CM-L) could completely reverse suppression.
Limiting dilution analysis (LDA) revealed the B cell
precursor as the
primary target for cortisol-induced immunosuppression of B cell responses. In
addition, cortisol was shown to down-regulate clone size. Such cortisol-induced
immunosuppression could be completely restored by the addition of CM-L,
whose partial characterization indicated that it contained an interleukin-like
factor(s) in the molecular weight of 10
bovine interleuldn-1
18 kd. Both recombinant human and
additions also demonstrated this restorative capacity;
bovine interleuldn-1 demonstrated the greatest restorative capacity.
The strict requirement for macrophages in TNP-LPS responses was revealed
using LDA. Macrophages were also found to produce soluble bioactive agents.
Pronephric
lymphocytes
were
physically
separated
immunoglobulin positive (slg +) and negative (slg -)
into
surface
cell populations. Both
mitogen and antigen profile analysis revealed their similarity to mammalian B
and T lymphocytes, respectively. These findings demonstrate that salmonids
140
represent excellent animal models to discern not
only the evolutionarly
conserved and therefore immunologically vital immune functions found in all
vertebrates, but perhaps also the phylogenetic origins of
interactions which are now known to
functions.
endocrine-immune
regulate many mammalian immune
141
BIBLIOGRAPHY
1. Miller,N.W, Sizemore,R.C. and L.W. Clem. Phylogeny of lymphocyte
heterogeneity: The cellular requirements for in vitro antibody responses of
channel catfish leukocytes. 1985.
J. Immunol. 134:2884.
2. Miller,N.W., Cuchens,M.A., Lobb,C.J. and L.W. Clem. Phylogeny of
lymphocyte heterogeneity: The cellular requirements for in vitro mitogenic
responses of channel catfish leukocytes. 1984. J. Immunol. 133:2920.
3. Miller,N., and L.W.Clem. Microsystem for in vitro primary and secondary
immunization of channel catfish
hapten-carrier conjugates. 1984. J.
(Ictalurus punctatus) leukocytes with
Immunol. Meth.72:367.
4. Cuchens,M.A. and L.W.Clem. Phylogeny of lymphocyte heterogeneity.II.
Differential effects of temperature on fish T-like and B-like cells. 1977.
Cell.Immunol. 34:219.
5. Faulmann,E., Cuchens,M.A., Lobb,C.J., Miller,N.W. and L.W.Clem. An
effective culture system for studying in vitro mitogenic responses of channel
catfish lymphocytes. 1983. Trans. Amer. Fish. Soc. 112:673.
6. Miller,N.W., Deuter,A. and L.W.Clem. Phylogeny of heterogeneity: The
cellular requirements for the mixed leukocyte reaction with channel catfish.
1986. Immunol. 59:123.
Bly,J.E., van Ginkel,C., Ellsaesser,C. and L.W. Clem.
Phylogeny of lymphocyte heterogeneity: identification and separation of
functionally distinct subpopulations of channel catfish lymphocytes with
7. Miller,N.W.,
monoclonal antibodies. 1987. Dev Comp. Immuno1.101:739.
8. Kaattari,S.L. and M.J.Irwin. Salmonid spleen and anterior kidney harbor
populations of lymphocytes with different B cell repertoires. 1985. Dev.
Comp. Immunol. 9:433.
9. Maule,A.G., Schreck,C.B. and S.L.Kaattari. Changes in the immune system
of coho salmon (Oncorhnchus kisutch)
during the parr-to-smolt transformation and after implantation of cortisol. 1987. Can. J. Fish.Aqua Sci.
44:161.
10. Kaattari,S.L. and R.A.Tripp. Cellular mechanisms of
glucocorticoid
immunosuppression in salmon. 1987. J. Fish. Biol. 31A:129.
11. Tripp,R.A., Maule,A.G., Schreck,C. and S.L.Kaattari. Cortisol-mediated
salmonid lymphocyte responses in vitro. 1987. Dev.
suppression of
Comp. Immunol.
11:565.
142
12. Kaattari,S.L., Irwin,M.J., Yui,M.A., Tripp,R.A. and J.S.Parkins. Primary in
vitro stimulation of antibody production by rainbow trout lymphocytes. 1986.
12:29.
Vet. Immunol. Immunopath.
13. Maule,A.G., Tripp,R.A., Kaattari,S.L. and C.B.Schreck Stress-induced
glucocorticoids alter immune function in chinook salmon
changes in
(Oncorhynchus tshawsycha).1988.
J. Endocrinol. (accepted for publication).
14. Jacobs,D.M. and Morrison,D.C. Stimulation of a T-independent primary
anti-hapten response in vitro by TNP-lipopolysaccharide (TNP-LPS). 1975. J.
Immunol. 144:360.
15. Ruben,L.N., Warr,G.W., Decker,J.M. and J.Marchalonis. Phylogenetic origins
of immune recognition: Lymphoid heterogeneity and the hapten/carrier effect in
the goldfish, (Carassius auratus). 1977. Cell.Immunol. 31:266.
16. Besedovsky,H.O. and E.Sorkin. Network of immuno-endocrine interactions.
1977. Clin. Exp. Immunol. 27:1.
17. Besedovsky,H.O., Del Ray,A.E. and E.Sorkin. Immuno-endocrine interactions.
J.Immunol. Supp. 135:750s.
1985.
18. Claman,N.H. Corticosterone and lymphoid cells. 1972. N. Engl.J. Med.
287:388.
Crabtree,G.R., Gillis,S., Smith,K.A. A.Munck. Mechanisms of
immunosuppression: Inhibitory effects on expression
glucocorticoid-induced
19.
of Fc receptors and production of T
cell growth factor. 1980. J. Steroid
Biochem. 12:445.
20. Snyder,D.S. and E.Unanue. Corticosteroids inhibit murine macrophage Ia
expression and interleukin 1 production. 1982. J. Immunol. 129:1803.
si 21. Kelso,A. and A.Munck. Glucocorticoid inhibition of lymphokine secretion
by alloreactive T lymphocyte clones. 1984. J. Immunol. 133:784.
22. Culpepper,J.A. and F.Lee. Regulation of interleukin 3 expression by
cloned murine T lymphocyte clones. 1985. J. Immunol.
glucocorticoids in
133:3191.
23. Gillis,S., Crabtree,G.M. and K.A.Smith. Glucocorticoid- induced inhibition
of T cell growth factor production. I. The effect on mitogen-induced lymphocyte
Immunol. 123:1624.
proliferation. 1979. J.
24. Fahey,J.V., Guyre,P.M. and A.Munck. Mechanisms of anti-inflammatory
actions of glucocorticoids. 1981. Adv. Inflamm. Res. 2:21.
143
25. Pickering,A.D. and J.Duston. Administration of cortisol to brown trout, Salmo
in
L., and its effects
on the suseptibility to saprolegnia infection and
furunculosis. 1983. J. Fish Biol.
23:163.
26. Pickering,A.D. Cortisol-induced lymphocytopenia in brown trout, Salmo
trutta L. 1984. Gen. Comp. Endocrinol. 53:252.
27. Grimm,A.S. Suppression by cortisol of the mitogen- induced proliferation
of peripheral blood leukocytes from plaice, Pleuronectes platessa L. 1985. In:
Fish Immunology,M.J. Manning and M.F. Tatner (Eds.). London: Academic
Press, p.263.
28. Chester Jones,I., Chan,D., Henderson,I.W. and J.Ball. The adrenocortisol
adrenocorticotropin and the corpuscles of Stannius. 1985. In: Fish
steroids,
Physiology, W.S. Hoar and D.J. Randall (Eds.). New York: Academic Press,
Vol.II,p.321.
29. Henderson,I.W. and H.O.Garland. The interrenal gland in Pisces. Part 2,
Physiology. 1980. In: General, Comparative and Clinical Endocrinology of the
Adrenal Cortex.I. Chester Jones and I.W. Henderson (Eds.), New York:
Academic Press, p.473.
30. Barton,B.A., Schreck,C.B., Ewing,R.D., Hemmingsen,A., and R.Patino.
Changes in plasma cortisol during stress and smoltification in coho salmon,
Comp. Endocrinol. 59:468.
Oncorhynchus kisutch 1985. Gen
31. Etlinger,H.M., Hodgins,H.O. and J.M.Chiller. Evolution of the lymphoid
Evidence for lymphocyte heterogeneity in rainbow trout revealed
system. I.
by the organ distribution of mitogenic responses. 1976. J. Immunol. 116:1547.
32. Specker,J.L. and C.B.Schreck. Stress responses to transportation and fitness
for marine survival in coho salmon (Oncorhyncus kitsuch) smolts. 1980. Can.
J. Fish. Aquat. Sci.
37:765.
33. Ellis,A.E. Stress and the modulation of defense mechanisms in fish. 1981. In:
Stress and Fish, A.D. Pickering (Ed.). London: Academic Press, p.147.
34. Mazeaud,M.M., Mazeaud,F. and E.M.Donaldson. Primary and secondary
effects of stress in fish: Some new data with a general review. 1977. Trans.
Amer. Fish. Soc. 106:201.
35. Zapata,A. Ultrastructural study of the teleost fish kidney. 1985. Dev. Comp.
Immunol. 3:55.
36. Irwin,M.J. and S.L.Kaattari Salmonid B lymphocytes demonstrate organ
dependent fuctional heterogeneity. 1986. Vet. Immunol. Immunopath. 12:39.
144
37. Sabbele,N.R., Van Oudenaren,A. and R.Benner. The effects of corticosteroids
distribution of "background"
number and organ
upon the
1983. Cell. Immunol. 77:308.
immunoglobulin-secreting cells in mice.
Monocytes as
accessory cells in fish immune responses. 1985. Dev. Comp. Immunol. 9:803.
38. Clem,L.W., Sizemore,R.C., Ellsaesser,C. and N.Miller.
Mishell,R.I., Grabstein,K.H. and S.M.Shiigi. Prevention of the
immunosuppressive effects of glucocorticosteroids by cell-free factors from the
adjuvant-activated accessory cells. 1980. Immunopharm. 2:233.
39.
supernatant recruits
lymphocyte precursors into an
Immunol. 128:720.
40. Kaattari,S.L. and M.B.Rittenburg. Concanavalin A
antigen-insensitive IgG memory B
antigen-sensitive precursor pool. 1982. J.
41. Levkovitz,I. Limiting Dilution Analysis of Cells of the Immune System
1979. Cambridge: Cambridge Univ. Press. p.257.
42. Warr,G.W. and R.C.Simon. The mitogen response potential of lymphocytes
rainbow trout (Salmo gairdneri) re-examined. 1983. Dev. Comp.
from the
Immunol. 7: 379.
43. Cupps,T.R., Gerard,T.L. ,Falkoff,R.J.M., Whalen,G. and A.S.Fauci. Effects
corticosteroids on B cell activation, proliferation, and
of in vitro
1985.
J. Clin. Invest. 75 :754.
differentiation.
44. Mishell,R.I., Lucas,A. and B.Mishell. The role of activated accessory cells
in preventing immunosuppression by hydrocortisone. 1977. J. Immunol.
119:118.
45. Munck,A., Guyre,P.M. and N.J.Holbrook. Physiological functions of
stress and their relation to pharmacological actions. 1984.
glucocorticoids in
Endocrine Reviews. 5:25.
46. Homo,F., Picard,F., Durant,S., Gagne,D., Simon,J., Dardienne,M. and
D.Duval. Glucocorticoid receptors and their functions in lymphocytes. 1980.
12:433.
J. Steroid Biochem.
47. Marx,M., Ruben,L.N., Nobis,C. and D.Duffy. Compromised T-cell regulatory
functions during anuran metamorphisis: The role of corticosteroids. 1987. Dev.
Comp. Immunol. 1:129.
48. Highet,A.B. and L.N.Ruben. Corticosteroid regulation of
I1-1 may be
responsible for deficient immune suppressor function during the metamorphisis
of Xenopus laevis, the South African clawed toad. 1987. Immunopharm. 13:149.
145
49. Barron,M.G. Endocrine control of smoltification in anadromous salmonids.
Endocrinol. 108:313.
1986. J.
50. Shavit,Y., Terman,G.W., Martin, F.C., Lewis,J.W., Liebeskind,J.C. and
opioid peptides, the immune system, and cancer. 1985. J..
P.Gale. Stress,
Immunol. 135: 834s.
51. Baxter,J.D. Glucocorticoid hormone action. 1976. Pharmac. Ther. vol. 2,
p.605.
1 52. Goodwin,J.S., Atluru,D., Sierakowski,S. and E.Lianos. Mechanism of action
glucocorticosteroids. Inhibition of T cell proliferation and interleukin
of
hydrocortisone is reversed by leukotriene B4 1986. J. Clin.
2 production by
Invest. 77:1244.
53. Richman,N.H., Nishioka,R.S., Young,G. and H.A.Bem. Effects of cortisol
and growth hormone replacement on osmoregulation in hypophysectomized coho
salmon (Oncorhynchus kisutch). 1987. Gen. Comp. Endocrinol. 67(2):194.
54. Calabrese,J.R., Kling,M.A. and P.W.Gold. Alterations in immunocompetence
during stress, bereavement, and depression: Focus on neuroendocrine regulation.
1987. Am. J. Psychiatry. 144(9):1123.
55. Sheridan,M.A. Effects of thyroxin, cortisol, growth hormone, and prolactin
on lipid metabolism of coho salmon, Oncorhynchus kisuch, during smoltification.
1986. Gen. Comp. Endocrinol. 64:220.
56. Richman,N.H. and W.S.Zaugg. Effects of cortisol and growth hormone on
osmoregulation in pre- and desmoltified coho salmon (Oncorhynchus kisuch).
1987. Gen. Comp. Endocrinol. 65:189.
57. Young,G. Cortisol secretion in vitro by the intrarenal of coho salmon
(Oncorhynchus kisuch) during smoltification: Relationship with plasma thyroxine
and plasma cortisol. 1986. Gen. Comp. Endocrinol. 63:191.
58. Sumpter,J.P., Dye,H.M. and Benfay,T.J. The effects of stress on plasma
ACTH, alpha-MSH, and cortisol levels in salmonid fishes. 1986. Gen. Comp.
Endocrinol. 62:377.
59. Strijbosh,L.W.G., Buurman,W.A., Does,R., Zinken,P. and G.Groenewegan.
Limiting dilution assays: Experimental design and statistical analysis. 1987.
J.Immunol. Meth. 133:133.
60. Vie,H., Bonnieville,M., Sondermeyer,P., Moreau,J. and J.Soulillou. Limiting
dilution analysis (LDA) of cells responding to recombinant interleukin -2 without
previous stimulation: Evidence that all responding cells are lymphokine- activated
potent effectors. 1986. Immunol. 57:351.
146
61. Hibi,T. and Dosch,H-M. Limiting dilution analysis of
the B cell
compartment in human bone marrow. 1986. Eur. J. Immunol. 16:139.
62. Vie,H. and R.Miller. Estimation by limiting dilution analysis of human Il
2-secreting T cells: Detection of Il 2 produced by single lymphokine- secreting
T cells. 1979. J. Immunol. 136:3292.
63. Yoshinaga,M., Yoshinaga,A. and B.H.Waksman.Regulation of lymphocyte
responses in vitro. I.Regulatory effect of macrophages and thymus-dependent (T)
cells on the response of thymus-independent (B) lymphocytes to endotoxin.
1972. J. Exp. Med. 136:956.
64. Ellis,A.E. Ontogeny of the immune response in Salmo salar. Histogenesis
of the lymphoid organs and appearance of membrane immunoglobulin and mixed
leukocyte activity. 1977a. In: Developmental Immunobiology (eds. J.B. Solomon
and J.D. Horton), Elsevier/North Holland Biomedical Press, Amsterdam. p.225.
65. Koch,G., Lok,B.D., van Oudnaren,A. and R.Benner. The
capacity and
mechanism of bone marrow antibody formation by thymus-independent antigens.
1982. J. Immunol. 128: 1497.
66.
Taswell,C.
Limiting
dilution
immunocompetent cell frequencies.
analysis
I. Data
for the determination of
analysis. 1981. J. Immunol.
126:1614.
67. Alderson,M.R., Pike,B.L. and J.V.Nossal. Single cell studies on the role of
B-cell stimulatory factor 1 in B cell activation. 1987.Proc. Natl. Acad. Sci.
84:1389.
68. Chiller,J.M., Hodgins,H.O. and R.S.Weiser. Antibody response in rainbow
trout (Salmo gairdneri). II. Studies on the kinetics of development of antibodyproducing cells and on complement and natural hemolysin. 1968. J. Immunol.
102:1202.
69. Maryanski,J., De Plaen,E. and J. Van Snick. A simple panning method for
the selection of cell surface antigen transfectants. 1985. J. Immunol. Meth.
79:159.
70. Hoover,M.L., Chapman,S.W. and M.A.Cuchens. A procedure for the isolation
of highly purified populations of B cells, T cells and monocytes from human
peripheral and umbilical cord blood. 1985. J. Immunol. Meth. 78:71.
71. Wysocki,L.J. and L.Sato. "Panning" for lymphocytes: A method for cell
selection. 1978. Proc. Natl. Acad. Sci. 75:2844.
72. Blazer,V.S., Bennett,R.O. and R.E.Wolke. The cellular immune response in
rainbow trout (Salmo gairdneri Richardson) to sheep red blood cells. 1984. Dev.
147
Comp. Immunol. 8:81.
73. Caspi,R.R., Shahrabani,R., Kehati-Dan,T. and R.R. Avtalion. Heterogeneity
of mitogen- responsive lymphocytes in carp (Cyprinus carpio). 1984. Dev.
Comp. Immunol. 8:61.
74. Chilmonczyk,S. In vitro stimulation by mitogens of the peripheral blood
lymphocytes from rainbow trout (Salmo gairdneri).1978. Annales D'Immunol.
129:3.
75. Bowen,D.L. and A.S.Fauci. Selective suppressive events of glucocorticoids
on the early events in the human B cell activation process. 1984. J. Immunol.
133:1885.
76. Rusthoven,J.J. and R.A.Phillips. Hydroxyurea kills B cell precursors and
markedly reduces functional B cell activity in mouse bone marrow. 1980. J.
Immunol. 124: 781.
77. Jelinek,D.F. and P.E.Lipsky. The role of B cell proliferation in the generation
of immunoglobulin-secreting cells in man. 1983. J. Immunol. 130:2597.
78. Ellis,A.E. The leukocytes of fish: A review. 1977. J. Fish. Biol. 11:453.
79. Martin,C.R. The glucocorticoids. IN: Fundamental Immunology. 1985. Oxford
University Press.
80. Ingle,D.J. Permissability of hormone action. A review.
1954. Acta
Endocrinol. 17:172.
81. Berczi,I. and K.Kovacs. Hormones and Immunity. 1987.
MTP Press,
Norwell, Ma.
82. Cupps,T.R. and A.S.Fauci. Corticosteroid-mediated
man. 1982. Immunol. Rev. 65:133.
immunoregulation in
83. Claman,H.N. and G.G.Kreuger. Site of action of cortisol
in cellular
immunity. 1973. J. Immunol. 110:880.
84. Bloom.A. Diabetes Explained. 1975. Medical and Technical Publishing Co.
LTD., Lancaster, England.
85. Cooper,E.L. Comparative Immunology. 1976. Prentice- Hall Inc., Englewood
Cliffs, N.J.
86. Paul,W.E. Fundamental Immunology. 1985. Raven Press, NY, NY.
87. Zettergen,L.D. Ontogeny and distribution of cells in B
lineage in the
148
American leopard frog (Rana pipiens). 1982. Dev. Comp. Immunol. 6:311.
88. Oppenheim,J.J. and D.L.Rosenstreich. Miiogens in Immunobiology. 1976.
Academic Press, NY, NY.
89. Nowell,P.C. Phytohemagglutinin: An initiator of mitosis in cultures of normal
human leukocytes. 1960. Cancer Research, 20:462.
90. Thorn,G.W. Clinical considerations in the use of corticosteroids. 1966. N.
Engl. J. Med. 274:775.
91. Fauci,A., Dale,D. and J.Barlow. Glucocorticoid therapy: Mechanisms of
action and clinical considerations. 1976. Ann. Intern. Med. 84:304.
92. Mishell,R.I. and R.W.Dutton. Immunization of normal mouse spleen cell
suspensions in vitro. 1966. Science. 153:1004.
93. Medawar,P. and E.Sparrows. The effects of adrenocortical hormones,
adrenocorticotrophic hormones and pregnancy on skin transplantation immunity
in Mice. 1956. J. Endocrinol. 14:240.
94. Gabrielsen,A. and R.Good. Chemical suppression of adaptive immunity. 1967.
Adv. Immunol. 6:91.
95. Smith,K.A., Crabtree,G., KennedyS. and A.Munck. Glucocorticoid receptors
and glucocorticoid sensitivity of mitogen stimulated and unstimulated human
lymphocytes. 1977. Nature. 267:523.
96. Neifeld,J., Lippman,M. and D.Tormey. Steroid hormone receptors in normal
and human lymphocytes. 1977. J. Biol. Chem. 252:2972.
97. Sorg,C. and A.Schimpl. Cellular and Molecular Biology of Lymphokines.
1984. Academic Press Inc., Orlando, Fl.
98. Smith,K. and F.Ruscetti. Advances in Immunology. 1981. H.Kunkel and
F.Dixon (eds). Academic Press Inc. NY, NY.
99. Gillis,S., Ferm,M., Ou,W. and K.Smith. T cell growth factor: Parameter of
production and a quantitative microassay for activity. 1978. J. Immunol.
120:2027.
100. Smith,K., Gilbridge,K. and M.Fauata. Lymphocyte activating factor promotes
T cell growth factor production by cloned murine lymphoma cells. 1980. Nature.
287:853.
101. Gillis,S. and S.Mizel. T cell lymphoma model for the
analysis of
interleukin 1-mediated T cell activation. 1981. Proc. Natl. Acad. Sci. 78:1133.
149
102. Isakson,P., Pure.E., Vitteta,E. and P.ICrammer. T cell derived B cell
differentiation factor(s). Effect on the isotype switch of murine B cells. 1982.
J. Exp. Med. 155:734.
103. Pure,E., Isakson,P., Kappler,J., Marrack,P., Krammer, P. and E.Vitteta. T
cell-derived growth and differentiation factor. Dichotomy between the
responsiveness of B cells from adult and neonatal mice. 1983. J. Exp. Med.
157:600.
104. Mosier,D.E. A requirement for two cell types for antibody formation in
vitro. 1967. Science. 158: 1573.
105. Schimpl,A. and E.Wecker. Replacement of T cell function by a T cell
product. 1972. Nature. 237:15.
106. Raff,M. Two distinct populations of peripheral
lymphocytes in mice
indistinguishable by immunofluorescence. 1970. Immunology. 19:637.
107. Cantor,H. and I.Weissman. Development and function of subpopulations of
thymocytes and T lymphocytes. 1978. Prog. Allergy. 20:1.
108. Scher,I., Sharrow,O. and W.Paul. B lymphocyte heterogeneity: Ontogenetic
development and organ distribution of B lymphocyte populations defined by
their density of surface immunoglobulin. 1976. J. Exp. Med. 14:494.
109. Horan,P. and L.Wheeless. Quantitative single cell analysis and sorting.
Rapid analysis and sorting of cells is emerging as an important new technology
in research and medicine. 1985. Science. 198:149.
110. Mage,M., McHugh,M. and T.Rothstein. Mouse lymphocytes with and
without surface immunoglobulin: Preparative scale separation in polystyrene
tissue culture dishes coated with specifically purified anti-immunoglobulin.
1977. J. Immunol. Methods. 15:47.
111. Boitard,C., Chatenoud,L-M. and M.Debray-Sachs. In vitro inhibition of
lymphocytes from diabetics with associated
autoimmune diseases: a T cell phenomenon. 1982. J. Immunol. 129: 2529.
pancreatic B cell function by
112. Miller,R. Age-associated decline in precursor frequency for different T
cell-mediated reactions with preservation of helper or ctotoxic effect per
precursor cell. 1984. J. Immunol. 132:63.
113. Feng,H., Glasebrook,A., Engers,H. and J.Louis. Clonal analysis of T cell
unresponsiveness to alloantigens induced by neonatal injection of F, spleen cells
into parental mice. 1983. J. Immunol. 131:2165.
114. Kanagawa,O., Louis,A., Engers,H. and J-C.Cerottini. Effects of sub-lethal
150
whole-body irradiation on subsequent secondary cytolytic T cell responses in
vitro. 1983. J. Immunol. 130:24.
115. Buszello,H., Pottmeyer,C. and W. Droge. Frequencies of alloreactive and
self-restricted cytotoxic T cell precursors during lymphatic regeneration. 1983.
J. Immunol. 131:548.
116. Andersson,J. and F.Melchers. 1974. Maturation of mitogen- activated bone
marrow-derived lymphocytes in the abscence of proliferation. 1974. Eur. J.
Immunol. 4:533.
117. Melchers,F., Andersson,J., Lernhardt,W. and M. Schreir. H-2 unrestricted
polyclonal maturation without replication of small B cells induced by
antigen-activated T cell help factors. 1980. Eur. J. Immunol. 10:679.
118. Grayson,J., Dooley,N., Koski,I. and R.Blaese. Immunoglobulin production
induced in vitro by glucocorticoid hormones. T cell-dependent stimulation of
immunoglobulin production without B cell proliferation in cultures of human
peripheral blood cells. 1981. J. Clin. Invest. 68:1539.
119. Miller,N., Bly,J., van Ginkel,F., Ellsaesser,C, and L.W. Clem. Phylogeny
of lymphocyte heterogeneity: Identification and separation of functionally
distinct subpopulations of channel catfish lymphocytes with monoclonal
antibodies. 1987. Dev. Comp. Immunol. 11:739.
120. Caspi,R. and R.Avtalion. The mixed leukocyte reaction (MLR) in carp:
Bidirectional and unidirectional MLR responses. 1984. Dev. Comp. Immunol.
8:631.
121. Jayaraman,S., Mohan,R. and VR. Muthukkaruppan. Relationship between
migration inhibition and plaque-forming cell response to sheep erythrocytes in
the teleost Tilapia mossambica. 1979. Dev. Comp. Immunol. 3:67.
functions of
122. Munck.A., Guyre,P. and N.Holbrook. Physiological
glucocorticoids in stress and their relation to pharmacological actions. 1984.
Endocr. Rev. 5:25.
123. Gabrielsen,A. and R.Good. Chemical suppression of adaptive immunity.
1967. Adv. Immunol. 6:91.
124. Schlesinger,M. and V.K. Golakai. Loss of thymic-distinctive serological
characteristics in mice under certain conditions. 1967. Science. 155: 1114.
and Claman,H. Thymus-marrow immunocompetence. V.
Hydrocortisone-resistant cells and processes in the hemolytic antibody response
of mice. 1971. J. Exp. Med. 133:1026.
125.
Cohen,J.
151
126. Weissman.G. and L.Thomas. Studies on lysosomes. H. The effect of
cortisone on the release of acid hydrolases from a large granule fraction of
rabbit liver induced by a excess of vitamin A. 1963. J. Clin. Invest. 42:661.
127. Zurrier,R. and G. Weissman. Anti-immunologic and anti-inflammatory
effects of steroid therapy. 1973. Med. Clin. North america. 57:1295.
128. Libertini,L., Waggoner,A. and P.Jost. Orientation of lipid spin labels in
lecithin multilayers. 1969. Proc. Natl. Acad. Sci. 64:13.
129. Howard,M., Farrar,J.,
Johnson,B.,Takatsu K., Hamaoka,T. and
W.Paul. Identification of a T cell-derived B cell growth factor distinct from
interleukin 2. 1982. J. Exp. Med. 155:914.
130. Okada,M., Sakaguchi,N. and T.Kishimoto. B Cell Growth Factor (BCGF)
and B Cell Differentiation Factor (BCDF) from human T hybridoma: Two
distinct kinds of BCGFs and their synergism in B cell proliferation. 1983. J.
Exp. Med. 157:583.
131. Yoshizaki,K., Nakagawa,T. and T.Kishimoto. Characterization of human B
Cell Growth Factor (BCGF) from cloned T cells or mitogen-stimulated T cells.
1983. J. Immunol. 130:1241.
132. DeLuca,D., Wilson,M. and G.Warr. Lymphodyte heterogeneity in the trout,
Salmo gairdneri, defined with monoclonal antibodies to IgM. 1983. Eur. J.
Immunol. 17:971.
133. Jentz.P., Chou,C., Szymanska,I., Alcala,R., Dubiski,S., and B.Cinader.
Immunoglobulin synthesis by thymus B cells. 1982. Immunol. 38:275.
134. Rosenstreich,D., Farrar,J. and S.Dougherty. Absolute
macrophage
dependency of T lymphocyte activation by mitogens. 1976. J. Immunol. 116:131.
135. Coutinho,A., Grorowicz,W., Bullock,W. and G.Moller. Mechanism of
thymus-independent immunocyte triggering mitogenic activation of B cells
results in specific immune responses. 1974. J. Exp. Med. 139:74.
136. Thomas,D. Discontinuous antibody secretion during the secondary response
to sheep erythrocytes in vitro. 1974. J. Immunol. 112:1602.
137. Yui,M. and S.L.Kaattari. Vibrio anguillarum antigen stimulates mitogenesis
and polyclonal activation of salmonid lymphocytes. 1987. Dev. Comp. Immunol.
11:539.
138. Cunningham,A.J. and A.Szenberg. Further improvements in 'the plaque
technique for detecting single antibody- forming cells. 1968. Immunol. 14:599.
152
139. Roess,D., Bellone,M., Ruth,M., Nadel,E.M. and E.Ruh. The effect of
glucocorticoids on mitogen-stimulated B lymphocytes: Thymidine incorporation
and antibody secretion. 1982. Endocrinol. 110:169.
140. Katsura,Y., Kina,T., Amagi,T., Tsubata,T., Hirayoshi K., Takaoki,Y.,
Sado,T. and S-C.Nishikawa. Limiting dilution analysis of the stem cells for T
cell lineage. 1986. J. Immunol. 137:2434.
141. Letvin,N.L., Benacerraf,B. and R.N.Germain. B lymphocyte responses to
trinitrophenyl-conjugated ficoll: Requirement for T lymphocytes and Ia-bearing
adherent cells. 1981. Proc. Natl. Acad. Sci. 78:5113.
142. Endres,R.O., Kappler,J.W., Marrack,P. and S.C.Kinsky. A requirement for
nonspecific T cell factors in antibody responses to "T cell independent" antigens.
1983. J. Immunol. 130:781.
143. Lee,K-C. and M.Wong. Functional heterogeneity of culture-grown bone
marrow-derived macrophages. II. Lymphokine stimulation of antigen-presenting
function. 1982. J. Immunol. 128:2487.
144. Pike,B.L. and G.J.V.Nossal. A reapprasial of "T-independent" antigens. I.
Effect of lymphokines on the
response of single adult hapten-specific B
lymphocytes. 1984. J. Immunol. 132:1687.
145. Morrissey,P.J., Boswell,H., Scher,I. and A.Singer. Role of accessory cells
in B cell activation. IV. Ia+
accessory cells are required for the in vitro
generation of thymic independent type 2 antibody responses to polysaccharide
antigens. 1984. J. Immunol. 127:1345.
146. Secombes,C.J., Van Groningen,J.J.M. and E.Ehberts.
Separation of
lymphocyte subpopulations in carp, Cyprinus carpio, by monoclonal antibodies:
Immuno- histochemical studies. 1983. Immunol. 48:165.
classes of
Freitas,A.A., Rocha,B. and A.Coutinho. Two major
mitogen-reactive B lymphocytes defined by lifespan. 1986. J. Immunol. 136:466.
147.
148. Sakai,D.K. Separation of lymphocytes from the peripheral blood of rainbow
trout and goldfish. 1981. Bull. Jap. Soc. Sci. Fish. 47:1281.
149. Smith,K.A., Crabtree,G.R., Kennedy,S.J. and A.Munck. Glucocorticoid
receptors and sensitivity of mitogen stimulated and unstimulated lymphocytes.
1977. J. Biol. Chem. 252:2972.
150. Mishell,R.I. and R.W.Dutton. Immunization of dissociated
cultures from normal mice. 1967. J. Exp. Med. 126:423.
spleen cell
151. Rittenburg,M. and K.Pratt. Anti-trinitrophenyl (TNP) plaque assay. Primary
153
response of BALB/c mice to soluable and particulate immunogen. 1969. Proc.
Soc. Biol. Med. 132:575.
152. Sinha,A.A., Guidos,C., Lee,K. and E.Diener. Functions of accessory cells
in B cell responses to thymus-independent antigens. 1987. J. Immunol. 138:4143.
153. Watkins,D. and N.Cohen. The phylogeny of interleukin 2.
1985. Dev.
Comp. Immunol. 9:819.
154. O'Garra,A., S.Umland,T.DeFrance and J.Christiansen. "B cell factors" are
pleiotropic. 1988. Immunol. Today. 9:45.
155. Luster,M.I., D.R.Germolec, G.Clark, G.Wiegand and G.J. Rosenthal.
Selective effects of 2,3,7,8-tetrachlorodi- benzo-p-dioxin and corticosteroid on
in vitro lymphocyte maturation. 1988. J. Immunol. 140:928.
156. Vandenabeele,P., B.Jayarum, R.Devos, A.Shaw and W.Fliers. Interleukin
la acts as an autocrine growth
factor for RPMI 1788, an Epstein-Barr
virus-transformed human B cell line. 1988. Eur. J. Immunol. 18:1027.
APPENDIX
154
APPENDIX
Cacodylate Buffer
Cacodylate buffer (0.28 M) was prepared by dissolving
acid in 1 L of distilled water.
the
38.2 g of cacodylate
Anhydrous sodium hydroxide (0.95 g) was added to
solution and the pH adjusted to 7.2, with 2 N HC1. The buffer was stored at
17°C for no longer than 3 months.
Modified Barbital Buffer (MBB)
A 5x preparation of MBB was made by dissolving 1 vial (0.05 moles sodium
barbital, 0.01 moles barbital) of
barbital buffer (Sigma, St. Louis, MO) in 1 L of
distilled water at ambient temperature. This solution was then
supplemented with
anhydrous calcium chloride (0.083 g/L) and magnesium chloride (0.508 g/L). The 5x
preparation was stored at 17°C for no longer than 6 months. MBB (lx) was prepared
by dilution of the 5x preparation with saline (0.85%), and the pH adjusted to 7.4
with 1 N HC1. Fresh lx MBB was prepared biweekly.
155
Phosphate Buffered Saline (PBS)
Phosphate buffered saline (0.7 M) was prepared by dissolving monobasic
potassium phosphate (KII2PO4,
(Na2HPO4,7 H2O, 17.8
1
g/L) and dibasic sodium phosphate-7 hydrate
g/L) in one liter of distilled water. To this solution sodium
chloride (NaC1, 8.5 g/L) were added, and the pH adjusted to 7.4 with 1 N HC1.
Alsever's Solution
Alsever's solution was prepared by dissolving 20.5 g dextrose, 8.0 g sodium
citrate dihydrate
(Na3C61-1507,2H20), 0.55 g citric acid monohydrate (113C,1150 and
4.2 g sodium chloride (NaC1) in 1 L of double-distilled water. The pH of the
solution was adjusted to 7.1 using 1 N NaOH, and
autoclaved at 250°C for 15
minutes. Upon cooling, an aliquot was aseptically removed, and mixed 1:1 with
sheep red blood cells (SRBC). SRBCs were stored at 17°C for no
longer than
three weeks.
Stock Cocktail
For the preparation of stock feeding cocktail, 10 ml of 200 mM L-glutamine,
10m1 of 200 mg/ml dextrose, 10 ml of 100x nonessential amino acids (M.A.
156
Bioproducts) and 20 ml of 50x essential amino acids (M.A. Bioproducts) are mixed
with 140 ml of RPMI 1640, and the pH adjusted to 7.2 using 10 N NaOH. 0.1%
gentamicin sulfate (Sigma) is added to the
solution,
and the mixture
is
filter-sterilized (0.44 u, Millipore, Bedford, MA), aliquoted in 15 ml tubes
(Corning), and stored at -10°C for no longer than 6 months.
Feeding Cocktail
Feeding cocktail is prepared by completely thawing and mixing one 15 ml
stock cocktail aliquot with 7.5 ml of fetal bovine serum, plus 1 ml of a 10 ug/ml
suspension of
adenosine, uracil, cytosine and guanosine (all from Sigma)
dissolved in RPMI 1640.
Binding Buffer
For the preparation of binding buffer, 31.4 g of premixed buffer solids (Biorad)
were reconstituted in 100 ml of distilled water, under constant stirring.
The
mixture was filtered, and the pH adjusted to 9.0+0.2 using 10 N NaOH.
Reconstituted binding buffer was held at 4°C for no longer than 3 months.
157
Elution Buffer
Elution buffer was prepared by dissolving 2.2 g of premixed solids (Biorad) in
100 ml of distilled water. The mixture was stirred for 10 minutes, filtered, and the
pH adjusted to 3.0+0.2 with 6N HC1. All reconstituted elution buffer was held at
4°C for no longer than 3 months.