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Endogenous regulatory factors in the inhibition and down-regulation of immune responses

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Endogenous regulatory factors in the inhibition and down-regulation of immune responses

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ENDOGENOUS REGULATORY FACTORS IN THE
INHIBITION AND DOWN-REGULATION OF IMMUNE
RESPONSES
by
Vidyalakshmi.A.Ganapathy
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(PHARMACEUTICAL SCIENCES)
August 2001
Copyright 2001 Vidyalakshmi.A.Ganapathy
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UNIVERSITY OF SOUTHERN CALIFORNIA
THE GRADUATE SCHOOL
UNIVERSITY PARK
LOS ANOELES. CALIFORNIA 90007
This dissertation, written by
VlD W L A K S m I • A- SAUAPATHy
under the direction of h.£X Dissertation
Committee, and approved by all its members,
has been presented to and accepted by The
Graduate School, in partial fulfillm ent of re
quirements for the degree of
DOCTOR OF PHILOSOPHY
Detn of Graduate Studies
Date
DISSERTATION COMMITTEE
O u i
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Vidyalakshmi.A.Ganapathy Dr. Sarah Hamm-Alvarez
ABSTRACT
ENDOGENOUS REGULATORY FACTORS IN THE INHIBITION
AND DOWN-REGULATION OF IMMUNE RESPONSES
Immune responses, like all biological responses, are controlled by a balance
of positive and negative signals. One of the mechanisms used by the immune system
to maintain homeostasis is inhibition and down-regulation of immune response
signals. In the immune system, T cells are turned off to prevent recognition of
self-antigens and to terminate responses to foreign antigens. This negative regulation
is mediated by various endogenous factors that include cytokines such as
transforming growth factor-P (TGF-P) and mediators of inflammation such as
prostaglandin-Ei (PGEi). The focus of this dissertation research has been to
understand the regulatory effects of PGE2 and TGF-P in immune responses.
PGEi is a proinflammatory mediator and has been shown to modulate
immune responses both in vitro and in vivo. PGE2 mediated effects on CD4+ T cells
have been extensively studied. However, there is no evidence regarding the effects of
PGEt in T cell receptor mediated regulation of CD8 + T cell responses. I was able to
demonstrate the inhibitory role of PGE2 on IFN-y release from CD8 + T cells. In
addition, I identified the receptor subtypes involved in the PGE2-mediated inhibition
(EP2 and EP4) and the factors that counteract or diminish the inhibitory effect (IL-2).
TGF-P is an immunoregulatory cytokine the deficiency of which is known to
be associated with immune dysregulation. TGF-P, like PGE2, is known for its ability
I
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to regulate the function and interaction of immune ceils in the development of both
humoral and cell-mediated immunity. Hence, my focus was to understand the
ligand-receptor interactions of TGF-p that lead to the inhibitory responses. I
performed structural studies using peptides derived from the TGF-P i sequence to
determine the contact sites between the ligand and the receptor. In addition, I
conducted kinetic analysis of ligand binding and found that a short period (20-30
minutes) o f exposure of the ligand was sufficient for signaling. I found the peptides
to have no inhibitory effect on signaling. Nevertheless, the results from our kinetic
study provided us with a useful model to study the kinetics of ligand-receptor
assembly.
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ACKNOWLEDGEMENTS
I would like to express my deep gratitude to my advisor, Dr. Hermann von
Grafenstein, for his guidance, understanding and support during the entire course
of my graduate studies and research. In addition, I would like to thank my
committee chair Dr. Sarah Hamm-Alvarez for her guidance during the completion
of this dissertation. I would also like to thank the rest of my committee members,
Dr. Ian Haworth, Dr. Minnie McMillan and Dr. Curtis Okamoto for their valuable
time in providing advice and helpful comments for the completion of this
dissertation. My sincere thanks also are due to Dr.Tatyana Gurlo for her patience
and understanding in guiding me through the course of my research. Finally, yet
most importantly, my sincere thanks go to my husband and daughter for their
support that made it possible for me to accomplish my goals.
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TABLE OF CONTENTS
ACKNOWLEDGMENTS............................................................................................ii
LIST OF FIGURES...................................................................................................... vii
LIST OF TABLES.........................................................................................................ix
LIST OF ABBREVIATIONS...................................................................................... x
CHAPTER ONE INTRODUCTION...................................................................... 1
1.1. The immune system-An overview.........................................................2
1.1.1. Components of the immune system........................................2
1.1.2. Selection of T cell repertoire and tolerance
induction....................................................................................................4
1.1.3. T cell antigen recognition and activation............................... 6
1.1.4. Cytokines...................................................................................7
1.2. Signal integration in T cells....................................................................8
1.2.1. Positive regulatory factors........................................................ 10
1.2.2. Negative regulatory factors...................................................... 1 1
1.3 Prostaglandin-Ei....................................................................................... 1 1
1.3.1. COX isoforms............................................................................13
1.3.2. PGE2 receptor isoforms.............................................................14
1.3.2.1. Structural homology of prostaglandin
receptors..............................................................14
1.3.2.2. Signal transduction pathways...............................15
1.3.2.3. Distribution of EP receptors.................................15
1.3.3. PGEt in immunity..................................................................... 16
1.3.3.1. PGE2 in humoral immune responses................... 16
1.3.3.2. PGEi in cell-mediated immune
responses.............................................................17
1.3.3.3. PGE2 in lymphocyte development...................... 17
1.3.3.4. Induction of an erg y ...............................................18
1.4. Transforming growth factor-P (TGF-P)...............................................19
1.4.1. Expression and regulation of TGF-P.......................................20
1.4.2. Biological actions of TGF-P....................................................20
1.4.3. TGF-P in immunity...................................................................20
1.4.3.1. Regulation of T cell development and
effector function ............................................... 2 1
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1.4.3.2. Regulation of B cell and dendritic
functions ............................................................ 2 1
1.4.3.3. Regulation of accessory cell functions............... 23
1.4.3.4. TGF-P in autoimmune diseases ..........................23
1.4.4. TGF-P receptor isoforms........................................................24
1.4.4.1. Structural features of TGF-P receptors .............. 24
1.4.4.2. Receptor-mediated TGF-P signal
transduction .......................................................25
1.5. T cell receptor signal transduction pathways.........................................25
1.5.1. PLC-y 1 mediated TCR signaling pathway............................27
1.5.2. Downstream signaling events regulating cytokine
gene expression.........................................................................................27
1.5.3. Function of cAMP in T cell receptor signaling
pathway......................................................................................................29
1.6 . Significance of PGE2 and TGF-P in type 1 diabetes mellitus............. 31
1.6.1. Type 1 diabetes mellitus, TID M ............................................. 31
1.6.2. Significance of PGE2 in TID M ............................................... 32
1.6.3. Evidence for role of TGF-P in TID M .....................................35
1.7.......................... Scope of the dissertation.................................................. 35
CHAPTER TWO EFFECT OF PGE2 ON IFN-y RELEASE FROM ISLET-
REACTIVE NOD CD8 + T
CELLS.......................................................................................38
2.1. Introduction............................................................................................... 39
2.2. Materials and Methods............................................................................. 46
2.2.1. Antibodies and reagents........................................................... 46
2.2.2. M ice...........................................................................................47
2.2.3. Islet reactive polyclonal CD 8 + T cells and clones................ 47
2.2.4. Stimulation of IFN-y release from T cells..............................48
2.2.5. Assay of IFN-y by ELISA........................................................49
2.3. Results........................................................................................................50
2.3.1. Dose-response study of IFN-y release from islet
reactive CD8 + T cell clones..................................................................50
2.3.2 Time-course of TCR-dependent EFN-y release by
islet reactive CD8 + clones...................................................................50
2.3.3 Inhibition of TCR-dependent IFN-y release by PGE2...........52
2.4. Discussion................................................................................................. 55
CHAPTER THREE ROLE OF PGE2 RECEPTOR SUBTYPES IN THE
DOWN-REGULATION OF CD 8 + T CELL
RESPONSES......................................................................... 58
3.1. Introduction............................................................................................... 59
3.2. Materials and Methods............................................................................ 6 6
3.2.1. Antibodies and reagents.......................................................... 6 6
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3.2.2. Agonists and Antagonists........................................................6 6
3.2.3. Stimulation of IFN-y release and IFN-y ELISA................... 6 6
3.2.4. Measurement of intracellular free calcium in P815
cells........................................................................................................67
3.2.5. Measurement of uterine contractions.................................... 67
3.2.6. Curve fitting and statistical analysis..................................... 6 8
3.3. Results........................................................................................................70
3.3.1. EP2 receptors mediate the inhibitory effect of PGE2
on IFN-y release....................................................................... 70
3.3.2. Role of EP 1/EP3 receptors in PGE2 mediated inhibition of
IFN-y release.............................................................................72
3.3.3. Contribution of EP4 receptors to the inhibitory effect of
PGE2 ..........................................................................................75
3.3.4. Analogs of cAMP mimic he inhibitory effect of PGE2 on
IFN-y release.............................................................................77
3.4. Discussion................................................................................................. 79
CHAPTER FOUR ROLE OF ACCESSORY SIGNALS IN
MEDIATING THE INHIBITORY EFFECT OF
PGE2 ON IFN-y RELEASE.................................................... 83
4.1. Introduction............................................................................................... 84
4.2. Material and methods...............................................................................87
4.2.1. Antibodies and reagents...........................................................87
4.2.2. Mice........................................................................................... 87
4.2.3. Preparation of polyclonal CD8 + T cells.................................. 87
4.2.4. Flow cytometry analysis to assess the purity of
polyclonal CD 8 + T cells................................................................. 8 8
4.2.5. Stimulation of IFN-y and IFN-y ELISA ................................. 8 8
4.3. Results........................................................................................................90
4.3.1. Effects o f T cell activation state on sensitivity of
cells to PGE2 .............................................................................90
4.3.2. Accessory signals: IL-2 and IL-12 have contrasting
effects on PGE2 -mediated inhibition of IFN-y release 93
4.3.3. The sensitivity of PGE2 depends on T cell receptor signal
strength..................................................................................... 97
4.4. Discussion................................................................................................. 99
CHAPTER FIVE TGF-P LIGAND-RECEPTOR INTERACTIONS..................102
5.1. Introduction................................................................................................103
5.2. Materials and methods............................................................................. 108
5.2.1. Peptides and reagents............................................................... 108
5.2.2. Mink lung epithelial cells........................................................ 109
5.2.3. Peptide inhibition studies......................................................... 109
5.2.4. Kinetic studies.......................................................................... 110
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5.2.5. Luciferase Assay......................................................................110
5.3 Results........................................................................................................112
5.3.1. Inhibition of TGF-P signaling by peptides derived
from TGF-P i ligand sequence................................................. 112
5.3.2. TGF-P kinetic studies...............................................................114
5.3.3. Inhibition of TGF-P signaling by soluble TGF-P type II
receptor...................................................................................... 114
5.4. Discussion................................................................................................ 118
CHAPTER SEX SUMMARY AND PERSPECTIVES.........................................121
6.1. Summary.................................................................................................... 122
6.2. Future directions......................................................................................126
REFERENCES............................................................................................................... 128
v i
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LIST OF FIGURES
Figure l.l. Signal integration in T cells.................................................................. 9
Figure 1.2. Biosynthetic pathway of Prostaglandin-E2 ..........................................12
Figure 1.3. T cell receptor signal transduction.......................................................28
Figure 1.4. Proposed model for interdependence of PKC and PKA
signaling pathways..................................................................................30
Figure 1.5. Role of PGE2 in T 1DM-A proposed model........................................34
Figure 2 .1. PGE2 release from islets........................................................................ 41
Figure 2.2. Effects of PGE2 on T cells....................................................................43
Figure 2.3. Dose-response and time-course studies EFN-y release.......................51
Figure 2.4. Inhibition of IFN-y release by PGE2 ....................................................53
Figure 3.1. Activation of protein kinase-A............................................................. 63
Figure 3.2 Effect of PGE2 agonists on TCR-dependent IFN-y release
from cloned CD8 + T c ells......................................................................7 1
Figure 3.3. Effect of EP 1/EP3 agonist sulprostone on IFN-y release from
cloned CD8 + T cells............................................................................... 73
Figure 3.4. Effect of sulprostone on [C a 2+]j release from P815 cells ................. 73
Figure 3.5. Effect of sulprostone on uterine tissue contractions......................... 74
Figure 3.6. Dose-effect analysis of PGE2 effect on IFN-y release from
cloned CDS^T cell, 8 D8 .........................................................................76
Figure 3.7. Effect of cAMP analogs on TCR-dependent IFN-y release
from cloned CD8 + T c ells..................................................................... 78
Figure 3.8. Proposed model for the convergence of TCR-cAMP
pathways..................................................................................................82
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Figure 4 . 1 . Schematic representation of the time-line stimulation of
polyclonal CD8 + T cells.........................................................................91
Figure 4.2. Effect of activation state on sensitivity to PGE2 .................................92
Figure 4.3. Partial reversal of PGE2-mediated inhibition by IL-2........................94
Figure 4.4. Effect of IL-12 on PGE2-mediated inhibition of IFN-y
release from polyclonal CD8 + T cells.................................................. 96
Figure 4.5. Effect of signal strength on sensitivity of polyclonal CD8 + T
cells to PGE2 ........................................................................................... 98
Figure 4.6. Modulatory effects of PGE2 and IL-12 on IFN-y release
from polyclonal CD8 + T cells................................................................101
Figure 5.1. Proposed model for extracellular TGF-p signaling........................... 102
Figure 5.2. Inhibition of TGF-Pi induced signaling by peptides.......................... 113
Figure 5.3. Kinetic analysis of TGF-P receptor complex assembly.....................115
Figure 5.4. Inhibition of TGF-Pi induced signaling by soluble TGF-P
type II receptor.........................................................................................116
Figure 5.5. Inhibition of TGF-Pi induced signaling following pulsing of
M vlLu cells with the peptides............................................................. 117
viii
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LIST OF TABLES
Table 3.1. Signal transduction mode of EP receptors............................................ 59
Table 3.2. EP receptor subtype selective agonists..................................................65
Table 5.1. Design of peptides derived from the TGF-P sequence ...................... 108
IX
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LIST OF ABBREVIATIONS
Ab: Antibody
AC: Adenylate cyclase
APCs: Antigen presenting cells
cAMP: Cyclic adenosine monophosphate
con-A: Concanavalin-A
COX: Cyclooxygenase
DAG: Diacylglycerol
EAE: Experimental autoimmune encephalomyelitis
ELISA: Enzyme-linked immunosorbent assay
FBS:Fetal bovine serum
FcR: Fc receptor
FITC: Fluorescein isothiocyanate
GM-CSF: Granulocyte-macrophage colony-stimulating factor
HRP: Horseradish perodixase
IFN-y: Interferon gamma
IL: Interleukin
iNOS: inducible nitric oxide synthase
IP3: Inositol 1,4,5-trisphosphate
ITAM: Immunoreceptor tyrosine-based activation motifs
LPS: Lipopolysaccharide
mAb: Monoclonal antibody
MHC: Major histocompatibility complex
MvlLu cells: Mink lung epithelial cells
NF-kB: Nuclear factor kB
NF-AT: Nuclear factor of activated T cells
NK cells: natural killer cells
NOD: Non-obese diabetic
NSAID: Non-steroidal anti-inflammatory drug
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OPD: O-phenylenediamine
PDE: Phosphodiesterases
PGE2: Prostaglandin-E:
PI: Phosphotidyl inositol
PIP2: phosphatidylinositol 4,5 bisphosphate
PKA: Protein kinase A
PKC: Protein kinase C
PLC-y 1: Phosphoiipase C-y 1
PMA: Phorbol myristyl acetate
TCM: Tissue culture medium
TCR: T cell receptor
TIDM: Type 1 diabetes mellitus
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CHAPTER 1
Introduction
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1.1. The immune system-An overview
The immune system evolved in response to the need of the body to defend
itself against invading pathogens. The process by which the immune system creates
protection against infectious pathogens is called immunity. Immunity can be broadly
classified into innate or natural immunity and adaptive or acquired immunity. Innate
immunity involves components that exist at birth. The principal components of the
innate immune system are physical and chemical barriers such as epithelia, as well as
blood proteins, and phagocytic cells such as neutrophils and natural killer cells.
Adaptive immunity develops as the immune system adapts to specifically defend
against the pathogens that escape the innate immune system. The characteristics of
the adaptive immune system are its excellent specificity for distinct molecules and its
ability to remember and respond to repeated exposure of the same microbe. The
elements of adaptive immunity are lymphocytes and their products such as
antibodies (Abbas K. A., 1997). Infectious pathogens can enter the host through
various entry points. Hence, the immune system is not confined to a particular organ
in the body. In fact, the cells that comprise the immune system are distributed all
over the body.
1.1.1. Components of the immune system
The immune system is a complex network of specialized cells and organs.
The cells of the immune system are mostly present as circulating cells in the blood
and lymph or as stationary cells in body tissues. All the blood cells originate from
pluripotent hematopoietic stem cells that later become committed to differentiate
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along a particular lineage, namely erythroid, granulocytic, monocytic and
lymphocytic. The cells responsible for immune specificity of the adaptive immune
system belong to a class of white blood cells called lymphocytes. However,
non-lymphoid cells or accessory cells, such as mononuclear phagocytes, dendritic
cells and several other cell populations also play a role in the induction of immune
responses. The two major classes of immune responses, namely humoral and
cell-mediated immune responses are mediated by different classes of lymphocytes.
B-lymphocytes, which in mammals develop in the adult bone marrow (the name “B”
cells referring to bone marrow-derived), develop into antibody producing cells. T
lymphocytes, whose precursors arise in the bone marrow and then migrate to and
mature in the thymus (the name “T” cells referring to thymus-derived) are
responsible for cell-mediated immunity (von Boehmer, 1988).
There are two main classes of T cells: helper T cells or CD4+ T cells and
cytotoxic T cells or CD8+ T cells. Helper and cytotoxic T cells recognize peptide
antigens that are non-covalently bound to major histocompatability (MHC) gene
products and expressed on the surface of antigen-presenting cells. Peptides derived
from cytosolic proteins are bound to class I MHC molecules and are recognized by
CD8 + T cells. Cytotoxic T cells lyse cells that are infected by viruses and other
intracellular microbes. Peptides derived from extracellular proteins are endocytosed
by specialized cell types in the immune system, termed antigen-presenting cells.
These extracellular peptides bind to class II MHC molecules and are recognized by
CD4+ T cells. In response to antigenic stimulation, helper T cells act on other cells
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promoting the proliferation, differentiation and activation B cells, macrophages and
T cell subsets.
The mononuclear phagocyte system is a non-lymphoid component of the
immune system. Like all cells of the immune system, mononuclear phagocytes
originate in the bone marrow and differentiate to various distinct cell types. These
are monocytes when they arc in peripheral blood and macrophages once they reach
and settle in tissues.
Additional components of the non-lymphoid immune system are the dendritic
cells and granulocytes. The dendritic cells play a role in mediating adaptive immune
responses and granulocytes play a role in mediating innate immune responses.
1.1.2. Selection of T cell repertoire and Tolerance Induction
In healthy individuals, immune responses usually lead to the destruction and
elimination of invading pathogens. It is crucial that the immune responses be
directed against molecules foreign to the host and not to those of the host itself. The
ability to distinguish between self and non-self is one of the fundamental features of
the immune system. The innate immune system is inherently self-tolerant.
Immunological self-tolerance of the adaptive immune system is acquired either
during the development of T and B cells in central lymphoid organs or in the
periphery. Failure to make this self-nonself distinction leads to destructive immune
responses against one's own molecules leading to autoimmune diseases.
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The T cell repertoire is the total set of T cell specificities in an individual.
The mature T cell repertoire is largely self-tolerant. Most of the immunocompetent
cells arise in the bone marrow. T-cells migrate to and mature in the thymus.
Interestingly, more than 95% of T cells that migrate to the thymus die before they
mature. This is due to a stringent selection process that operates on developing cells,
involving positive and negative selection of T cells (Benoist and Mathis, 1992; von
Boehmer, 1992). Positive selection is thought to be due to weak recognition of
self-peptide-MHC complexes presented by thymic epithelial cells (Jameson et al.,
1995). Negative selection is most likely due to a strong interaction of peptide-MHC
complexes with the antigen receptor of developing T cells. The inability to mount
immune responses against self-antigens as a result of negative selection in the
thymus is called central tolerance. Central tolerance occurs principally by clonal
deletion, a process of activation-induced cell death (Nossal, 1994; Sebzda et al.,
1999).
The deletion of self-reactive T cells in the thymus cannot delete all
potentially self-reactive T cells, as some self-molecules are not presented in the
thymus. Peripheral tolerance is the mechanism by which self-reactive T cells in the
periphery are rendered tolerant. Principal mechanisms of peripheral tolerance are
clonal deletion and functional inactivation without cell death, termed clonal anergy.
Even in the absence of these types of peripheral tolerance, self-antigens may be
ignored by potentially self-reactive T or B cells. Clonal ignorance or lack of response
to self-reactive cells is another way by which tolerance could be achieved. If these T
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cells become activated, their activity can be down regulated by a variety of
mechanisms (Miller and Morahan, 1992).
Autoimmunity results from the failure or breakdown of mechanisms that
normally maintain self-tolerance (Crispe, 1988). Loss of self-tolerance could be a
result of abnormalities in the selection or regulation of self-reactive T cells. It could
also result from abnormalities in the way self-antigens are presented. Autoimmunity
could result from failure of the selection processes leading to a failure in central
tolerance (Nossal, 1994). However, not much is known regarding this hypothesis.
Alternatively, peripheral tolerance may take over once central tolerance fails. There
is indeed evidence supporting the idea that autoimmunity results from the failure of
peripheral tolerance. In addition, conditions that lead to breakdown of T cell anergy
could lead to autoimmunity. Conditions such as infections, tissue necrosis and local
inflammation that activate tissue antigen presenting cells may break T cell tolerance
by enhancing the expression of costimulators and the production of cytokines (Miller
and Morahan, 1992). This may result in T cell proliferation and differentiation of T
cells into tissue-injurious pro-inflammatory effectors leading to autoimmune
reactions against the tissue.
1.1.3. T cell antigen recognition and activation
A general property for activation of lymphocytes is the need for two distinct
signals. The first signal is provided by the interaction of the T cell receptor with the
peptide-MHC complex. The second signal is provided by the interaction of specific
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costimulatory molecules on accessory cells with specific receptors on T cells
(Isakov, 1988).
T cell antigen recognition leads to several biological responses:
1. Proliferation of T cells occurs primarily through an autocrine growth pathway.
The principal autocrine growth factor for most T ceils is interleukin (IL-2 and
IL-4). The result of the proliferative response is clonal expansion, which
generates enough antigen-specific T cells to handle the invading pathogen.
2. Differentiation is the process that leads to the development of effector cells.
3. Effector functions made possible by the above two sequences of events are the
various biological functions that contribute to successful immune responses. The
major effector function of CD4+ T cells is the secretion of cytokines and that of
CD8 + T cells is lysis of virus infected cells (Favero and Lafont, 1998).
1.1.4. Cytokines
T cell activation leads to the release of protein products called cytokines.
Cytokines are soluble factors and have multiple effects on target cells. Cytokine
secretion is usually rapid and the effect could be either autocrine as in the case of
interleukin-2 (“IL” referring to Interleukin), IL-4, etc. or paracrine as in the case of
interferon-y (IFN-y), IL-5, etc. Of primary interest in this dissertation is IFN-y.
IFN-y is produced by activated CD4+ and CD8 + T cells and NK cells. IFN-y
(a) provides the means by which T cells activate macrophages, (b) induces the
synthesis of iNOS (inducible nitric oxide synthase), (c) increases class I MHC
molecule expression, (d) promotes functional differentiation of naive CD4+ T cells,
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(e) promotes maturation of CD8 + cytolytic cells (Ijzermans and Marquet, 1989;
Pawelec et al., 1989).
1.2. Signal integration in T cells
Immune responses, like all biological responses, are controlled by a balance
of positive and negative signals. Activation and subsequent differentiation of T cells
is mediated by endogenous positive regulatory factors that include IL-2, CD28-B7
interactions etc. One of the mechanisms used by the immune system to maintain
homeostasis is inhibition and down-regulation of immune response signals. In the
immune system, T cells are turned off to prevent recognition of self-antigens and to
terminate responses to foreign antigens (Long, 1999). This negative regulation could
be mediated by various endogenous factors that include cytokines such as
transforming growth factor-P (TGF-P) and mediators of inflammation such as
prostaglandin-Ei (PGEi). Positive and negative regulatory signals can interact in
complex ways. There is a hierarchy of signaling interactions and unraveling the
hierarchies of this signaling network is essential for understanding immune
regulation and autoimmunity.
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Signal I
IL-2
T Cell
CD28-B7
Activation Anergy/
Cell death
PGE2
TGF-P
Figure 1.1. Signal Integration in T cells
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1.2.1. Positive regulatory factors
In addition to the antigen itself, a number of endogenous factors contribute to
T cell activation resulting in cytokine production, cytokine receptor expression,
proliferation and ultimately to the activation of effector function of activated cells.
The activation of T cells could be a result of direct cell to cell contact or of the
release of soluble mediators (Fitch et al., 1993) that act on cells following interaction
with specific receptors on the cell surface. During the course of this research the role
of two such soluble factors were examined.
IL-2, originally termed T cell growth factor, is an autocrine/paracrine growth
factor. IL-2 is produced mostly by CD4+ T cells and in lesser quantities by CD8+ T
cells. Some of the paracrine functions of IL-2 include: (a) stimulation of cytokines
such as IFN-y, (b) rescue from antigen-specific T cell anergy; when inadequate
amounts of IL-2 is produced anergy is induced, (c) stimulation of antibody synthesis
from B cells (Gomez et al., 1998; Higuchi et al., 1997).
IL-12 is a mediator of immune -mediated inflammation. IL-12 is primarily
synthesized by activated mononuclear phagocytes and dendritic cells. IL-12 is a: (a)
potent inducer of EFN-y production by T cells as well as NK cells, (b) promotes
differentiation of CD4+ T cells into IFN-y producing Thi cells, (c) enhances cytolytic
functions of activated NK cells and CD8+ T cells (Adorini, 1999; Gately et al.,
1998).
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1.2.2. Negative regulatory factors
Growth inhibition, anergy, apoptosis and down-modulation of receptor
signals are inhibitory tools employed by the immune system to maintain homeostasis
and prevent injurious side effects of lymphocyte activation (Long, 1999). In addition,
negative regulatory signals may down-modulate unwanted effector functions in
certain instances like cytokine release and activation of cytolysis. Negative
regulation is mediated by various pathways and molecules in the immune system. Of
interest in this dissertation are prostaglandin-E2 (PGE2 ) and transforming growth
factor-p (TGF-P).
1.3. Prostaglandin-E2 (PGE2 )
Prostaglandins are cyclooxygenase products derived from C-20 unsaturated
fatty acids. They are classified based on their chemical structure into types A to I of
which types A, B and C are believed to be produced only during artificial extraction
procedures. The major steps of arachidonic acid metabolism are shown in Figure 1.2.
The metabolism involves hormone-induced release of arachidonic acid from
phosphoglyceride, conversion of the arachidonate to the prostaglandin endoperoxides
PGG2 and PGH2, and isomerization of PGH2 to thromboxanes (TX) or
prostaglandins (PG). Prostaglandins and thromboxanes thus formed are released to
the outside of the cells immediately after synthesis. Prostaglandins G, H, and I are
chemically unstable and are degraded into inactive products under physiological
conditions. PGE2 and PGD2 , though chemically stable, are metabolized quickly and
ll
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Arachidonic Acid
COOH
Cyclooxygenase
PGGi
O
COOH
— OH
p g h 2
PGE->
HO
O
COOH
PGI Synthase
TX Synthase
T X A
PGF
Synthase
PGD
Synthase
PGE
Synthase
COOH
Figure 1.2. Biosynthetic pathway of Prostaglandin-E:
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hence it is believed that they work locally, acting only near the site of their
production (DeWitt, 1991; Samuelsson, 1978). Of the prostaglandins, we are
primarily interested in Prostaglandin-E2, PGEj.
PGE2 is a lipid that is synthesized and released upon activation of a variety
of cells. Prostaglandin E2 mediates and regulates a number of physiological
processes. Furthermore, as PGE2 is usually produced at sites of inflammation it may
be an important regulator of inflammatory and immune responses.
1.3.1. COX isoforms
COX, Cyclooxygenase, (Prostaglandin G/H synthase; Prostaglandin
endoperoxide synthase) is the enzyme that converts membrane phospholipid derived
arachidonic acid (AA), to prostaglandins (PGs). Two isoforms of COX exist. COX-1
is the constitutive form, whose products are thought to play a role in modulating
physiological, basal activities in tissues in the absence of inflammation. COX-2, the
regulated form, is associated with inflammatory or stimulated events in tissues and
its activity is upregulated in response to specific inducers. COX-1 is present in most
tissues and is selectively localized in the endoplasmic reticulum. Prostaglandin
synthesis by COX-1 occurs minutes after exposure to appropriate stimuli (e.g.: free
arachidonic acid). Synthesis by COX-2 is delayed by several hours due to the
requirement for de novo COX-2 mRNA and protein synthesis. Under normal
physiological conditions, COX-2 is usually present in low concentrations and is
found primarily in the nuclear envelope (Goetzl et al., 1995). COX-2, by virtue of its
nuclear localization, may produce prostanoids that act at the level of the nucleus in
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cellular differentiation, replication and growth. In pathological conditions such as
inflammation, COX-2 is responsible for high levels of prostanoid production. COX-2
induction is mediated by factors such as T lymphocyte derived cytokines and
activated macrophages. Studies by Sorli et al. demonstrated the dominant basal
expression of COX-2 in Syrian hamster and human pancreatic islets (Sorli et al.,
1998).
1.3.2. PGE2 receptor isoforms
PGE2 produces a broad range of pharmacological effects in different tissues
through binding to cell surface receptors on the plasma membrane. The diversity of
PGE2 effects in the body is attributed to the molecular diversity of the various EP
receptors mediating these responses. Four subtypes of pharmacologically active
PGE2 receptors differing in their mode of signal transduction have been
characterized.
1.3.2.1. Structural homology of prostaglandin receptors
PGE2 receptors, EP1-EP4, belong to the family of seven transmembrane
domain G-protein coupled receptors. Sequence homology studies revealed them
G-protein coupled rhodopsin-type receptors. Despite the presence of conserved
sequences the overall homology among the EP receptors is quite limited, ranging
from -20 to 30% (Narumiya, 1994).
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1.3.2.2. Signal transduction pathways
The four different EP receptors are coupled to different intracellular
signaling pathways. EP| receptors activate phosphatidyl inositol (PI) turnover and
Ca2 + release via a poorly characterized G-protein mediated mechanism. The increase
in free calcium is dependent on the availability of extracellular Ca2 + and is
accompanied by a barely detectable PI response. EP2/EP4 receptor activation is
mediated by a stimulatory G-protein (Gs) and leads to an increase in intracellular
cAMP production. EP3 receptor couples to an inhibitory G-protein (Gi) and inhibits
adenylate cyclase (Narumiya, 1997; Negishi et al., 1993). There have been a few
reports of the coupling of EP3 to other signaling pathways. However, EP3 coupling to
Gi is described as the major pathway (Negishi et al., 1995). Namba et al.
characterized four isoforms of EP3 by alternative splicing of EP3 mRNA in bovine
adrenal medulla. The four isoforms (EP3A , EP3B , EP3C , and EP3D ) couple to different
signaling pathways. EP3A couples to Gi, EP3b and EP3C activate Gs, EP3D couples to
Gi, Gs, and Gq (another member of the G-protein family) to evoke a pertussis
toxin-insensitive PI response (Namba et al., 1993).
1.3.2.3. Distribution of EP receptors
Among the four EP receptors, EP3 and EP» are widely distributed throughout
the body. In situ hybridization studies show that EP3 receptors are widely distributed
throughout the central nervous system. EP3 and EP4 receptor expression is also found
in kidney, stomach and uterus. The expression of EP| receptor is restricted to the
lungs, kidney and stomach. The EPi receptor is the least abundant among the EP
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receptors. However, EP: receptor expression is effectively induced in response to
stimuli. For example, EP: receptor expression was upregulated in response to LPS in
a macrophage cell line and this increase was completely inhibited by the addition of
IFN-y (Negishi et al., 1995).
1.3.3. PGE: in immunity
PGE: has multiple functions in regulating normal immune responses. One
such function is the feedback inhibition of cellular immune responses. Antigen
presenting cells (APCs) such as macrophages and follicular dendritic cells are among
the major producers o f PGE:. PGE: regulates both humoral and cellular immune
responses and plays a role in lymphocyte development. PGE: can also facilitate the
induction of anergy in T cells (Roper and Phipps, 1994).
1.3.3.1. PGE2 in Humoral Immune Responses
B-lymphocytes do not express either of the COX isoforms. The cells of the B
lymphocyte microenvironment (mostly APCs) produce PGEs constitutively and
increase production in response to stimuli such as IL-1, tumor necrosis
factor-a (TNF-a) and lipopolysacharide (LPS). PGE: modulates humoral responses
at multiple levels. PGE: inhibits activation of cell enlargement, class II expression
and IgM production in mature B cells. On the other hand, PGE: enhances
differentiation of mature B cells and synthesis of IgGi, IgG^, and IgE at various
stages of B-cell differentiation (Fedyk et al., 1996). PGE: enhances immunoglobulin
16
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(Ig) synthesis via stimulating IL-4 mediated- Ig class switching (Roper and Phipps,
1994).
1.3.3.2. PGE2 in cell-mediated Immune Responses
PGEt modulates adaptive immune responses by inhibiting T cell activation
events including IL-2R gene expression. One of the most common mechanisms by
which PGE2 mediated responses occur is through the synthesis of cAMP and binding
of the intracellular second messenger cAMP to its intracellular receptor protein
kinase A (PKA). Upon binding to one of its G-protein coupled receptors PGE2
activates adenylate cyclase, which then activates downstream signaling events
leading to cAMP synthesis and activation of protein kinase A (PKA) (Goetzl et al.,
1995). This kinase-mediated activation has been shown to lead to inhibition of IL-2R
expression, IL-2 production, and T cell proliferation (Paliogianni et al., 1993). The
effects of PGEt in T cell responses will be addressed in greater detail in Chapter 2 of
this thesis.
1.3.3.3. PGE2 M lymphocyte development
Prostaglandins of the E-series, in particular, PGE2 have been implicated to
play a role in lymphocyte differentiation. Katamura et al. have studied the role of
PGE2 in the acquisition of cytokine-producing ability of naive CD4+ T cells. Their
results suggest that PGE2, present at the time of primary stimulation or at the time of
priming of naive T cells, facilitates the differentiation of naive CD4+ T cells into Th2
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cells in vitro. Furthermore, these naive CD4+ T cells seem to maintain the Th2
phenotype once acquired (Katamura et al., 1995).
Deletion of autoreactive double positive thymocytes occurs by apoptosis in
the thymus. This process of negative selection in the thymus is required for the
maintenance of self-tolerance. Lee et al. observed that exogenous corticosteroids and
cAMP inhibited activation-induced apoptosis of thymocytes (Lee et al., 1993). This
suggested a possible role of endogenous prostaglandin mediators in the regulation of
thymocytes. Goetzl et al. described cAMP mediated inhibition of activation-induced
apoptosis of human double positive (CD4+ CD8+ ) thymocytes by PGE2. Either the
EP2 or the EP4 receptor subtype could mediate this cAMP mediated inhibition.
Goetzl et al. also found that thromboxane A2 (a COX metabolite) enhanced
activation-induced apoptosis of double positive thymocytes (Goetzl et al., 1995).
Thus, negative and possibly positive selection is maintained by the ratio of
prostaglandins and other endogenous mediators present during thymocyte
development.
1.3.3.4. Induction of Anergy
Anergy is marked by a long-lasting blockade o f IL-2 production. PGE2, as
described in the previous section, has the ability to inhibit IL-2 production and hence
may induce anergy of effector T cells. In autoimmune encephalomyelitis, Mannie et
al. have reported that PGE2 may promote disease remission by inducing anergy in
helper T cells. These studies show that the presence of PGE2 during activation elicits
long lasting anergy in myelin basic protein specific T cells. Anergy was reversed by
1 8
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continued exposure to IL-2 of PGE2 treated cells. It has been proposed that PGE2
may induce anergy by inhibition of costimulatory pathways and converting an
inflammatory site into an anergy-inducing microenvironment. Signal transduction
pathways activated by PGE2 may act in concert with the antigenic signals to express
anergy-inducing genes. This might be explained by the positive coupling of PGE2
receptors to Gs protein and the subsequent increase in intracellular cAMP (Mannie et
al., 1995). From the results of this study, the authors conclude that CD4+ T cell
mediated recruitment of macrophages into inflammatory sites initiates a negative
feedback loop by which inflammatory products such as prostaglandins anergize the
primary inducers of macrophages i.e. CD4+ T cells.
1.4. Transforming growth factor-beta (TGF-p)
The transforming growth factor-P family of proteins are a set of pleiotropic
secreted signaling molecules with unique and potent immunoregulatory properties.
The biologically active forms of TGF-P related factors are disulfide-linked
homo-dimers containing subunits of 110-140 amino acids. All members of the
TGF-P family show sequence similarity to the prototype, TGF-P 1. The degree of
identity between the mature TGF-P sequences ranges from 64%-82% (Massague,
1990).
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1.4.1. Expression and regulation of TGF-p
TGF-P is ubiquitously expressed by many cell types and the expression is
tightly regulated throughout embryonic development into adulthood. Numerous cell
types can respond to TGF-P and its related polypeptides, and TGF-P expression and
activity are controlled by three mechanisms. The control of activity is achieved by (i)
regulation of TGF-P gene transcription, (ii) conversion of the latent form of TGF-p
to its active form, and (iii) sequestration of the activated molecule by the
extracellular matrix and circulating proteins. TGF-Pi is released from cells as part of
an inactive complex, which upon exposure to extreme pH, heat or glycosidases
releases the active factor (Massague, 1990; Yinglinget al., 1995).
1.4.2. Biological actions of TGF-P
TGF-P and related polypeptides regulate cell growth, differentiation, motility,
and death. The nature of a cell’s response to TGF-P is dictated by cell-specific
factors. The members of the TGF-P family also have a unique and essential role in
regulating immune function.
1.4.3. TGF-P in immunity
TGF-P initiates a series of signaling events resulting in diverse cellular
responses. In the immune system, effects of TGF-P are, in part, a function of their
cellular state of differentiation and are often altered by the state of their activation
signals. Endogenous production of TGF-P occurs in B cells, monocytes and
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macrophages. In T cells, TGF-P production is thought to occur in a subpopulation of
activated cells.
1.4.3.1. Regulation of T cell development and effector function
TGF-P effects on T cells are similar to the effects of PGEi on CD4+ T cells
discussed in detail in Chapter 2. Exogenous TGF-P has been shown to inhibit IL-2
dependent proliferation and IFN-y release from T cells. In contrast, TGF-P has been
shown to have a growth enhancing effect on naive T cells. TGF-P has also been
shown to favor Thl differentiation in naive CD4+ T cells but in primed CD4+ T cells,
it inhibits secretion of a Th 1 cytokine profile. By virtue of its reciprocal relationship
with both IFN-y and IL-12, TGF-P exposure favors Th2 development. This
antagonistic relationship between these cytokines may be a factor in the induction of
peripheral tolerance (Letterio and Roberts, 1998).
Analogous to its role in CD4+ T cells, TGF-P plays a stimulatory role in the
generation of naive CD8+ T cells. TGF-P is reported to induce CD8 expression and
together with TNF-a favors the development of CD8+ T cells in the thymus. TGF-p
has been shown to inhibit IFN-y release from mature CD8+ T cells (Lee and Rich,
1991).
1.4.3.2. Regulation of B cell and dendritic cell functions
Activation of B cells leads to production of either latent or bioactive TGF-Pi
depending on the nature of the signal. As in other immune cell populations, the
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response to TGF-p in B cells is a consequence of their state of differentiation and the
activation signals involved. The autocrine induction of TGF-P in B cells likely serves
as an important regulatory feedback loop to limit the expansion of an activated
population of B cells. Studies in some B lymphoid malignancies have shown this
autocrine inhibitory loop to be intact and resulting in reduced proliferation of
malignant cells and slow rate of progression. In addition to its effect on B cell
proliferation, TGF-P also regulates the expression of cell surface molecules such as
IgM, IgD, IgA and the induction of MHC class II expression. TGF-P is known for its
ability to inhibit immunoglobulin synthesis and the secretion of all classes of Igs.
However, under certain conditions TGF-P has been shown to enhance production
and secretion of certain immunoglobulins. For example, in LPS-activated cultures
the addition of an anti-TGF-P blocking antibody led to a decrease in secretion of
IgGl, IgG2a, IgG3 and IgE, without any effect on IgM (Kehrl et al., 1986).
Dendritic cells function as antigen presenting cells in the activation of T cell
responses. In dendritic cell subpopulations TGF-P both regulates their development
and mediates their effects. TGF-P has been shown to play an important role, along
with other cytokines such as TNF-a, GM-CSF etc., in the differentiation of dendritic
cells. Studies that show the ability of TGF-P to inhibit antigen-induced rescue of
B-cells, suggests a specific yet undetermined role for TGF-P in dendritic cells. The
surface of follicular dendritic cells (a subpopulation of dendritic cells) is the site for
primary selection events in B cells that have undergone activation and clonal
expansion. Antigens trapped on the surface of follicular dendritic cells provide an
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effective survival signal for cells with high affinity surface membrane
immunoglobulin and TGF-P is known to interrupt this signal (Letterio and Roberts,
1998).
1.4.3.3. Regulation o f accessory cell functions
Monocytes and macrophages secrete TGF-P, which can also regulate a
spectrum of their activities ranging from chemotaxis to pathogen destruction. Effects
of TGF-P on the proliferation of macrophages can be either stimulatory or inhibitory
depending on mode of stimulation, a feature characteristic of all immune cells. The
most prominent effect of TGF-P on macrophages is its ability to limit the production
of iNOS. TGF-P is recognized as an important immunoregulator and mediator of
parasite escape mechanism in parasitic infections (Letterio and Roberts, 1998).
1.4.3.4. TGF-P in autoimmune diseases
TGF-P has been implicated in the pathogenesis of autoimmune diseases.
TGF-P null mice have been shown to develop autoimmune disease suggesting a role
for TGF-p in the maintenance of self-tolerance. Endogenous TGF-P has been shown
to have a suppressive effect on experimental allergic encephalomyelitis (EAE) and
collagen-induced arthritis (CIA) progression (Kuruvilla et al., 1991). A protective
role for TGF-P has been suggested in T1DM and will be discussed in Section 1.6.3.
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1.4.4. TGF-P receptor isoforms
TGF-P exerts its effect by binding to specific cell surface receptors on target
cells. Two glycoproteins (receptors I and II) of 53 and 70-85kDa respectively, were
found to be components of a multimeric receptor. The third isoform, is a membrane
proteoglycan, designated betaglycan that was formerly termed type III receptor
(Yingling et al., 1995).
1.4.4.1. Structural features of TGF-P receptors
The type I TGF-p receptor is a 50-60kDa glycoprotein and is expressed in
most mammalian cells and tissues. The type I receptor belongs to the transmembrane
serine/threonine kinase receptor family. The receptor contains a putative 22 amino
acid signal sequence, a 101 amino acid hydrophilic extracellular domain, a 23 amino
acid transmembrane domain, and a 355 amino acid intracellular domain. The
extracellular domain is cysteine rich and the cytoplasmic domain has been shown to
contain an intrinsic kinase activity (Attisano et al., 1994).
The type II receptor is a 75-85 kDa glycoprotein ubiquitously expressed in
most cells and tissues. The human type II receptor contains a signal sequence, a 136
residue hydrophilic extracellular domain, a single transmembrane domain and a 376
amino acid long intracellular domain. The extracellular domain, like that of the type I
receptor, is cysteine rich and the cytoplasmic domain has intrinsic kinase activity
(Attisano et al., 1994).
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The type III receptor, alternatively known as betaglycan, is the most abundant
TGF-P binding protein expressed on cell surface of many types of cells. It is a
280-330 kDa transmembrane protein that binds TGF-Pi, TGF-Pa, and TGF-p3 with
equal affinity. Unlike the type I and II receptors, the type III receptor lacks any
known signaling motifs in its cytoplasmic domain. However, the stringent
conservation of this domain among different species suggests that it serves an
important role in TGF-P signal transduction (Massague, 1990; Massague and Weis-
Garcia, 1996).
1.4.4.2. Receptor-mediated TGF-P signal transduction
Studies with chemically induced epithelial cell mutants selected for resistance
to TGF-P action show that the type I and II receptors are involved in signaling and
type III receptors regulate access of TGF-P to the signaling receptors. Studies have
shown that the type II receptor is critical for ligand binding. The type I and type II
receptor-ligand complex facilitates TGF-P mediated signaling (Wrana et al., 1994).
The TGF-P signaling cascade will be discussed in detail in Chapter 5.
1.5. T cell receptor signal transduction pathways
As discussed in Section 1.1.3, two signals are required for the activation of T
cells. The first signal is elicited by way of interaction of the T cell antigen receptor
(TCR) with a specific complex of a MHC molecule and an antigenic peptide
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complex. The second signal is not antigen specific and is generated by accessory
molecules present on the surface of antigen presenting cells (APCs).
The TCR/CD3 complex contains the variable TCR a and P subunits in a
noncovalent association with the invariant chains of the TCR complex: the TCR £
chains and the y, 8 and e chains of the CD3 antigen complex. Antigen specificity of
the T cell receptor is mediated by the a and p subunits. The capacity of the TCR to
transduce signals across the T cell membrane is mediated by the cytoplasmic
domains of the subunits of the CD3 complex and the C , chains (Clevers et al., 1988).
The earliest biochemical event subsequent to the ligation of TCR with the
MHC-peptide complex is the activation of protein tyrosine kinases (PTKs). There are
many different cellular substrates for the PTKs leading to different signaling
cascades (The TCR subunits, ZAP-70, phospholipase-y 1 are a few of these
substrates). Two PTKs found in association with the TCR/CD3 complex, namely, lek
and fyn catalyze the phosphorylation of IT AMs (immunoreceptor tyrosine-based
activation motifs). ITAMs are a part of the CD3 and £ chains of the TCR complex.
The tyrosine kinase ZAP-70 is activated subsequent to TCR engagement. ZAP-70
plays a critical role in the signaling cascade that is set in motion by the TCR
recognition of antigen. TCR-regulated tyrosine kinases induce inositol phospholipid
breakdown and activate Ras signaling pathways (Cantrell, 1996). A schematic
representation of the TCR signaling pathway is shown in Figure 1.3.
□
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1.5.1. PLC-y 1 mediated TCR signaling pathway
The PLC-y I signaling cascade was the first characterized TCR signaling
cascade. Activation of PLC-y 1 leads to the hydrolysis of phosphoinositol
biphosphate into inositol triphosphate (IP3) and diacylglycerols (DAG). DAG
activates protein kinase C (PKC), which participates in activation of mature T cells.
IP3 leads to an increase in intracellular calcium and to the activation of the
serine/threonine phosphatase calcineurin (Garcia et al., 1999). Activation of
calcineurin results in the dephosphorylation of cytoplasmic nuclear factor of
activated T cells (NF-AT) which triggers its translocation to the nucleus (Baksh and
Burakoff, 2000). The nuclear component of NF-AT then leads to the activation of
cytokine genes.
1.5.2. Downstream signaling events regulating cytokine gene expression
The inositol phospholipid pathway and the Ras pathway both stimulate the
expression of cytokine gene expression required for T cell effector function and
clonal expansion. This is accomplished mostly by the activation of transcription
factors that lead to cytokine gene expression. Several transcription factors play a role
in cytokine gene expression, namely, AP-1, NF-kB, Oct-1 and nuclear factor of
activated T cells (NF-AT). The nuclear component of NF-AT acts in synergy with
AP-1 to transactivate a variety of cytokine genes. AP-1 is a transcription factor
composed of two Fos monomers and Jun. Fos is activated by the Ras pathway and
Jun expression is induced by the Rac pathway (Cantrell, 1996).
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T C R /I T A M S C D 2 8
Lck
Lck/fyn
ZAP-70
Ras
PLCyl
Ras/Raf
MEK
MAPK/JNK/ERK
Pathway
Ras Pathway
PIP:
IP3 + DAG
Ca2
PKC
Calcineurin
NF-ATc
Nucleus J u n / F o s
NF-kB
NF-AT -4 API F-AT 4 ----------- API NF' kB
\ 1 /
Cytokine genes (e.g. IL-2)
Figure 1.3. T cell receptor signal transduction
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1.5.3. Function o f cAMP in T cell receptor signaling pathway
Intracellular elevation of cAMP leads to a subsequent activation of
cAMP-dependent protein kinase, protein kinase A (PKA). Thus, the ligation of
TCR/CD3 complex initiates not only stimulatory (such as NFAT-dependent
signaling pathways) but also inhibitory (such as PKA-dependent signaling pathways)
signal transduction pathways. PKA has been shown to abolish the translocation of
NFAT from cytoplasm to nucleus, which is essential for IL-2 gene expression
(Sheridan and Gardner, 1998; Tsuruta et al., 1995).
The mechanism for receptor-mediated activation of PKA involves a G protein (Gs)
interaction. However, in T cells the TCR/CD3 complex does not appear to interact
with a Gs protein to activate the PKA pathway during receptor-mediated T cell
activation. One of the proposed models, as shown in Figure 1.4. suggests that the
PI/Ca2 + /PKC and the cAMP/PKA pathways may interact in T cells. This preliminary
model suggests that the signals generated by the TCR/CD3 complex result in the
activation of PKC isozymes, as discussed in detail in Section 1.5.1. The PKC
isozymes then translocate to the plasma membrane. At the plasma membrane the
isozymes either phosphorylate a G protein subunit or the adenylate cyclase catalytic
subunit. Activation of adenylate cyclase leads to cAMP production (discussed in
detail in Chapter 3). The rapid occupancy of the nearby regulatory subunits of PKA-I
by cAMP would then activate PKA-I. PKA-I subsequently phosphorylates proteins
in the T cell plasma membrane and this may in part regulate the early events of T cell
activation (Laxminarayana and Kammer, 1996).
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CD3/TCR Complex
IL-l R CD4/8 CD45
PKA-I
[ ] ZAP
ATP cAMl
PLC-y 1
PKC
DAG
FIGURE 1.4. Proposed model for interdependence of PKC and
PKA signaling pathways
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1.6. Significance of PGE2 and TGF-P in Type 1 diabetes mellitus
1.6.1. Type 1 diabetes mellitus, T1DM
T1DM or Type 1 Diabetes is an autoimmune disease characterized by T cell
mediated destruction of islet 3-cells. The Non-Obese Diabetic Mouse (NOD), is an
excellent animal model for the study of the pathogenesis of T1DM because it shares
many features with the human disease. In NOD mice and in humans, the etiology of
T1DM is considered to be multifactorial with disease development requiring the
interactions of a multitude of cells, which coordinate their function by means of
contact-dependent and soluble factors. Some of these factors, and the cells that
produce them, are thought to be disease promoting while others are considered
protective. T lymphocytes are known to be critical for the unfolding of the disease
but the role of particular T cell subsets is not clear. It has been suggested that CD8+ T
cells initiate the disease process (Wang et al., 1996) and CD4+ T cells are
predominant during the early stages of the disease and both CD8+ and CD4+ T cells
are required for maximum destruction of 3 cells (Atkinson and Maclaren, 1994).
Evidence from the NOD mouse model suggests a disease promoting role for
Thl cytokines such as IFN-y and a disease-protecting role for Th2 cytokines such as
IL-4 and systemic IL-10 and TGF-3 (Rabinovitch, 1998). During the disease
initiation process, CD8+ T cells are one o f the major sources of IFN-y (Gurlo et al.,
1999). IFN-y has been implicated to be a key cytokine in priming islet 3-cells for T
cell mediated destruction. Protocols that diminish IFN-y expression seem to protect
mice against developing T1DM further confirming the important role of IFN-y in
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progression of T1DM. Although the Thl-Th2 dichotomy may be an
oversimplification, an imbalance of positive and negative regulatory signals may
influence the development of autoimmune disease. It is therefore essential to identify
and characterize not only factors and mechanisms that promote disease, but those
that limit or delay anti-islet immunity and have the potential to protect against
disease development.
Among the numerous fundamental questions related to T1DM that remain to
be explored in the NOD mouse model is the following: What factor(s) is responsible
for the down-regulation of the autoimmune response, and what is the mechanism of
action?
1.6.2. Significance of PGE2 in T1DM
Autoimmunity results from failure of self-tolerance in T lymphocytes.
Development of autoimmunity is normally prevented by ( I ) the selection processes
operative during lymphocyte maturation that result in apoptosis of self-reactive
clones and by (2) mechanisms that maintain peripheral tolerance.
PGE: is a candidate protective factor because it leads to the down-regulation
of T cell-dependent immune responses. It has been proposed that one of the
functions of PGE: is to protect against immunity to self-antigens when released
during tissue injury and inflammation (Roper and Phipps, 1994) and PGE: is thought
to mediate some of the immunosuppressive effects of tumors. PGE: has also been
shown to inhibit negative selection of immature thymocytes by protecting them from
apoptosis. The same reasons may argue for a protective role of PGE: against
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spontaneous autoimmune disease, although this possibility has not been addressed in
detail.
Corbett et al. have shown cytokine-induced stimulation of PGE2 production
in rat and human islet of Langerhans. Dominant expression of COX-2 will lead to
increased PGE2 production in the local environment of the islets (Corbett et al.,
1996). The implications of the dominance of COX-2 in the islet on islet function and
pathogenesis of T1DM is not clear. It is possible that on the one hand dominance of
COX-2 and the subsequent cytokine-induced overproduction of PGE2 will inhibit
insulin release, worsening the developing hyperglycemia. On the other hand,
enhanced cytokine-induced PGE2 production by virtue of being able to inhibit insulin
secretion in P-cells may represent a mechanism by which PGE2 protects from
T1DM.
The evidence available from studies so far led us to propose a model for the
role of PGE2 in TIDM (Figure 1.5.). There exists a delicate balance between disease
promoting and disease-protective factors in Type I diabetes. One among the
numerous known disease-promoting factors are cytokines such as IFN-y that is
released by CD8+ and CD4+ T cells and IL-ip released from macrophages. Studies
have shown that IFN-y, in combination with IL-ip, can induce i-NOS synthase in rat
islets which intum can induce the expression of COX-2 (Corbett et al., 1996). It is
possible that COX-2 facilitates the conversion of arachidonic acid to PGE2 . PGE2, by
virtue of its ability to down-regulate cellular immune responses could be a candidate
protective factor in TIDM. Furthermore, it has been shown that rat and human islets
33
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can produce PGE2. The possibility that NOD mouse islets could be a source for
PGE2 in the islet microenvironment is an area yet to be explored.
CD8+ ,CD4+
Macrophages
Disease (T1DM)
Promoting factors
(IFN-Y, IL-lP)
Protecting factors
(PGE2)
i-NOS
Synthase
Arachidonic
acid
Figure 1.5. Role of PGE2 in TIDM- A proposed model
34
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1.6.3. Evidence for role of TGF-P in T1DM
TGF- P is a known immunosuppressive agent, which has been proven to be a
protective factor in various experimental autoimmune diseases. In vivo
administration of TGF-P was found to be protective in experimental autoimmune
encephalomyelitis, collagen-induced arthritis and in streptococcal ceil wall-induced
arthritis (Kuruvilla et al., 1991).
TGF-P, by virtue of its ability to inhibit the expression of IFN-y, could be a
potential disease protective factor in T1DM. Earlier studies done with pancreatic
beta cells of BB/Wor rats suggest that TGF-p can prevent the autoimmune
destruction of beta cells. Studies also show that adoptive transfer of TGF-pi
producing islet-reactive CD4+ T cells prevents diabetes in NOD mice (Han et al.,
1997). However, transgenic mice producing active TGF-P in their islets developed
insulitis suggesting immunoprotection may depend on the levels achieved locally or
systemically and/or on the state of TGF-P activation. Evidence also suggests that
TGF-Pi somatic gene therapy can prevent T1DM in NOD mice. Studies show that a
vector encoding mouse TGF-Pi cDNA that is expressed in skeletal muscle cells
reduced the incidence of diabetes in NOD mice (Piccirillo et al., 1998).
1.7. Scope of the dissertation
The ability to distinguish between self and non-self is one of the
fundamental features of the immune system. Failure to make this distinction leads to
detrimental immune responses against one's own molecules leading to autoimmune
35
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diseases. The focus of this research was to elucidate the role played by endogenous
negative regulatory factors in the inhibition and down-regulation of immune
responses with special emphasis on CD8 + T cells. The ultimate goal of this research
would be to understand the relevance of these factors in T1DM. The focus of the
research was on (a) The effects of PGE2 on CD8 + T cell responses and (b)
Ligand-receptor interactions that lead to TGF-P mediated CD8 + responses. The
research pertaining to this is discussed in four chapters.
1. What effect, if any, does PGE2 have on CD8 + T cell responses? (Chapter 2)
PGE2 mediated effects on CD4+ T cells have been extensively studied.
However, there is no evidence regarding the effects of PGE2 in T cell receptor
(TCR) mediated regulation of CD8 + T cell responses. The effects of PGE2 were
determined in NOD-derived clones and polyclonal CD8 + T cells. The effect of
PGE2 on IFN-y release subsequent to TCR stimulation in these cells was studied.
2. Four subtypes of EP receptors exist. Which of these subtypes mediate the effect
of PGE2 on IFN-y release? (Chapter 3)
The diversity of PGE2 effects in the body is attributed to the molecular
diversity of the various EP receptors mediating these responses. In order to
determine the role of one or more receptors in the PGE2-mediated effect, receptor
specific agonists were used and cells stimulated through the TCR in the presence of
these agonists. In addition, since three out of the four EP receptors signal by changes
in intracellular cAMP levels, cAMP analogs were used to further confirm the
findings.
36
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3. The role of accessory signals, if any, in altering the PGEj-mediated effect.
(Chapter 4)
T cells integrate multiple signals and modify their response to antigenic
stimulation, some of which may counteract the inhibitory effect of PGEi. The role of
two such signals IL-2 and IL-12 was studied in clones and/or polyclonal CD8 + T
cells to determine their role in the PGE2-mediated effect. In addition, signal
integration by T cells can occur by tuning o f signaling thresholds for antigenic
stimulation. The sensitivity of T cells to PGE2 over time for a period of 15 days after
stimulation was investigated. In addition, the TCR signal strength was varied at a
time when the cells were insensitive to PGE2 and the effect of signal strength on
PGE2-mediated effect studied.
4. Ligand-receptor interactions of TGF-p. (Chapter 5)
The effects of TGF-P on immune cells and CD8 + T cells have been
extensively studied and reviewed in numerous studies. However, an understanding of
the ligand-receptor interactions is still under investigation. In an effort to better
understand these interactions we designed peptides derived from both the TGF-P
sequence and from the type-II receptor sequence and performed peptide inhibition
studies. In addition, we studied the kinetics o f TGF-P receptor interactions to
facilitate the structural studies with the peptides.
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CHAPTER 2
Effect of PGE2 on IFN-y release from islet-reactive CD8+ T cells
38
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2.1. Introduction
Prostaglandin^ (PGE2) is an immunomodulatory agent produced primarily
by macrophages in the immune system. The ability of PGE2 to down-regulate T cell
mediated responses has been extensively studied (Roper and Phipps, 1994). One
example illustrating this ability is the influence of PGE2 on tumor growth. Recently,
NSAIDs such as aspirin have been reported to play a role in the inhibition of cancer
growth (Arber and DuBois, 1999; Baron and Sandler, 2000; Castelao et al., 2000).
Furthermore, it has been shown that the NSAIDs’ antitumor effects are a result of
their inhibition of cyclooxygenase-2 (COX-2) (Hida et al., 2000; Kokawa et al.,
2001; Ochiai et al., 1999; Simmons and Moore, 2000). This suggests that one of the
factors involved in tumor immunosurveillance could be PGE2. PGE2, by virtue of
being an immunosuppressive agent, could possibly protect the tumors from being
recognized by attacking immune cells. The same reasons may argue for a protective
role of PGE2 against spontaneous autoimmune disease, although this possibility has
not been addressed in detail. In experimental autoimmune encephalomyelitis (EAE),
an increase in PGE2 levels was found to be associated with disease remission
(Harbige et al., 2000). PGE2, could be a candidate protective factor in type 1 diabetes
mellitus (T1DM). However, the role of PGE2 in T1DM has not been studied
extensively.
During development of diabetes in the non-obese diabetic (NOD) mouse, the
islets of Langerhans become infiltrated with mononuclear cells. This phenomenon,
39
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also known as islet inflammation or insulitis, exhibits a large degree of heterogeneity
at any one time. Heavily inflamed islets and islets undergoing destruction co-exist
with seemingly healthy islets in the same pancreas (Atkinson and Maclaren, 1994;
Bach et al., 1997; Bach and Mathis, 1997). PGEi is an inflammatory mediator
produced at sites of inflammation and it is likely that inflamed NOD mice islets
produce this mediator. Studies by Sorli et al. demonstrated a dominant expression of
cyclooxygenase-2 (COX-2), both under basal and IL-1 stimulated conditions in
Syrian hamster and human pancreatic islets (Sorli et al., 1998). COX-2 is one of the
enzymes that catalyzes the conversion of arachidonic acid to PGE?. Cytokines such
as interleukin-la (IL-1) have been known to stimulate PGE? in rat islets
predominantly through the activation of COX-2 gene expression (Hughes et al.,
1989). Recent studies have suggested cytokine-induced synthesis of PGEt in the
local environment of islets in humans and in rats (Corbett et al., 1996). Studies were
conducted in our laboratory to determine whether NOD mouse islets produce PGE2.
Figure 2.1 shows that inflamed islets produce significant amounts of PGEi, in
contrast to islets that do not show signs of inflammation. PGEt production by these
islets was catalyzed by the inducible form of cyclooxygenase, namely COX-2, as it
was inhibited by NS-398, a specific inhibitor of this enzyme. Evidence from studies
thus far suggest that PGE2 might play diverse roles in regulating the islet beta-cell
destruction in T1DM. A more detailed understanding of PGE2’s multiple effects on T
cell subsets and their function is clearly necessary before an understanding of its role
can be reached.
40
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700 i
□ NOD
600 -
■ NODinf
500 ■
< 5
(A
Z 4 0 0 -
Q.
O)
O- 300 -
1 1 1
0
CL 200 -
100 -
0 -
d b E±]
LPS - +
NS-398
- -
+
+
Figure 2.1. PGEi release from islets. Islets from NOD mice without
any sign of infiltration (NOD) or severely infiltrated NOD islets (NODinf)
were cultured for 40 h in the presence or absence of LPS (10 |ig/ml) as
indicated. The specific COX-2 inhibitor NS-398 (10 |lM) was added as
indicated. Data are the mean + SEM of three measurements. These results
were part of Hilde O.Jarstadmarken’s Master’s thesis and are based on work
done in our laboratory.
41
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Most of the general information about the effect of PGE2 on T cell development and
function that is available supports a potential protective role of PGE2 in T1DM. The
effects of PGE2 on T cell functions is schematically represented in Figure 2.2.
Betz et al. showed that in murine Th clones, PGE2 inhibited the production of
IL-2 and IFN-y production whereas that of IL-4 was not inhibited and IL-5 synthesis
was slightly enhanced (Betz and Fox, 1991). The inability of PGE2 to inhibit IL-4
and IL-5 was not due to the cell’s unresponsiveness to PGE2, because it led to an
increase in cAMP production in Th2 cells. Furthermore, direct activation of
adenylate cyclase with froskolin failed to inhibit IL-4 and IL-5 production. A
possible explanation for this phenomenon could be that Th2 cells, by virtue of their
higher levels of constitutive cAMP than Thl cells, are more refractory to elevation of
cAMP. Alternatively, cAMP response elements (CRE) may enhance or
down-regulate cytokine production depending on the specific cytokine gene. In some
cells, an increase in cAMP can activate the transcription of certain cytokine genes.
The cAMP response element or CRE, that mediates this transcriptional activation, is
a short DNA sequence found in the regulatory region of genes that are activated by
cAMP. This sequence is recognized by a specific gene regulatory protein called
CRE-binding (CREB) protein. When CREB is phosphorylated by protein kinase-A it
is activated to turn on the transcription of these genes (Alberts B, 1994).
The development of a Thl or Th2 response may be determined in part by
APCs. Macrophages are one of the major sources of PGE2 . Macrophages, when
42
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CD44
Thl
Proliferation Th2
IFN-y,IL-2 IL-4, IL-5
PGE
CD84
IFN-y
Figure 2.2. Effects of PGE2 on T cells
43
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stimulated to produce PGE2, may favor the development of Th2 cells (Betz and Fox,
1991). Thus, an inflammatory process that leads to an increase in PGE2 might also
skew an immune response towards dominant production of Th2 associated cytokines.
Snijdewint et al. described the differential effect of PGE2 in human helper T cells.
The differential effects of PGE2 were correlated with intracellular cAMP levels in
CD4+ T cell clones (Snijdewint et al., 1993). Information about differential cytokine
modulation by PGE2 is very useful in understanding the immunopathogenesis of
allergic disorders where a predominant Th2 response is observed. Hilkens et al.
observed that PGE2 down-regulated IFN-y irrespective of the mode o f stimulation
(Hilkens et al., 1995). The fact that PGE2 can act at distinct signaling steps in the T
cell activation pathway has been studied extensively. Chouaib et al. demonstrated
that PGE2 could inhibit signaling at a late stage of T cell response (Chouaib et al.,
1985).
In contrast to the considerable amount of information that is available on the
influence of PGE2 on CD4+ T cell function, little is known as to how PGE2 affects
CD8 + T cells. In previous studies it has been shown that IL-2 driven proliferation of
CTLL-2 cells, a cell line derived from CD 8 + T cells, could be inhibited by PGE2
(Gurlo et al., 1998). However, the role of PGE2 in the regulation of TCR mediated
CD8 + T cell responses such as release of IFN-y is not known.
Earlier studies have proposed that CD8 + T cells are an important source of
IFN-y during the development of T1DM (Gurlo et al., 1999). Evidence from the
NOD mouse model suggests a disease-promoting role for Thl cytokines such as
44
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IFN-y and a disease-protecting role for Th2 cytokines such as IL-4 (Rabinovitch,
1998). During the disease initiation process, CD8 + T cells are considered to be one of
the major sources of IFN-y (Wang et al., 1996). IFN-y has been implicated to be an
essential cytokine in priming islet p-cells for T cell mediated destruction
(Rabinovitch et al., 1990; von Herrath and Oldstone, 1997). Protocols that diminish
IFN-y expression in islets seem to protect mice against developing T1DM further
confirming the essential role of IFN-y in progression of T1DM (von Herrath and
Oldstone, 1997).
In view of the data with the endogenous production of PGE2 by NOD
inflamed islets and evidence that suggest a possible protective role for PGE2 in CD4+
T cells, we address a critical question in this chapter. What effect, if any, does PGE2
have on CD8 + T cell immune responses, specifically IFN-y release? To address this
question we used NOD-derived CD8 + T cell clones and polyclonal CD8 + T cells and
determined the effect of PGEi on T cell receptor-dependent IFN-y release from both
the NOD-derived clones and polyclonal CD8 + T cells.
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2.2. Materials and Methods
2.2.1. Antibodies and Reagents
Prostaglandin-Ei (PGEi), avidin-HRP, o-phenylenediamine (OPD) and
murine rlFN-y were obtained from Sigma Chemical Co. (St. Louis, MD). Murine
rIL-7 was purchased from Life Technologies (Gaithersburg, MD).
TCX6310 cells were kindly provided by Dr. F. Melchers at the Basel Institute
for Immunology. Hybridomas H57-597 (anti-apTCR) was purchased from the
American Type Culture Collection (ATCC, Rockville, MD) and hybridoma
YCD3-1 (anti-CD3e) was a kind gift from Dr. C. A. Janeway, Jr. at Yale University.
Anti-aPTCR mAb was purified from hybridoma culture supernatant using
GammaBind Plus Sepharose (Pharmacia Biotech, Piscataway, NJ) columns.
Anti-CD3 mAb was used in the form of diluted hybridoma supernatant.
The tissue culture medium (TCM) used for cell culture and for all
experiments was based on Click's medium (Irvine Scientific, Santa Ana, CA) which
was supplemented with 4 mM L-glutamine, 100 U/ml penicillin, 100 pg/ml
streptomycin (Life Technologies, Gaithersburg, MD), 40 |J.M p-mercaptoethanol
(Sigma, St. Louis, MD) and 10% FBS (HyClone, Logan, UT). For routine T cell
culture, we used the supernatant of TCX6310 cells (Karasuyama and Melchers,
1988) as a source of IL-2.
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2.2.2. Mice
Non-obese diabetic (NOD) mice were obtained from The Jackson Laboratory
(Bar Harbor, ME) and were bred and maintained in the USC animal facility under
pathogen-free conditions. The spontaneous incidence of diabetes in our NOD colony
reaches 65-70% in female mice by 20 wk of age and diabetes usually commences by
13 weeks of age. For experiments, 8-12 week old mice were used.
2.2.3. Islet reactive polyclonal CD8+ T cells and clones
The islet reactive polyclonal CD 8 + T cells and clones used in this research
were generated by Dr. Tatyana Gurlo in our laboratory. The generation of
islet-reactive polyclonal CD8 + T cells and cloning of CD8 + T cells is described in
detail elsewhere (Gurlo et al., 1999). Briefly, spleen cells from newly diabetic female
NOD mice were cultured with NOD islets of Langerhans in the presence of IFN-y
(10 U/ml), IL-2 (10 U/ml) and IL-7 (10 ng/ml) for 5 days. T cell clusters surrounding
disintegrating islets were picked using a pipette and pooled. Cells were re-stimulated
with islets or the P-cell line NIT-1 (Hamaguchi et al., 1991) 4 times at 7-10 day
intervals. Polyclonal CD8 + T cells obtained this way potently destroyed islets and
were used for cloning. For cloning by limiting dilution, T cells were stimulated using
anti-CD3 mAb and mitomycin C-treated NOD spleen cells as described (Gurlo et al.,
1999). In this study, we used CD8 + T cell clone 8 D8 , which was one among the 18
clones obtained.
For routine maintenance, polyclonal CD8 + T cells and clones were stimulated
with anti-CD3 mAb every 3-4 weeks. T cells (l-5xl05) were cultured with
47
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mitomycin C-treated spleen cells (5xl06) in 5 ml of TCM in the presence of YCD3-1
(anti-CD3e mAb) cell culture supernatant diluted 1 in 20. After 48 h, T cells were
washed and treated with 40 U/ml IL-2 and 10 ng/ml IL-7. Two days later cells were
fed once more with the same medium, and after that every three to four days with
TCM alone or, during every other feeding cycle, with TCM supplemented with IL-2
and IL-7 (10 U/ml and 10 ng/ml respectively). In all experiments, T cell clones were
used fifteen days post stimulation if not otherwise indicated. Dead cells were
removed using LSM® (Lymphocyte Separation Medium, Organon Teknika Corp.,
Durham, NC).
2.2.4. Stimulation o f IFN-y release from T cells
Unless otherwise indicated, IFN-y release from T cells was stimulated using
anti-apTCR mAb H57-597 immobilized in tissue culture plates (Falcon, 96 well, flat
bottom plate coated with 1 (ig/ml mAb in PBS at 4°C overnight). Clonal T cells were
seeded in Ab coated plates at a density of lxlO4 cells per well and polyclonal CD8 +
T cells at 3xl04 cells per well. Immediately after seeding the cells, PGE2 was added
to the culture at the indicated concentrations. After 24 h, supernatant was collected
and stored at -80°C. PGEi was dissolved in DMSO and further dilutions were made
in TCM. The final concentration of DMSO in culture was less then 0.18% and at that
concentration did not have any influence on IFN-y release from T cells (data not
shown).
48
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2.2.5. Assay of IFN-y by ELISA
The concentration of IFN-y in the culture supernatant was measured by
sandwich ELISA using paired anti-cytokine mAbs (PharMingen, San Diego, CA),
following protocols recommended by the manufacturer. Primary mAb was
immobilized in Immulon-4 plates (Dynatech, Boston, MA) and incubated overnight
at 4°C. The following day the non-specific binding sites were blocked with
phosphate-buffered saline + 10% FBS (Hyclone, Logan, UT) for 2h. Subsequently,
samples and recombinant IFN-y were added and the plates incubated overnight at
4°C. The following day biotin-labeled anti-rat IFN-y (Pharmingen, USA) was added
and incubated for lh followed by incubation for 30 minutes with avidin-HRP
(Sigma, St.Louis, MD). Finally, the substrate, O-phenylenediamine, (Sigma,
St.Louis, MD) was added and color development quantified colorimetrically using a
microtiter plate reader. During the entire assay the plates were washed rigorously
with phosphate-buffered saline + .1% Tween-20, (Sigma, St.Louis, MD) as per
manufacturer’s instructions. The sensitivity of the assay was 1 U/ml (67 pg/ml).
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2.3. Results
2.3.1. Dose-response study of IFN-y release from islet-reactive C D S'*' T cell
clones
Before studying PGE2 effects on IFN-y release, it was important to determine
the time course and dose range of anti-TCR Ab for triggering IFN-y secretion from
the CD 8 + T cell clone 8 D8 . Two weeks after expansion with soluble anti-TCR Ab
and splenocytes, 8 D8 cells were stimulated with immobilized anti-TCR antibody in
the absence of splenocytes. To do this, 8 D8 cells were added to tissue culture plates
coated with varying concentrations of anti-TCR antibody. A concentration of
1 |ig/ml o f anti-TCR Ab (as shown in Figure 2.3A) in the coating buffer generated a
half-maximal response and we chose this concentration for studies of PGEo effects.
2.3.2. Time course of TCR-dependent IFN-y release by islet-reactive CD8+
clones
W e next performed time course studies of IFN-y release from the
TCR-activated clone 8 D8 . Two weeks after expansion with soluble anti-TCR Ab and
splenocytes, clones were stimulated with immobilized anti-TCR antibody in the
absence o f splenocytes. To do this, 8 D8 cells were added to tissue culture pates
coated with lflg/ml of anti-TCR antibody. After 24 h, IFN-y release began to level
off and had reached a level one half of that released by 96 h (as shown in Figure
2.3B). We chose this time interval to study the PGEi-dependent modulation of TCR
triggered IFN-y release.
50
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A
8000
_ 6000
” 4000
L L
2000
0 *
0 3 6
[Anti-apTCR], jjg/rrf
B
10000
8000
6000
4000
2000
0
24 0 48 72 9 6
Time, h
Figure 2.3. (A) IFN-y release from clone 8 D 8 in response to
immobilized anti-aPTCR Ab. Cloned CD8 + T cells were stimulated with the
indicated concentrations of Ab for 24 h.(B) Time-course o f TCR-dependent
IFN-y release from clone 8 D8 . Cloned T cells were stimulated with 1 (ig/ml
of immobilized anti-afJTCR Ab for the indicated periods o f time.
51
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2.3.3. Inhibition of TCR-dependent IFN-y release by PGEi
Throughout this study, the NOD CD8 + T cell clone 8 D8 was used which have
been described earlier in Section 2.2.3. These clones destroy islets in vitro and
release IFN-y in response to islets. PGE2 produced by inflamed NOD islets is likely
to affect the function of cells in the inflammatory infiltrate, including CD 8 + T cells.
The following experiments were conducted to investigate the effects of PGEi on
islet-reactive CD8 + T cells.
To determine the effect of PGEi on IFN-y release from cloned islet-specific
CD 8 + T cells, 8 D8 cells were activated with the half-maximal concentration of
immobilized anti-aPTCR antibody that had been determined in Section 2.3.1.
Various concentrations of PGE2 were added at the same time. The cells were
cultured for optimal IFN-y release for 24h as determined in Section 2.3.2. As shown
in Figure 2.4 A, PGE2 inhibited TCR-dependent IFN-y release in a dose-dependent
manner. PGE2 inhibited IFN-y release from a concentration of 10' 5 M to a
concentration of 10 10 M. At a concentration of approximately 2X10' 8 M a
half-maximal inhibition of IFN-y was achieved. PGE2 abolished the release of IFN-y
from 8 D8 cells at a concentration of 10 6 M.
PGE2 inhibited polyclonal CD 8 + T cells (Figure 2.4 B) with similar potency
as clone 8 D8 . The inhibition of IFN-y release was observed over a wide range of
PGE2 concentrations ranging from 10'9 M to 10' 5 M. The half-maximal inhibitory
concentration was between 10’8 M and 10' 9 M. At a concentration of 10 6 M the
52
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_ 150 -
.E
D
rp 100 -
z
UL
50 -
.E
D
T
z
Li-
10000
8000 * l
6000 - '
4000 -
2000 -
■ 9 8 •7 ■ 6 5
0
log1 0 ([PGE2 ]/M )
Figure 2.4. PGEi inhibits IFN-y release from clone 8D8 (A) and polyclonal
CD8+ T cells (B). T cells were stimulated with 1 pg/ml of immobilized anti-
aPTCR Ab in the presence o f indicated concentrations of PGE? for 24h.
53
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IFN-y release from polyclonal CD8+ T cells was abolished.
This indicates that this effect is a general characteristic of islet-reactive NOD
CD8 + T cells, and is not restricted to the clonal cell line used in this study.
Furthermore, the inhibitory effect of PGEi was not due to toxicity. This was
confirmed by determining the number of viable cells subsequent to the culture of
cells with PGEi. Trypan blue is a stain that diffuses into dead cells and hence marks
dead cells for microscopic determination. Earlier studies done in our laboratory have
shown that the number of trypan blue excluding cells do not decline, but rather
increase slightly, during culture with PGEi.
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2.4. Discussion
Our studies have shown that PGE2 inhibits IFN-y release from cloned NOD
CD8 + T cells and polyclonal CD8 + T cells. The results of our study were obtained
with islet-reactive CD8 + T cell clones and polyclonal CD8 + T cells isolated from
diabetic NOD mice which are as outlined in Chapter 1.
In NOD mice, the development of T1DM is characterized by a mononuclear
cell infiltrate in the pancreatic islets, which is followed by beta cell destruction.
Benign or non-destructive insulitis develops after weaning in all NOD mice. Onset of
insulitis in the islet perimeter is believed to be the initial phase of the disease. The
second phase is characterized by mononuclear invasion of the islet interior, islet
destruction and finally the appearance of the clinical signs of diabetes. It has been
suggested that the early benign infiltrate is dominated by IL-4 producing Th2 T cells
whereas the destructive phase is characterized by a dominance of Thl T cells
producing IL-2 and IFN-y (Rabinovitch, 1998).
PGEi, synthesized in islets, may be one of the factors that influences the
development and function of T cell subsets in the islet infiltrate. Earlier studies have
proven that PGE2 plays a role in the polarization of Thl-mediated immune responses
(Miao et al., 1996). Recently, a similar dichotomy of T cell subsets has been
described for CD8 + T cells and the subsets corresponding to Th 1 and Th2 cells have
been termed Tel and Tc2 cells (Croft et al., 1994). The polyclonal cell line and
clones used in our study, being potent producers of IFN-y, could be considered Tel
cells. The PGEi-mediated inhibitory effect described in this study would thus be
55
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analogous to the inhibitory effect of Thl cell functions in CD4+ T cells. Thus, in the
pancreatic islets PGE2 might act by inhibiting both Thl - and CD8 + -mediated effects.
As discussed in the introduction of this chapter, the data from our laboratory
show that large amounts of PGEi are produced in inflamed islets. Given the
inhibitory effect of PGEi on CD8 + T cells and the negative regulatory role of PGE2
in both CD8 + T cells and Thl cells, the question arises as to how disease can still
develop. Earlier studies have shown that PGE2-mediated effects can be overcome by
factors such as IL-2 (Rincon et al., 1988). Furthermore, it has also been shown that
effects of PGE2 on T cells depend on the mode of activation (Sunder-Plassmann et
al., 1991). Hence, it is likely that there are other accessory signals present in the islet
microenvironment that overcome or counteract the effects of PGE2. We will describe
the role of several such signals in detail in chapter 4.
The discussion above was based on studies performed with NOD-derived
clonal cells and polyclonal cell lines. The NOD mouse spontaneously develops
autoimmune disease and this could in part be due to abnormalities in the integration
of positive and negative regulatory signals. To address this question studies were
conducted in our laboratory to compare the influence of PGE2 on CD8 + T cells
derived from both NOD and BALB/c mice. The data from this study suggest that
PGE2 may be a less effective negative regulator of CD8 + T cells in NOD mice than
in BALB/c mice. The results of studies on the sensitivity differences to PGE2 in
different strains of mice were published subsequent to our findings. The data from
that study show that BALB/c mice were highly sensitive to the suppressive effects of
PGE2 on IFN-y release compared to their counterparts C57BL/6 mice. These results
56
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suggest that PGE2 plays an important role in polarizing Th2 responses in BALB/c
mice (Kuroda et al., 2000).
The therapeutic significance of the protective nature of PGEi in T 1DM would
have implications on the use of anti-inflammatory drugs. If PGE2 was indeed a
disease-protecting factor then the use of anti-inflammatory drugs in individuals
susceptible to T1DM could be detrimental. Many of the currently used non-steroidal
anti-inflammatory drugs block the increase of PGE2 synthesis during inflammation
and may prevent important immunomodulating actions of PGE2. Considering, the
negative regulation of CD4+ and CD8 + T cell function by PGE2, reducing PGE2
levels over a long period of time could lead to an imbalance of positive and negative
control of T cell function. This would lead to adverse effects associated with this
lack of control. However, further investigations are needed to give us a better
understanding of the effects of PGE2 in T1DM.
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CHAPTER3
Role of PGE2 receptor subtypes in the down-regulation of CD8+ T
cell responses
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3.1. Introduction
PGE2 mediates a broad range of physiological effects in different tissues. The
diversity of PGE2 effects can be attributed to the molecular diversity of receptors for
PGE2 . Four subtypes of pharmacologically active PGE2 receptors differing in their
mode of signal transduction have been characterized (Table 3.1.). All known PGE2
receptor subtypes, termed EP, through EPj, are coupled to intracellular signaling via
heterotrimeric GTP binding proteins (G-proteins). EP| receptors activate
phosphatidylinositol turnover and intracellular Ca2 + release via a poorly
characterized G-protein mediated mechanism. EP2 and EP4 receptors are coupled via
Gs protein to an increase in intracellular cAMP production (Narumiya, 1997;
Narumiya, 1994; Negishi et al., 1995; Negishi et al., 1993). EP3 receptors exist in
multiple forms that differ in their cytoplasmic tail that couple the receptor to
different intracellular signaling pathways, but have the same extracellular ligand
binding characteristics. EP3 receptors couple to either Gj or Gs or Gq depending on
the isoform expressed (Irie et al., 1993; Kotani et al., 1995; Negishi et al., 1995).
Receptor Subtype Signal Transduction Mode
EP, Ca2 + mobilization and increase in PI response
e p 2 Increase in cAMP
e p 3 Decrease in cAMP
e p 4 Increase in cAMP
Table 3.1. Signal transduction mode of EP receptors
59
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Fedyk et al. (Fedyk et al., 1996) attributed the varying effects of PGEi in B
cells to the varying receptor subtypes on B cells. Molecular analysis of PGE2
receptors on B cells revealed the presence of all four receptor subtypes. Based on this
study it was proposed that the EP2/EP4 receptors mediate inhibition of B cell
activation and PGE2-enhanced isotype switching since both inhibition of activation
and isotype switching require an increase in intracellular cAMP. On the other hand,
EPi and EP3 may serve to counterbalance the EP2/EP4 receptor effects and return B
cells to the resting state, maintaining homeostasis.
Most studies on intracellular signaling events following PGE2 receptor
activation have focused on EP2/EP4 receptors expressed by CD4+ T cells and events
downstream of receptor-induced cAMP production such as inhibition of IL-2 gene
expression (Paliogianni and Boumpas, 1996; Paliogianni et al., 1993). However,
little is known about the role of PGE2 in regulating IFN-y production in CD 8 + T cells
and any receptors and signaling mechanisms that might mediate such PGE2 induced
effects.
The four EP receptors exhibit different affinities for PGE2 and its analogs and
inhibitors (Coleman et al., 1994; Kiriyama et al., 1997). The intracellular signaling
events subsequent to ligand-binding dictate the PGE2-mediated responses in T cells.
Hence, a better understanding of the receptors involved in mediating the inhibition
by PGE2 is crucial.
One of the most important signal transduction pathways in TCR signaling is
the PI/Ca2 + /PKC pathway, as discussed in Chapter 1. Another signal transduction
60
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mechanism of relevance in T cell activation is the adenylate cyclase-cAMP pathway.
Activation o f adenylate cyclase in T cells is triggered by autocoids such as PGE2,
cholera toxin or treatment with agents such as froskolin (Alava et al., 1992). As
mentioned earlier, PGEi activates adenylate cyclase by binding to its EP2/EP4 type
surface receptors. Studies have shown that an increase in intracellular cAMP and
subsequent activation of PKA could be coupled to the PI/Ca27PKC pathway in T
cell receptor signaling (Laxminarayana and Kammer, 1996).
Cyclic adenosine monophosphate (cAMP), a major intracellular second
messenger, is synthesized from ATP by adenylate cyclase (AC). The G-proteins that
couple transmembrane receptors for various biological stimuli to adenylate cyclase
are heterotrimers and consist of a, (3 and y subunits. The a subunit of the
heterotrimeric complex varies depending on the nature of the G-protein. The O s
subunit is associated with the stimulatory Gs and oq is associated with the inhibitory
Gj. Ligand binding alters the receptor’s conformation, allowing it to associate with a
G protein. Interaction of the G protein with the activated receptor promotes the
exchange of GDP bound to the a subunit for GTP. Subsequently, the a subunit
dissociates from the (3y heterodimer. The Ga-GTP then binds to adenylate cyclase
and modulates its activity (Gs stimulates cAMP production and Gj inhibits cAMP
production). The cAMP thus produced binds to protein kinase A (PKA) resulting in
the activation of PKA. The binding of cAMP promotes the dissociation of the
holoenzyme releasing active catalytic (C) subunits and dimers of the regulatory (R)
subunits as shown schematically in Figure 3.1. (Alberts B, 1994). Different functions
6 1
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of cAMP are thought to be mediated by specific isozymes o f PKA. Two major types
of PKA have been described, namely PKA-I and PKA-II. The intracellular
localization of the two isoforms in T cells is distinct (Hasler et al., 1992). Studies
have shown that PKA-I is predominantly localized to the plasma membrane in
human T cells (Laxminarayana et al., 1993). In addition, association of the PKA-I
isozyme with the TCR/CD3 complex has been studied in resting and activated T
cells. The results from that study show that PKA-I colocalizes with the antigen
receptor in T cells and completely abolishes mitogenic responses in T cells
(Laxminarayana et al., 1993). Furthermore, it has been determined that the activation
of PKA-I but not PKA-II is sufficient to mediate the inhibitory effect of cAMP on T
cells (Skalhegg et al., 1992).
This observation together with the fact that triggering of the TCR/CD3
complex leads to cAMP production suggests that PKA activation represents a
negative feedback control mechanism. In support of this, impaired PKA-I activity
has been reported in T cells from patients with systemic lupus erythematosus,
possibly contributing to persistent immune activation in these patients by lack of
inhibition (Kammer et al., 1994). In addition to this, it has been demonstrated that
dysfunction of T cells isolated from patients with HIV (human immune deficiency
virus) and CVI (common variable immunodeficiency) could be reversed by addition
of PKA-I antagonists. Specifically, a combination of PKA-I selective antagonists and
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GTP
i
X blosGDPC
DP
r )
I f ^ G a ,.G T P ^ A C ^
AC: Adenylate cyclase
C and R: Catalytic and regulatory
subunits of protein kinase A
ATP cAMP
i
Figure 3.1. Activation of protein kinase-A.
63
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IL-2 normalize immune function of T cells from all patients (Aandahl et al., 1999;
Aandahl et al., 1998; Aukrust et al., 1999). This implies an important role of cAMP
in regulating antigen receptor induced proliferation and clonal expansion of
lymphoid cells and testifies to the role of PKA-I in mediating these effects.
The hydrolysis of cAMP to biologically inactive 5' monophosphate
nucleosides is catalyzed by cyclic nucleotide phosphodiesterases (PDEs). The level
of cAMP and corresponding cellular responses are regulated by both the rates of
cAMP synthesis by cyclases and cAMP degradation by PDEs. Seven different gene
families of PDEs have been isolated so far. The PDEs expressed by CD4+ and CD8 +
T cells have been identified as the PDE3, PDE4 and the PDE7 isoenzymes.
Furthermore, PDE7 is thought to be detected only in T cells suggesting a role for
PDE7 in T cell activation. It has been proposed that changes in cAMP modulated by
different PDE isozymes work at different phases of the T cell cycle (Li et al., 1999).
In this study we determined:
1. Which of the four EP receptors (EP|, EPi, EP3 or EP4) are involved in the
PGEi-mediated inhibition of IFN-y release?
2. What intracellular signals mediate the effects of PGEi?
To address the first question, we stimulated IFN-y release from the CD8 + T
cell clone 8 D8 in the presence of various PGE2 receptor subtype specific agonists,
such as misoprostol, 19-OH PGEi, and sulprostone as described in Table 3.2.
Misoprostol is an EP2/EP3/EP4 selective agonist. 19-OH PGE2 is an EP2 selective
agonist and sulprostone is an EP1/EP3 selective agonist.
64
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In order to determine the intracellular signaling pathways mediating the PGE2
effect, we used two cAMP analogs, 8 -Bromo-cAMP (8 -Br-cAMP) and Sp-adenosine
3’5’- monophosphothioate (Sp-cAMPS). 8 -Br-cAMP, is a membrane-permeable
cAMP analog having greater resistance to hydrolysis by phosphodiesterases than
cAMP. Sp-cAMPS is an analog of cAMP in which one of the two exocyclic oxygen
atoms bonded to the phosphate moiety is replaced by a sulfur. It is lipophilic,
resistant to the action of PDE and does not have a butyrate, a potential cell toxin, as
one of its metabolites (Yusta et al., 1988). It activates both PKA-I and PKA-II and
exhibits greater specificity than other analogs.
Agonist R eceptor Subtype Specificity
PGE, EP,, EP2, EP3, EP4
19-OH PG E2 e p 2
M isoprostol e p 2, e p 3, e p 4
Sulprostone EP,, EP3
Table 3.2. EP receptor subtype selective agonists
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3.2. Materials and Methods
3.2.1. Antibodies and reagents
Avidin-HRP, o-phenylenediamine (OPD), murine rIL-2 and murine rIFN-y
were obtained from Sigma Chemical Co. (St. Louis, MD). Murine rIL-7 was
purchased from Life Technologies (Gaithersburg, MD). Indo-l-AM was obtained
from Molecular Probes (Eugene, OR).
The tissue culture medium and anti-aPTCR antibody and anti-CD3e antibody
were obtained from sources mentioned in Chapter 2.
3.2.2. Agonists and Antagonists
Prostaglandin-Ei (PGEi) and 8 -bromo-cAMP were obtained from Sigma
Chemical Co. (St. Louis, MD). Misoprostol and sulprostone were obtained from
Cayman Chemicals (Ann Arbor, MI). Sp-cAMPS and l9(R)OH-PGEi were obtained
from BIOMOL (Plymouth Meeting, PA).
3.2.3. Stimulation of IFN-y release and IFN-y ELISA
IFN-y release from T cells was stimulated as described in Materials
and Methods in Chapter 2. Immediately after seeding the cells, PGE2, PGE2 agonists
or cAMP analogs were added to the culture at the concentrations indicated in figures.
After 24 h, supernatant was collected and stored at -80°C. PGE2, misoprostol and
sulprostone were dissolved in DMSO and dilutions were made in tissue culture
medium. The final concentration of DMSO in culture was less then 0.18% and at that
66
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concentration did not have any influence on IFN-y release from T cells (data not
shown).
The concentration of IFN-y in the culture supernatant was measured
by sandwich ELISA using paired anti-cytokine mAbs (PharMingen, San Diego, CA),
following protocols recommended by the manufacturer. Primary mAb was
immobilized in Immulon-4 plates (Dynatech, Boston, MA) by non-specific
adsorption from a solution of 2 pg/ml in buffer containing sodium di-phosphate
(pH:8.2). The sensitivity of the assay was 1 U/ml (67 pg/mi).
3.2.4. Measurement of intracellular free calcium in P815 cells
P815 cells, known to express EP3 receptors, coupled to an increase of
intracellular free calcium, were used as a control for the efficacy of the EP3 receptor
agonist sulprostone (Sugimoto et al., 1992). To load P815 cells with Ca indicator
dye, they were incubated with 3 mM of indo-I-AM for 30 minutes at 37°C. The cells
were then washed and kept in the form of a pellet at 4°C until assayed. The cell pellet
was resuspended in serum free tissue culture medium prior to the assay. Indo-l-AM
loaded cells (5xl06) were stimulated with PGEi, sulprostone (10‘6 M), or incubated
with digitonin to determine maximal intracellular free calcium. The fluorescence
intensity was recorded online with a fluorescence spectrophotometer.
3.2.5. Measurement of uterine contractions
Uterus contraction, known to respond to EP3 receptor stimulation was used as
an additional control for the biological activity o f EP3 receptor agonists. Uteri were
67
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excised from NOD-scid mice, cut in half, and mounted in a tension transducer. The
bath solution was Hank's Buffered Salt Solution, kept at 37°C and bubbled with 95%
O2 and 5% C 0 2. Various concentrations of PGE2 and sulprostone were added and
tension development was monitored.
3.2.6. Curve fitting and statistical analysis
Theoretical dose-effect curves were fitted to the data as described earlier.
Briefly, the following expression was used for the concentration-dependent effect of
PGE2:
Ii(L) =
ktL
1 + k,L
I2(L) = oil
kiL
1 + k ,L
+ (1-aO
k -> L
1 + k.L
r „ v kiL k2L k3 L
I3 (L) = a i ------------ + a 2 + a 3 a i+ a 2 + a 3 =l
1 + k|L 1 + k2L 1 + k3 L
In(L) = a ^ with = I
i= | 1 + kiL i = |
In (L) ranges from 1 (no inhibition) to 0 (full inhibition). L (ligand) refers to
the concentration of PGE2 , n is the number of theoretical binding sites, and kj are
68
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individual binding constants of these sites, otj is the relative contribution of site i to
the total inhibitory effect when it is fully occupied by ligand. The sum of all these
contributions is I (i.e. 100%). For fitting of In(L) to the experimental data, the sum of
square deviations of all data points from the theoretical curve was set to a minimum
by varying the binding constants kj, as well as otj. For each experiment a scaling
factor was used that allowed the data to be fitted to a curve ranging from 1 to 0 .
Statistical significance of a second or third binding constant was assessed by a
calculating the F-ratio and corresponding p-values due to the introduction of the
additional constant as compared to a simpler model without it (Maxwell and
Delaney, 1990). A p-value of 0.001 or below was considered to be significant.
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3.3. Results
3.3.1. EP2 receptors mediate the inhibitory efTect of PGE2 on IFN-y release
The EP2/EP3/EP4 receptor agonist misoprostol inhibited IFN-y release (Figure
3.2A). The inhibitory effect of misoprostol occurred within the concentration range
that would be expected from published binding constants for this agonist to EP2
(2.5xl0 ' 7 M) and EP4 (6.7xl0 ‘8 M) receptors (Coleman et al., 1994; Kiriyama et al.,
1997). To distinguish between EP2 and EP4 receptors we used 19(R)OH-PGE2 , an
agonist that is selective for EP2 receptors. Dose-effect curve for 19(R)OH-PGE2 ,
showed that it was an effective inhibitor (Figure 3.2B) and the K < i value was
comparable to a published value (1.5xl0‘8 M) for this agonist (Woodward et al.,
1993). These data provide definitive support for EP2 receptors being involved in the
effect of PGE2. The inhibition by EP2/EP3/EP4 receptor agonist misoprostol shows a
broader dose response than that of the EP2 receptor selective agonist. This suggests
that EP3 and EP4 receptors may also play a role in the inhibition by PGE2. To our
knowledge, there is no known EP4 selective agonist freely available. Hence, we used
an EP1/EP3 receptor specific agonist to determine the role of EP3 and a potential
involvement of EPi receptors.
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0 -10 -9 -8 -7 -6 -5
logi0 ([Misoprostol]/M)
® 4 0 0
300 -
.E
D
' 200 -
2
U_
100 -
o -10 -9 -8 -7 -6 -5
logio([19-OH PGEjJ/M)
Figure 3.2. Effect of PGEi agonists on TCR-dependent IFN-y
release from clone 8 D8 . Cloned T cells were stimulated with
immobilized anti-apTCR mAb (1 |ig/ml) in the presence of the
indicated concentrations of the EP2/EP3/EP4 agonist misoprostol (A),
and the EP2 agonist 19(R)OH-PGE2 (B). Data are the mean + SEM of
four experiments.
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3.3.2. Role of EPj/EPj receptors in PGE2 mediated inhibition of IFN-y release
As shown in Figure 3.3, sulprostone, an EP1/EP3 receptor agonist, was
ineffective up to a concentration of 10' 5 M. However, it is possible that sulprostone
was ineffective because it was inactive under the culture conditions used. In order to
exclude this possibility positive control experiments were performed using systems
that are known to respond to sulprostone.
As mentioned earlier, sulprostone is known to transduce signals through an
increase in intracellular calcium. The mastocytoma cell line, P815, known to express
EP3 receptors on their surface (Sugimoto et al., 1992) was used as a positive control
to check the efficacy of sulprostone. As shown in Figure 3.4. sulprostone increased
the intracellular free calcium concentration in P815 cells. PGEi elicited a higher
response than sulprostone, possibly because of its higher affinity to EP3 receptors.
Sulprostone has been shown to induce uterine smooth muscle contractions in
guinea pigs (Coleman et al., 1994). Hence, to verify that sulprostone was active
during these experiments, the agonist was cultured in the same medium and for the
same period of time as in experiments with CD8 + T cells. When removed after this
culture period, sulprostone potently induced contraction of uterine smooth muscle
excised from NOD mice as shown in Figure 3.5.
The data confirm that sulprostone was active and indicate that EP 1/EP3
receptor subtypes are not involved in the PGEi-mediated inhibition of IFN-y release.
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400 -i
^ 200 -
1 1 .
100 -
0 -10 -9 -8 -7 -6 -5
logio([Sulprostone]/M)
Figure 3.3. Effect of EP 1/EP3 agonist sulprostone on TCR-dependent
IFN-y release from clone 8D8. Cloned T cells were stimulated with
immobilized anti-apTCR mAb (1 (ig/ml) in the presence of the indicated
concentrations of sulprostone.
c
c
B
100 -
0 5
80 -
b
0 60 -
^ 0
40 -
20 -
0 -
n
PGEj 10'° 10 10
1 0 * , M < r
Sulprostone, M
B
3.20 -PGE2
• Sulprostone
Control
3.00 -
2.80
2.60
20 40 60 80
Time, Sec
100 120
Figure 3.4. Effect o f PGE2 and sulprostone on [C a2+]j release from P815
mastocytoma cells. A. Fluorescence intensity, in response to the addition of
PGEi and varying concentrations of sulprostone. B. PGE2 and sulprostone (10^
M) were added at the indicated time (as indicated with the arrow). A trace of an
average of 5 experiments for PGE2, three for sulprostone, and one of the control
is as shown.
73
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Figure 3.5. Effect of sulprostone, 10'6 M, on uterine tissue
contraction. A. Sulprostone was incubated at 37°C for 16 h to mimic
culture conditions and uterine muscle contraction was studied. B. Fresh
sulprostone was added at a concentration of 10~ 6 M and uterine muscle
contraction measured
74
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3.3.3. Contribution of EP4 receptors to the inhibitory effect of PGE;
The data in Section 3.3.1. show the definite involvement of the EP2 receptor
and suggest a possible participation of EP3/EP4 receptors in the inhibition of IFN-y
release. Furthermore, it was clear from the data in Section 3.3.2. that EP1/EP3
receptors are not involved in the PGE2 effect. In order to evaluate whether the effect
of PGEi binding was due to more than one receptor subtype, we pooled the data
from three experiments and performed curve-fitting analysis of the dose-effect
curves. Because the above findings have left only EP2 and EP4 receptors as candidate
mediators of PGE2 effects, any evidence for two active sites would implicate EP4
receptors in addition to EP2 receptors. Our analysis showed that a model with two
distinct PGE2 receptors fitted the data better than a model with one receptor (Figure
3.6.). The introduction o f the second active binding site was highly statistically
significant as judged by the F-ratio (F = 19.97) and p-value (p = 1.4xl0'7) of the two
receptor model versus the one receptor model. The calculated binding constants for
the two sites are 3.4xl0 ' 8 M (range 1.5 to 6.8xl0' 8 M) and l.OxlO' 9 M (range 0.6 to
2.8xl0 ' 9 M). These are (reasonably) close to 1.2xl0' 8 M (range 0.9 to l.5xl0 ‘8 M)
and 1.9xl0' 9 M (range 1.5 to 2.5xl0 ' 9 M), the reported binding constants of PGE2 for
EP2 and EP4 receptors respectively (Kiriyama et al., 1997). This analysis provides
evidence in support for the involvement of EP4 in addition to EP2 receptors. It also
allows an estimate of the contribution of both receptor subtypes to the total inhibitory
effect of PGE2 . The EP4 receptor accounts for approximately 57% (+6 % S.E.M.) of
the effect with EP2 receptors contributing the remaining 43%.
75
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1.2 T
®
c 0 .8 - -
o
a .
« 0.6 - -
£
® 0.4 - -
| 0.2 -
oc
0 --
-0.2
Figure 3.6. Dose-effect analysis of the PGE2 effect on IFN-y
release from clone 8 D8 . Data from three experiments were pooled and
theoretical curves corresponding to a one receptor model (interrupted
line) or a two receptor model (solid line) were Fitted to the data. Data
from individual experiments were multiplied with a constant to fit on
a scale ranging from 1 (no inhibition) to 0 (maximal inhibition).
Residual IFN-y release at 105 M PGE2 was considered non-
inhibitable and was subtracted from the data before curve fitting.
Non-inhibitable IFN-y release was consistently less than 10% of
maximal IFN-y release.
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3.3.4. Analogs of cAMP mimic the inhibitory effect of PGEi on IFN-y release
The above experiments with PGE2 agonists implicate EP2 and EP4 receptor
subtypes in the effect of PGE2 on IFN-y release, both of which are coupled to
adenylate cyclase. As discussed earlier, activation of adenylate cyclase leads to an
increase in intracellular cAMP levels. These results imply that the effect of PGE2 is
caused by an increase of the concentration of intracellular cAMP. Therefore, cAMP
analogs should inhibit IFN-y release from cloned CD8 + T cells.
To test this prediction and to better understand the signaling events associated
with the inhibitory effect of PGE2 we used the cAMP analogs 8 -Br-cAMP and
Sp-cAMPS. The data (Figure 3.7.A) show that both 8 -Br-cAMP and Sp-cAMPS
inhibited IFN-y release. This inhibition was half-maximal at concentrations below
lO" 4 M and complete at 10° M. Identical results were obtained when experiments
were performed with polyclonal CD8 + T cells (Figure 3.7.B). These data, together
with those on PGE2 receptor subtype specific agonists, strongly suggest that
cAMP-coupled receptor subtypes (namely EP2/EP4) are involved in PGE2 -mediated
inhibition of IFN-y release.
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A 8-Br-cAM P
100 * .
Sp-cAMPS
£ 60 -
£ 20 -
logio([cAMP analogJ/M)
B
8-BrcAMP
Sp- cAMPS
100 T
$ 80-
ni
®
£ SO
X '
£ 40 -
o
20 -
log1 0 ([cAMP anatog]/M)
Figure 3.7. Effect of cAMP analogs on TCR-dependent IFN-y release
from clone 8D8 (A), and from polyclonal CD8+ T cells (B). T cells were
stimulated with 1 |Xg/ml of immobilized anti-oc|5TCR in the presence of
indicated concentrations of 8-Bromo-cAMP (circles) and Sp-cAMPS (triangles).
Data are the mean + SEM of three experiments.
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3.4. Discussion
The data in this chapter show that sulprostone, an EP1/EP3 selective agonist,
does not inhibit the PGEi-mediated inhibition of IFN-y release. By measuring
intracellular calcium levels from P815 cells and measuring uterine contractions in
mice, we were able to prove that the agonist itself was effective. Thus, we were able
to conclusively exclude the involvement of EPi and EP3 receptors.
Using the EP2/EP3/EP4 selective agonist misoprostol and the EP2 selective
agonist 19-OH PGE2 we determined the involvement of EP2 and/or EP4 receptors.
Subsequently, we were able to conclude from the curve fitting analysis that both EP2
and EP4 receptors are involved in the PGE2-mediated inhibition of IFN-y release.
Both, EP2 and EP4 receptors signal through the heterotrimeric GTP binding protein
Gs by elevating the intracellular concentration of cAMP (Coleman et al., 1994;
Ichikawa et al., 1996; Narumiya, 1997; Narumiya, 1994; Negishi et al., 1993;
Nishigaki et al., 1995). Our finding that two different membrane permeable cAMP
analogs mimic the effect of PGE2 receptor signaling confirmed a link between a rise
in intracellular cAMP and inhibition of TCR triggered IFN-y release in NOD CD8 + T
cells. Furthermore, it was determined that this effect was not restricted to the CD8 + T
cell clone used in this study, since a similar effect of the cAMP analogs was also
seen in polyclonal CD8 + T cells.
What is the interdependence between the TCR signaling and cAMP
pathways? As mentioned in our earlier chapter, cAMP is thought to play a negative
regulatory role in the early phases of T cell activation. It has been proposed that T
79
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cell activation in the presence of a costimulatory signal such as rIL -la stimulates
PKA-I phosphotransferase activity and phosphorylation of membrane associated
proteins. The model proposed by Laxminarayana (Laxminarayana and Kammer,
1996) for cross talk between the PI/Ca2 + /PKC and AC/cAMP/PKA pathways entails
that the signals derived from TCR/CD3 and IL-IR merge at the level of PKC. The
signal derived from ligand binding to the TCR/CD3 complex would result in the
activation of CD45 and dephosphorylation of protein tyrosine kinases, activation of
PLC-yl, production of inositol triphosphate and diacylglycerol and release of Ca2 +
and activation of PKC isozymes. These PKC isozymes would then translocate to the
plasma membrane where they would either phosphorylate a G protein subunit or an
AC catalytic subunit. Activation of AC would subsequently activate PKA-I.
Activated PKA-I, as seen earlier, would then participate in T cell regulatory
functions.
However, in our experimental system, T cell activation and IFN-y release
occur in the absence of a second signal. It seems therefore likely that cAMP
interferes with some downstream event of TCR signaling. One of the possible
mechanisms of cross talk between the TCR/CD3 and cAMP pathway is through
phosphorylation of NF-AT. Another possible mechanism could be that cAMP
signaling would inhibit the calcineurin-regulated pathway, blocking the translocation
of NF-AT to the nucleus.
T cell receptor generated calcium signals lead to activation of calcineurin.
Calcineurin, a Ca2 + /calmodulin-dependent serine/threonine phosphatase is an
80
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essential enzyme required for activation of IL-2 transcription. Studies by Paliogianni
et al. have shown that agents that increase intracellular cAMP such as PGE2 reduced
IL-2 promoter activity that was induced by constitutively active calcineurin
(Paliogianni and Boumpas, 1996). The overexpression of this active form of
calcineurin rendered the IL-2 promoter more resistant to PGE2. The reduced IL-2
promoter activity suggests that the effects of cAMP elevating agents may be exerted
at more distal sites in the calcineurin-regulated pathways.
Calcineurin directly dephosphorylates NF-ATC (nuclear factor of activated T
cells) leading to the nuclear import of NF-AT. Nuclear translocation o f NF-AT leads
to the activation of immune response genes such as those encoding IL-2, IL-4,
CD-40 ligand etc. (Beals et al., 1997). Recent studies have proposed IFN-y
transcription to be mediated by NF-AT binding sites within introns of the IFN-y gene
(Sica et al., 1997). The transcriptional activator NF-AT is dephosphorylated by
TCR-dependent activation of calcineurin but is phosphorylated by cAMP-activated
protein kinase A (Li et al., 1999; Sheridan and Gardner, 1998; Tsuruta et al., 1995).
NF-AT may therefore be one of several possible downstream points where TCR and
cAMP signaling intersect. The mechanisms mentioned above are schematically
represented in Figure 3.8.
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CD3/TCR Complex
IL-1R
Lck/Fyn •ZAP-70
ATP cAM,
PKC
DAG
Calcineurin
N F A T c
N F - A T „
Cytokine genes (e.g. IL-2)
Figure 3.8. Proposed model for convergence of TCR-
cAMP signaling pathways
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CHAPTER 4
Role of accessory signals in PGE2 -mediated inhibition of IFN-y
release
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4.1. Introduction
There is accumulating evidence that autoimmune diseases may result from,
among other determinants, preferential activation of T cells with an aberrant, disease
promoting cytokine profile at the expense of T cells with a protective cytokine
profile. The cytokine secretion patterns of T cells can be modulated by physiological
factors present during the activation of T cells. Products from antigen presenting
cells (APCs) and/or accessory cells are likely candidates to play a role in this respect,
since they contribute to the microenvironment in which the T cells are activated.
Hence, it is possible that such factors will skew the nature of immune responses by
modulating the cytokine production of responding T cells.
PGE2 is one such factor that has been shown to differentially modulate
cytokine secretion in CD4+ T cells. PGEi inhibits IL-2 and IFN-y secretion from Th 1
cells whereas IL-4 and [L-5 secretion by Th2 cells is not affected (Betz and Fox,
1991; Hilkens et al., 1995). However, the differential modulation of cytokines by
PGEt depends on the mode of stimulation of T cells. For example, IL-5 expression
stimulated with anti-CD3 alone and in combination with anti-CD28 was inhibited by
PGEt and IL-5 expression stimulated with concanavalin A (con A) and phorbol
myristyl actetate (PMA) was upregulated by PGE2 (Borger et al., 1998).
Sunder-PlaBmann et al. showed that exposure of T cells isolated from human
peripheral blood mononuclear cells to different stimuli exhibited different sensitivity
to PGE2 (Sunder-Plassmann et al., 1991). In addition, the effect of PGE2 on T cells is
thought to be modulated by various extracellular signals such as costimulatory
84
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factors and cytokines such as IL-2. Costimulatory signals have been shown to
modulate the inhibitory effects of cAMP-elevating agents on proliferation and
interleukin-2 (IL-2) secretion. Bartik et al. showed that costimulatory signals can
alter the immunosuppressive effects o f PGEt in human T cells (Bartik et al., 1994).
Hilkens et al. showed that the differential modulation patterns of PGE2 were a
direct result of differences in the availability of IL-2. High IL-2 levels was shown to
overcome the selective inhibitory effect of PGEi (Hilkens et al., 1995). Gurlo et al.
showed that in a CD4+ T cell line, HT-2, high concentrations of IL-2 partially
reversed the inhibitory of PGE2. However, IL-2 dependent proliferation of the CD8+
T cell line, CTLL-2, was inhibited by PGE2 even in the presence of high
concentrations of IL-2 (Gurlo et al., 1998). These results suggest that the net
modulatory effect of PGE2 on the activation of T cells is critically dependent on the
availability of IL-2. In addition, it is known that IL-2 stimulates IFN-y release from T
cells (Su et al., 1998; Theze et al., 1996). Since our study (Chapters 2 and 3) was the
first to determine the effects of PGE2 on IFN-y release from CD8+ T cells, we were
also interested in determining the role of exogenous IL-2 in the PGE2 mediated
effect. To address this issue, we added IL-2 to our cultures at the time of culture
initiation along with an inhibitory concentration of PGE2.
IL-12 is an inflammatory cytokine secreted by neutrophils, macrophages,
microglia, and dendritic ceils in response to different stimuli. IL-12 enhances
proliferation and cytotoxicity of T cells and enhances IFN-y release from resting and
activated T cells (Hilkens et al., 1996). PGE2 and IL-12, both being accessory cell
85
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derived factors, may be present concurrently during T cell activation. The combined
modulatory effects of PGEt and IL-12 in CD4+ T cell activation have been
extensively studied. Hilkens et al. showed that the PGE2-mediated inhibition of
IFN-y release from CD4+ T cells was not reversed by IL-12. Likewise, PGE2 did not
have any effect on the IL-12 mediated enhancement of IFN-y release. IFN-y levels in
the presence of IL-12 were higher than the controls without IL-12 even with the
maximum inhibitory concentration of PGE2 (Hilkens et al., 1996). These
observations suggest that PGE2 and IL-12 modulate the production of IFN-y from
CD4+ T cells independent of each other. In order to explore the possibility of a
similar modulation in CD8+ T cells, we studied the effect of IL-12 and PGE2 in
combination on IFN-y release from CD8+ T cells.
Sunder-PlaBmann et al. showed that in proliferation assays the sensitivity of
naive/resting CD4+ T cells to PGE2 was lower than that of in vivo preactivated
memory T cells (Sunder-Plassmann et al., 1991). Our results, as discussed in Chapter
2 of this research thesis, show that PGE2 inhibits IFN-y from T cells two weeks after
expansion. It would be interesting to determine if the sensitivity of T cells to PGE2
changes based on the strength of the activation signal and the time point within the
activation cycle of CD8+ T cells. To address this issue, we evaluated the effect of
PGE2 from CD8+ T cells at different time points after stimulation. Furthermore, we
also determined if the signaling strength would alter the sensitivity of CD8+ T cells
to PGE2.
86
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4.2. Materials and Methods
4.2.1. Antibodies and reagents
Prostaglandin-Ej, avidin-HRP, o-phenylenediamine (OPD), murine rIL-2 and
murine rlFN-y were obtained from Sigma Chemical Co. (St. Louis, MD). Murine
rIL-7 was purchased from Life Technologies (Gaithersburg, MD). Murine rIL-12
was obtained from PharMingen (SanDeigo, CA). Anti-CD3, anti-CD4 and anti-CD8
mAbs were used in the form of diluted hybridoma supernatant. FITC-goat F(ab')2
anti-rat IgG Ab was obtained from Caltag Laboratories (South San Francisco, CA).
Hybridomas GKI.5 (anti-CD4) and 3.155 (anti-CD8) were purchased from the
American Type Culture Collection (ATCC, Rockville, MD). The tissue culture
medium and the anti-apTCR antibody were obtained from sources as described
under Materials and Methods in Section 2.2.1.
4.2.2. Mice
Non-obese diabetic (NOD) and BALB/c mice were obtained from The
Jackson Laboratory (Bar Harbor, ME). The mice were bred and maintained as
discussed in the Materials and Methods section of Chapter 2 of this dissertation
thesis.
4.2.3. Preparation of polyclonal CD8+ T cells
For full activation of T cells, spleen cells (l-2xl06) were stimulated with
anti-CD3 mAb (YCD3-1 hybridoma culture supernatant diluted 1 in 20) in the
presence of mitomycin C-treated spleen cells (3xl06) in 5 ml of TCM. Spleen cells
87
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contain a mixture of antigen presenting cells and lymphocytes. After 48 h, cells were
washed and treated with 40 U/ml IL-2 and 10 ng/ml IL-7. Two days later cells were
fed with the same medium, and feeding with TCM alone was repeated at three to
four day intervals.
To purify CD8+ T cells, polyclonal cells were incubated with anti-CD4 mAb
(supernatant from hybridoma GK1.5, diluted 1 in 3) for 30 min on ice, washed and
treated with diluted (1:10) pooled rabbit complement (ICN, Irvine, CA) for 45 min at
37°C. The resulting cell preparation was analyzed by flow cytometry and was found
to contain >95% of CD8+ T cells.
4.2.4. Flow cytometry analysis to assess the purity of polyclonal CD8+ T cells
For detection of CD8 expressing cells, the cells were washed once with
staining buffer, and incubated with 50 p .1 of anti-CD8 mAb 3.155 for 30 minutes on
ice. Subsequently the cells were washed three times with staining buffer and stained
with 50 pi of FrrC-Ffab’)! goat anti-rat IgG as second step Ab for 30 min on ice in
the dark. The cells were then washed three times. Cells were analyzed using a
FACStar flow cytometer (Becton Dickinson, San Jose, CA).
4.2.5. Stimulation of IFN-y and IFN-y ELISA
Unless otherwise indicated, IFN-y release from T cells was stimulated using
anti-aPTCR mAb H57-597 immobilized in tissue culture plates (Falcon, 96 well, flat
bottom plate coated with 1 |!g/ml mAb in PBS at 4°C overnight). Polyclonal CD8+ T
cells were added to 96 well microtiter plates at a density of 3xl04 cells per well.
88
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Immediately after seeding the cells, PGEi, were added to the culture at the indicated
concentrations (10'5M - 10loM). In order to determine the effect of IL-2 on
PGEo-mediated inhibition of IFN-y release, IL-2 (lOU/ml) was added at the time of
stimulation along with an inhibitory concentration (10'7 M) of PGEi. For the
dose-response study of IL-12 various concentrations of IL-12 was added at the time
of stimulation as indicated in figure legends. To study the modulation of IFN-y
release by the combination of PGE2 and IL-12, IL-12 at a concentration of lU/ml
was added along with an inhibitory concentration (10‘7 M) of PGE2 at the time of
culture initiation. After 24 h, supernatant was collected and stored at -80°C.
IFN-y ELISA was done as per manufacturer's instructions using paired
anti-cytokine antibodies (PharMingen, San Diego, CA). The primary or capture
antibody was immobilized in Immulon-4 plates (Dynatech, Boston, MA). The
sensitivity of the assay as described by the manufacturer (PharMingen, San Diego,
CA) is 1 U/ml (67 pg/ml).
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4.3. Results
4.3.1. Effect of T cell activation state on sensitivity of cells to PGEj
The inhibitory effect of PGE2 discussed in Chapter 2 was observed in CD8+ T
cells two weeks after their expansion. We wanted to determine if the sensitivity of
CD8+ T cells to PGE2 remained the same irrespective of their activation state.
To address this question, we compared the sensitivity of NOD CD8+ T cells
to PGE2 at different time points after full activation. Full activation is achieved by
using splenocytes in addition to anti-TCR antibody. The role of splenocytes in this
protocol is two-fold, to crosslink the TCR by binding anti-TCR Ab via Fc receptors,
and to provide costimulatory signals. The contribution of splenocytes is essential for
full activation since stimulation with anti-TCR Ab alone, soluble or immobilized,
does not trigger proliferation, although effectively eliciting IFN-y release. After 48 h,
stimulation was arrested by washing out the anti-TCR Ab in solution. The
stimulatory effect of any remaining anti-TCR Ab bound to Fc receptors of
splenocytes does not last beyond 48 h, because most APCs in splenocytes die within
48h. A schematic representation of the time-scale for expansion is shown in Figure
4.1.
Figure 4.2. shows that at 5-7 days after initiation of expansion, i.e., 3-5 days
after arrest of stimulation, PGE2 has very little effect on IFN-y release from CD8+ T
cells. At eight to nine days after expansion, CD8+ T cells remain rather insensitive to
PGE2. The concentration o f PGE2 that is required for ~70% inhibition was close to
90
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10'5 M and half-maximal inhibition occurred at ~10'6 M. In contrast, at 15 days after
expansion, the sensitivity to PGE2 was restored. The dose for half-maximal
inhibition had shifted by two orders of magnitude to ~10‘8 M. Therefore, the
sensitivity to PGE2 is critically dependent on the time after proliferation inducing
signaling. Resting cells are more sensitive to PGE2 than recently expanded CD8+ T
cells.
Full
activation
TCR +
splenocytes
-2 0
t
Times of observation
Time
^ ( D a y s )
Cells are fully
activated
Cells return to resting
state
15
f
Cells are
rested
Figure 4.1. Schematic representation of the stimualtion time-line of
polyclonal CD8+ T cells
91
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8-9 d
12-15 d
100
80 -
60 -
— 4 0 -
20 -
0
10 -9 -8 -7 -6 -5
togio([PGE2],M)
Figure 4.2. Resting CD8+ T ceils are more sensitive to the inhibitory
effect of PGE2 than activated CD8+ T ceils. Five to seven (circles), eight to
nine (squares) or twelve to fifteen (triangles) days after expansion with anti-
TCR Ab and splenocytes, polyclonal CD8+ T cells were re-stimulated with
immobilized anti-apTCR antibody (1 (ig/ml) in the presence of the indicated
concentrations of PGE2. The data are the mean ± SEM of three experiments.
Note: The author performed one representative experiment towards
the completion o f this research. Replicates of the initial data were performed
by Dr.Tatyana Gurlo in our laboratory. We found that the data were in
agreement with the initial experiment performed.
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4.3.2. Accessory signals: IL-2 and IL-12 have contrasting effects on
PGEi-mediated inhibition of IFN-y release
T ceils integrate multiple signals and modify their response to antigenic
stimulation, and some of these signals may counteract the inhibitory effect of PGEi.
IL-2 is among the signals that are known to modulate IFN-y production by CD8+ T
cells. Although IFN-y producing CD8+ T cells, such as those used in this study,
frequently are poor producers of IL-2, they may receive this cytokine from other
cells, particularly Thl CD4+ T cells in the islet inflammatory infiltrate or in
lymphoid tissue. It was therefore of interest to determine the role of exogenous IL-2
in PGEi-mediated inhibition of IFN-y release from CD8+ T cells.
To address this question, we added IL-2 to our culture at the time of
stimulation through the TCR together with a concentration of PGE2 that caused
half-maximal inhibition of IFN-y release. For this experiment, cloned CD8+ T cells
(clone 8D8) and polyclonal CD8+ T cells were used two weeks after expansion. In
two types of controls, either PGE2 or IL-2 was omitted. As shown in Figure 4.3 we
found that IL-2 at a concentration of 10 U/ml largely reversed the inhibitory effect of
PGE2 .
IL-12 is a cytokine that is known to exert pleiotropic effects on NK cells and
T cells. Similar to IL-2, IL-12 is a potent inducer and enhancer of IFN-y production
by resting and activated T cells. Furthermore, as discussed earlier IL-12 did not
prevent the PGE2 mediated downregulation of IFN- y in CD4+ T cells. In the light of
93
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□ No IL -2
< o 80
8D8 Polyclonal
Figure 4.3. IL-2 largely reverses the inhibitory effect of PGE: on
TCR-dependent IFN-y release from clone 8D8 and polyclonal CD8+ T
cells. Cells were stimulated with immobilized l|ig/ml anti-a|3TCR mAb
in the presence or absence o f lxlO'7 M PGE2. IL-2 (10 U/ml) was added
at the time of stimulation. The data are the mean + SEM of three
experiments.
94
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this data, we wanted to determine if IL-12 had any effect on the PGE: mediated
inhibition of IFN-y release from CD8+ T cells.
To address this question we first determined the optimal concentration of
IL-12 required to enhance T cell receptor (TCR) triggered IFN-y release from
polyclonal CD8+ T cells. We performed dose-response studies, where different
concentrations of IL-12 were added at the time of stimulation of T cells through the
T cell receptor. IL-12 at a concentration of 1 U/ml enhanced the TCR-triggered
IFN-y release somewhat above the half-maximal range and we chose this
concentration range to study the PGE^-mediated effects (Figure 4.4A).
In an effort to understand the modulation of IFN-y release from polyclonal
CD8+ T cells by PGE2 and IL-12 we added lU/ml of IL-12 and PGEi at a
concentration of 10'7M to polyclonal cells at the time of culture initiation. We found
that IL-12, unlike IL-2, had little or no effect on PGEi-mediated inhibition of
IFN-y. Furthermore, the modulatory effect of IL-12 on PGEi-mediated inhibition of
IFN-y release were similar in both NOD and BALB/c cells. For this study, the cells
from both strains were used two weeks after expansion (Figure 4.4 B).
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200 -
160
120
80
40
0 ------------------------------------------
0.001 0.01 0.1 1 10 100
log([IL-12], U/m l)
□ NoL-12
NOD BALB/c
Figure 4.4. (A) NOD CD8+ T cells were stimulated in the presence of
l|ig/m l of immobilized anti-aPTCR antibody. Varying concentrations of IL-
12 were added and IFN-y release dose-response was studied. (B) IL-12 has
little or no effect on the inhibitory effect o f PGEi on TCR-dependent IFN-y
release from polyclonal CD8+ T cells. Cells were stimulated with
immobilized l|ig/m l anti-aPTCR mAb in the presence or absence of IxlO7
M PGE2. IL-12 (1 U/ml) was added at the time of stimulation. The data are
the mean ± SEM of three experiments.
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4.3.3. The sensitivity to PGE2 depends on T cell receptor signal strength
Signal integration by T cells can occur by tuning of signaling thresholds for
antigenic stimulation. For example, costimulatory signals reduce the signal strength
that is required for proliferation whereas inhibitory signals may increase the TCR
signaling threshold. As seen earlier, at a time when proliferation-inducing signals are
active, the responsiveness to PGE2 is diminished. However, this effect disappears
with time as the proliferation-inducing signals wear off.
To investigate whether the diminished sensitivity to PGE2 after expansion of
CD8+ T cells was due to such tuning of the TCR signaling threshold, we reduced the
TCR signal strength at a time when the cells were still insensitive to PGE2. In our
earlier section, we determined that the cells were still insensitive to PGE2 at 8-9 days
after stimulation. We stimulated the cells 8-9 days after expansion with different
concentrations of immobilized anti-TCR antibody. Figure 4.5 shows that reducing
the anti-TCR concentration to 0.1 (ig/ml restored the sensitivity to PGE2 even at 9
days after expansion. Therefore, signals that stimulate proliferation do not abolish
sensitivity to PGE2, but alter the threshold at which TCR signals become sensitive to
PGE2.
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1 0 0in t ^ 1 ^ m i
< d t ri ® 01
c o 80
©
2
^ 60 H
“ 4 0 H
x
©
J 20-
'•S
* O-l
*9 -8 “ 7 -6 -5
log1 0 ([PGE2]/M )
Figure 4.5 The inhibitory effect of PGE2 on IFN-y release from CD8+
T cells depends on the strength of stimulation through the TCR. At eight to
nine days after expansion with anti-TCR Ab and splenocytes, polyclonal
CD8+ T cells were stimulated with 0.1 |ig/ml (closed bars) or 1 |ig/ml (open
bars) of immobilized anti-apTCR mAb in the presence o f the indicated
concentrations of PGE2. The data are the mean + SEM of three experiments.
Note: The author performed one representative experiment towards
the completion o f this research. Replicates of the initial data were performed
by Dr.Tatyana Gurlo in our laboratory. We found that the data were in
agreement with the initial experiment performed.
98
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4.4. Discussion
Our results show that the responsiveness of CD8+ T cells to PGE2 depends on
the activation state of CD8+ T cells. Typically, resting cells were found to be more
sensitive to PGEi than recently activated cells. Furthermore, we observed that the
TCR signal strength determines the sensitivity of PGEi to recently expanded cells.
This change in sensitivity could be due to activation-dependent alterations of
intracellular signal integration or to alterations of PGE2 receptor expression. In
addition, the inhibitory effect of PGEi could be overcome by signals such as IL-2.
IL-2 partially reversed the inhibitory effect of PGEi in our polyclonal CD8+ T cells.
On the other hand, IL-12, an IFN-y enhancing signal had little or no effect on the
PGEi-mediated effect.
Our earlier results (Chapter 2) suggest a potential disease-protecting role for
PGE2 in T1DM. In addition, studies done in our laboratory have shown an enhanced
production of PGE2 in inflamed NOD islets. One of the possible disease protective
mechanisms of PGEi could be that it delays or alters the timing of islet destruction.
In fact, progression of type I diabetes is very slow, possibly due to the delaying
influence of protective factors such as PGE2. However, ultimately the disease
progresses from a benign to a malignant form and beta cells get destroyed. This
could be orchestrated by the time-dependent interplay between disease-promoting
and disease-protective factors.
It has been suggested that Thl-type cytokines play a pathogenic role in
T1DM whereas Th2-type cytokines play a preventive role in the disease (Atkinson
99
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and Maclaren, 1994; Falcone and Sarvetnick, 1999). In the islet microenvironment
expression of a variety of cytokines have been found. Expression of type 1 cytokines
such as IFN-y and IL-2 correlated with P-cell destructive insulitis and that of type 2
cytokines such as IL-4 and IL-10 and the type 3 cytokine TGF-p correlated with
benign insulitis (Rabinovitch, 1998).
In addition to these cytokines, IL-12, a type-1 inducing cytokine, has been
implicated as a disease-promoting cytokine (Rabinovitch, 1998). In NOD mice
administration of IL-12 has been shown to accelerate the onset of TI DM (Trembleau
et al., 1995). However, the accelerating effects of IL-12 were not associated with a
direct toxicity of IL-12 towards islets. Rather, IL-12 acts mainly by promoting a
P-cell destructive Thl response towards beta-cell destruction (Rabinovitch et al.,
1996). Furthermore, an IL-12 antagonist has been shown to protect from disease by
diverting CD4+ T cells from a Thl to a Th2 phenotype. It has also been shown that
this skewing should be attempted before the onset of insulitis since IL-12 targeting is
not sufficient to divert cells to the Th2 pathway when the pancreas already contains
polarized Thl cells (Trembleau et al., 1997). The destruction of islet Pcells and
disease progression may depend on how the finely tuned balance of the
immunoregulatory cytokines is tipped. Hence, certain factors in the islet cytokine
milieu may counteract the inhibitory effect of PGEi on CD4+ and CD8+ T cell
responses.
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In the light of the above data, the inhibitory effect of endogenous PGEt in
islets may be offset by cytokines such as IL-2 and other proliferation-inducing
signals. However, IL-12 had little or no effect on the inhibitory effect of PG E2. We
observed that even at the highest inhibitory concentration of PGE2, IL-12 yielded an
elevated level of INF-y compared to controls (Figure 4.6). The effect of the
combination of PG E 2 and IL-12 on EFN-y release from CD8+ T cells are in
concordance with earlier studies in CD4+ T cells. In the context of T1DM, it is thus
important to uncover the mechanisms that lead to the delicate balance between the
disease-promoting and disease-protective factors.
□ NOL -12
450 - b l -12
400 -
350 -
g 300 -
^ 250 -
£ 200 -
U_
= 150 -
100 - _
: j j j j
PGE2, M 10‘ 5 10* 10'7 10* Control
Figure 4.6. Modulatory effects of PGE2 and IL-12 on IFN-y release from
polyclonal CD8+ T cells. Cells were stimulated with l|lg/ml of immobilized anti-
aPTCR Ab. IL-12 (3U/ml) and P G E 2 at the indicated concentrations were added
at the time o f stimulation.
101
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CHAPTER 5
TGF-p ligand-receptor interactions
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5.1. Introduction
The immune system has developed mechanisms whose principal role is to
inhibit the activation and effector functions of lymphocytes. These mechanisms play
an important role in limiting the magnitude of immune responses and in facilitating
the immune system’s ability to remain tolerant to self-antigens. One of the inhibitory
mechanisms in the immune system is the induction of regulatory and suppressor T
cells (Abbas K. A., 1997). The inhibitory effects of this subpopulation of T cells is
thought to be mediated by cytokines with inhibitory functions. One such cytokine is
transforming growth factor-p (TGF-p) which can inhibit immune responses. TGF-P
is produced by many tissues including tumors to protect themselves against immune
attack. Thus, TGF-P, like PGE2, may be an active indicator of “self’.
Transforming growth factor-P, TGF-p, is a potent immunomodulatory
cytokine that influences a wide variety of cellular processes. TGF-P mediated effects
have been extensively studied in most cells of the immune system. TGF-pi is
capable of certain stimulatory effects in the immune system. However, a majority of
studies thus far have reported negative immunoregulatory functions of TGF-P such
as growth suppression, inhibition of cytokine secretion etc (Letterio and Roberts,
1998). The effects of TGF-P on macrophage, monocyte and B cell populations were
briefly discussed in Chapter 1.
TGF-p mediated effects have been studied in great detail in both CD4+ and
CD8+ T cells. In both subsets of T cells, TGF-p 1 inhibits T cell clonal expansion,
103
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particularly affecting events that occur subsequent to IL-2 production and IL-2
ligand-receptor interaction (Bright et al., 1997). TGF-P also interferes with cytokine
production and expression of lytic activity in CD8+ T cells (Lee and Rich, 1993).
However, TGF-P is not merely inhibitory. Studies have suggested a bifunctional
regulatory role for TGF-P, dictated in part by the differentiation state of responsive T
cells. Initial studies have shown that exposure of naive CD4+ T cells to TGF-P i
enhances growth and polarizes naive CD4+ T cells towards a Thl phenotype.
However, further studies conclusively demonstrated that TGF-P i exposure leads to
Th2 differentiation (Gray et al., 1994). In CD8+ T cells, TGF-Pi enhances the growth
of naive cells and depresses their lytic activity. TGF- P costimulated CD8+ T cells
exhibit elevated IL-2 and IFN-y levels but unchanged lytic activity upon
restimulation in the absence of TGF-P (Lee and Rich, 1993). This suggests that
TGF-P could be an important stimulus in the maturation and growth of a CD8+ T cell
subset and could possibly modulate protective immune responses. TGF-P released
following natural killer cell (NK cell) and CD8+ T cell interaction has been shown to
activate a subpopulation of suppressor CD8+ T cells (Gray et al., 1994).
The ability of TGF-P to suppress immune responses suggests a potential
protective role for TGF-P in autoimmune diseases. Studies have indeed shown that
TGF-P can protect against collagen-induced arthritis and can delay the progression
of relapsing experimental allergic encephalomyelitis (Kuruvilla et al., 1991).
Furthermore TGF-P i has been extensively studied in type I diabetes (TIDM). These
104
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studies have shown that transgenic paracrine TGF-P i protects NOD mice, which is
the animal model of T1DM, from disease (Moritani et al., 1998).
The regulation of CD4+ and CD8+ T cell functions by TGF-P may be affected
by the expression level and function of TGF-P receptors on these cells. Studies have
shown the presence of high-affinity receptors on both resting and activated T ceils.
In addition, cellular activation was shown to result in a five to six fold increase in the
number of TGF-P receptors on a per cell basis (Kehrl et al., 1986). Since the
regulatory effects of TGF-P in T cells have been extensively studied, our emphasis
during the course of this research was primarily to understand the ligand-receptor
interactions that mediate TGF-P induced effects.
TGF-P signals through a heterotrimeric receptor complex consisting of the
type I and type II transmembrane serine/threonine kinases (Massague and Weis-
Garcia, 1996). As discussed in Chapter 1, most mammalian cells express three
TGF-P receptors namely, the type I, type II, and type III receptors. TGF-P type I and
type II receptors are glycoproteins (53 kDa and 70kDa respectively) and are
ubiquitously expressed in most cells and tissues. Both the type I and II receptors
contain cysteine rich extracellular domain and a single transmembrane region. The
cytoplasmic domain of both the type I and II receptors contains intrinsic kinase
activity that has been shown to be essential for TGF-P signaling. The type I and II
receptors are thought to be critical for TGF-P signaling. The type III receptor, or
betaglycan, is a membrane-bound proteoglycan with a short cytoplasmic tail that has
105
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no signaling motif. The proposed role of type III receptor is to bind and present the
TGF-P ligand to the signaling receptors (Yingling et al., 1995).
Studies in chemically mutagenized cell lines showed that TGF-P t binds to the
type II receptor with high affinity in the absence of the type I receptor. Binding of
TGF-P i to the type I receptor requires the presence of the type II receptor. Studies
with cloned TGF-P and activin type I receptors have confirmed the inability of the
ligand to bind the type I receptor in the absence of the type II receptor. The type II
receptor has a constitutively autophosphorylated kinase domain and ligand binding
has no appreciable change in this activity. It appears that ligand binding facilitates
the recruitment of the type I receptor into a complex with the type II receptors. The
type II receptor transphosphorylates the cytoplasmic domain of the type I receptor
(Wrana et al., 1994). Phosphorylation of the kinase domain of the type I receptor, in
turn, is thought to propagate the signal to substrates downstream of the TGF-P
receptor complex (Rodriguez et al., 1995). In support of this model, it has been
shown that the biological response to ligand in the TGF-P system is specified
primarily by the type I receptor engaged in the complex. In addition, it has been
shown that the heterodimers of the type II and type I receptors exist even in the
absence of the ligand. This implies that the ligand probably acts to further stabilize
the type I and II receptor interactions (Massague, 1996). A schematic representation
of the proposed formation of the TGF-P signaling complex is as shown in Figure 5.1.
To test the proposed model of ligand-receptor assembly and to gain a better
understanding of the interactions resulting in TGF-P mediated effects, we designed
106
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peptides based on the TGF-Pi sequence (Table 5.1) and monitored inhibition of
TGF-P signaling by these peptides. The peptides were chosen based on the crystal
structure of TGF-p( visualized using the program RASMOL. The choice was made
based on the residues that are surface exposed and hence would potentially have
access to the type II receptor. To facilitate the structural studies with the peptides, we
also determined the kinetics of TGF-p ligand-receptor assembly in order. These
studies were done using the mink lung epithelial cell line, MvlLu, which has been
known to express all three types of TGF-p receptors (Yingling et al., 1995).
n
> TGF-p
I
II
I
Figure 5.1. Proposed model for extracellular TGF-p signaling.
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5.2. Materials and Methods
5.2.1. Peptides and reagents
Peptides 1 through 6 were derived from the TGF-Pi sequence and were
synthesized at the University of Southern California microchemical core facility (Los
Angeles, CA.). Human rTGF-Pi was purchased from PharMingen (San Diego, CA).
Human rTGF-PsRII was obtained from Calbiochem (LaJolla, CA). “Enhanced
luciferase assay” kit was purchased from PharMingen (San Diego, CA).
The tissue culture medium (TCM) used for cell culture was based on
Dulbecco’s modified Eagle’s medium (DMEM) with high glucose content which
was supplemented with 100 U/ml penicillin, 100 fig/ml streptomycin (Life
Technologies, Gaithersburg, MD), 10% FBS (Gibco, Rockville, MD) and 200 pg/ml
geniticin (Life Technologies, Gaithersburg, MD). For experiments, the same medium
was used as above except that 200 (ig/ml geniticin was omitted. For trypsinization of
cells, trypsin-EDTA-lX (Life technologies, Gaithersburg, MD) was used.
Peptide Residue number Sequence
1 1 - 1 6 A L D T N Y C F S S T E N K C C
2
2 1 - 3 9 Y I D F R K D L G W K W I H E P K G Y
3 6 2 - 7 6 L A L Y N Q H N P G A S A A P
4 8 1 - 8 9 Q A L E P L P I V
5 8 5 - 1 0 1 P L P I V Y Y V G R K P K V E Q L S N
6 9 5 - 1 0 3 K P K V E Q L S N
Table 5.1. Design of peptides from the TGF-P sequence
1 0 8
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5.2.2. Mink lung epithelial cells
For the assay of TGF-0 receptor signaling Mink lung epithelial cells (MvlLu
cells) transfected with a plasminogen activator inhibitor-1 (PAI-1) luciferase
construct (a kind gift from Dr. David Warburton, Children’s hospital, Los Angeles,
CA) was used. The downstream intracellular signaling events following TGF-Pi
binding, such as SMAD-3 phosphorylation, is known to stimulate the expression of
PAI-1 (Padgett, 1999). The luciferase gene is coupled to the PAI-1 promoter and
TGF-Pi induced signaling was detected as a change in luciferase activity in the
transfected MvlLu cells.
For routine maintenance, cells were grown to confluence in DMEM + 10%
FBS supplemented with 200 pg/ml of geniticin. Cells were passaged every three to
four days by trypsinization of adherent cells. Cells were trypsinized by initially
aspirating the medium and washing the cells in 5-10 ml of 10% PBS
(Phosphate-buffered saline). Trypsin-EDTA (IX) solution was added to washed cells
and cells cultured at 37°C for 5-10 minutes. Once the cells started to detach, they
were washed with cold TCM by suspension and centrifugation. Subsequently, the
cell pellet was resuspended in warm TCM to which 200 |ig/ml geniticin was added.
Cells were cultured until further use or passage.
5.2.3. Peptide inhibition studies
Mv lLu cells were cultured in DMEM + 10% FBS at a cell density of 160,000
cells/ml for approximately 3h at 37°C. After 3h, the medium was aspirated and
109
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varying concentrations of the peptide were added to the cell monolayer. Cells were
incubated for 30 minutes and rTGF-Pi (lOpM) was added. The cells were then
incubated again for approximately 14h and cell lysis performed in preparation for the
luciferase assay.
For experiments involving pulsing with peptides, cells were cultured in tissue
culture medium (DMEM + 10% FCS) for approximately 24h. After 24h, cells were
incubated with peptides for 30 minutes and with rTGF-Pi for 20 minutes. The cells
were then washed and incubated for 14h in TCM. Dilutions of peptides and rTGF-Pi
were made in medium containing DMEM + 1% FCS + 0.1% BSA.
5.2.4. Kinetic studies
Mink lung epithelial cells were cultured in tissue culture medium
(DMEM+10%FBS) for approximately 24h. At the indicated time points (6h - Oh),
rTGF-Pi was added and ceils incubated at 37°C. Dilutions of rTGF-Pi were made in
medium containing DMEM + 1% FCS + 0.1% BSA. At the end of the time-course,
cells were washed and cultured for an additional 14h at 37°C. After 14h, ceils were
lysed. Luciferase assay was performed with the cell lysate, as described in Section
5.2.5.
5.2.5. Luciferase Assay
The high sensitivity and lack of endogenous luciferase activity in most
mammalian cell types makes luciferase an excellent reporter enzyme. For the assay,
the luciferase assay kit, “Enhanced luciferase assay kit” (PharMingen, San Diego,
110
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CA) was used. The cells were washed in phosphate-buffered saline following
incubation for 14h, as mentioned in Section 5.2.4. The cells were then incubated in
the cell lysis buffer (PharMingen, San Diego, CA) for 20 minutes at 4°C to minimize
protease degradation. The cell lysate was centrifuged to remove cell debris. Then,
20-100 (il of the extract was assayed by adding the reagents necessary for the
luciferase reaction (Substrate A). The light signal generated by the luciferase enzyme
present in the lysate is then immediately measured by the addition of solution
containing luciferin (Substrate B). The light signal was measured using a
luminometer, as relative light units. Substrate A (100|il) was added first and
substrate B (lOOp.1) was added immediately before measurement was taken. The cell
lysate and the substrates were mixed by vortexing the contents. The time between
addition of substrate B and the start of the measurement was kept as short as
possible.
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5.3. Results
5.3.1. Inhibition of TGF-P signaling by peptides derived from TGF-Pi ligand
sequence
In order to study the ligand-receptor interactions of TGF-P we wanted to
initially identify the contact sites between the receptor subunits and also those with
the ligand. We decided to first study the contact sites between the type II receptor
subunits and the TGF-P ligand. To address this issue, we synthesized a panel of
peptides derived from the TGF-Pi sequence. The effect of these peptides on
inhibition of TGF-p signaling in MvlLu cells was monitored. As shown in Figure
5.2, the peptides were mostly ineffective. Even when effective, the inhibitory effect
was very weak. The weak inhibitory effect observed could be due to the long time
period during which the ligand, TGF-pi was present in the culture. In addition, the
peptides had no effect in the absence of TGF-Pi (data not shown). One of the reasons
could be that these conditions might not be optimal to study the effect of the
peptides. Moreover, the peptide probably may inhibit the initial steps leading to the
TGF-P receptor-1 igand assembly and may be ineffective to compete with the
TGF-P ligand for binding sites on the receptor. Hence, we decided to perform a
kinetic analysis of the ligand-receptor interaction as discussed in the following
section.
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a >
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C L
2 -
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I°9 io([95-103], M)
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log1 0 ([62-76], M)
Figure 5.2. Inhibition o f TGF-P, induced signaling by peptides [95-103]
(A), [1-16] (B), [85-101] (C). [21-39] (D), [81-89] (E), and [62-76] (F). TGF-p,
induced signaling was detected as an increase in luciferase activity, in MvlLu cells
and is expressed as relative light units (RLU).
113
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5.3.2. TGF-p kinetic studies
To investigate whether the long incubation time required for the intracellular
signaling assay would reduce the ability of the peptides to compete with the ligand,
we performed a kinetic analysis o f the ligand-receptor interaction. We observed that
the time required for the signal to develop is very short. At a ligand concentration of
100 pM, a high signal is seen as early as 20-30 minutes (Figure 5.3). Furthermore,
the signal developed much faster when the ligand concentration was 1000 pM. This
suggests that the limiting step in the kinetic study is ligand diffusion rather than
ligand binding. On the other hand, it is possible that the ligand binds the receptor
quickly but the intracellular signaling complex formation takes a longer time.
5.3.3. Inhibition of TGF-P signaling by soluble TGF-p type II receptor
Our hypothesis that the time required for signaling might have an effect on
the degree of peptide inhibition was confirmed with our studies with the soluble type
II receptor. To investigate this, we incubated our cells with the complex of type II
soluble receptor and TGF-p. As shown in Figure 5.4, the inhibitory effect was more
pronounced when the cells were pulsed with the soluble receptor for a short period of
time. Based on this study we could design peptides that span the sequences of the
soluble receptor to study the ligand-receptor interaction. Furthermore, this
observation presents us with an experimental system in which pulsing of the cells
with the relevant peptide might produce detectable inhibition. However, as shown in
Figure 5.5, pulsing with the peptides that were used in Section 5.3.1 did not cause
any appreciable change in the inhibition of TGF-P induced signaling. Nevertheless,
114
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the kinetic study has provided us with a model to study the kinetics of TGF-P
ligand-receptor assembly.
6
5
3
2
C C
1
0
0.1 0.2 0.3
Time, h
0.4
1000pM
100pM
♦
4
Time, h
Figure 5.3. Kinetic analysis of TGF-p receptor complex assembly.
MvlLu cells were pulsed with indicated concentrations of TGF-Pi for varying
periods. Time shown is hours of pulsing with TGF-P|. TGF-Pi was washed from
cells and cells further incubated for approximately 14h. Signaling was detected as
an increase in luciferase activity. Results from a single representative experiment
are shown.
115
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100
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40 -
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20 -
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logioflTGF-psRII], M )
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0 -12 -11 -10 -9 -8 -7
togio([TGF-psRII, M ])
Figure 5.4. Inhibition of TGF-p i induced signaling by soluble TGF-P
type II receptor. The complex of rTGF-PsRII and I5pM TGF-P i was present at
all tiems in the culture (A). MvlLu cells were pulsed with indicated
concentrations of rTGF-psRII and 15pM TGF-P i for 30 minutes (B). The
complex was washed from cells and cells were further incubated for
approximately 14h. Signaling was detected as an increase in luciferase activity.
Data in panel B are mean + SEM of 5 experiments.
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1 .2
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Figure 5.5. Inhibition o f TGF-P 1 induced signaling following
pulsing of MvlLu cells with the peptides [81-89] (A), and [62-76] (B) for
30 minutes. Cells were washed following pulsing and incubated for an
additional 14h. Signal was detected as an increase in luciferase activity.
117
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5.4. Discussion
Synthetic oligopeptides with an amino acid sequence identical to part of a
parent molecule can be inhibitors or antagonists of the parent protein molecule. In
order to understand the ligand-receptor interactions we designed peptides that were
expected to inhibit TGF-P i induced signaling. The purpose of this study was to
elucidate the contact sites between the ligand and the receptor. The amino acid
sequences of a few of these peptides overlap one another. In all of the peptides, the
amino acid sequences of these peptides cover most of the TGF-P i sequence, which
has 112 amino acid residues. We found that the six peptides under investigation had
no or very little inhibitory effect on TGF-P induced signaling even at a concentration
of 10'9 M. Previous studies had shown that six pentacosapeptides (21-45; 31-55;
51-75; 61-85; 71-75; and 81-105) derived from the human TGF-Pi sequence had no
effect on i25I-TGF-Pi binding to TGF-P receptors in mink lung epithelial cells. In
addition, these studies showed that the peptide spanning regions 41-65 had a
profound inhibitory effect on the i2 5 I-TGF-Pi binding to TGF-P receptors. They
suggest that this region needed to contain the WSXD motif (52-55 amino acids), a
good candidate site because it corresponds to a surface exposed loop (Huang et al.,
1997).
We hypothesized that one of the reasons for the weak inhibitory effect could
be that the time required for the signaling assay was long, thus diminishing the
ability o f the peptide to compete with the ligand. In our kinetic analysis, we found
that pulsing the cells for a very short period yielded more that 80% of the signal. In
118
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addition, our results also showed that the required pulsing time was even shorter with
a higher concentration o f the ligand. This suggests that the time limiting step is
probably ligand diffusion rather than ligand binding. The ligand may bind type II
receptor very rapidly and the complete complex formation may occur slowly. A
second possibility is that TGF-Pi binds to type III receptor almost instantaneously
and then the process of the type III receptor presenting the ligand to the type II
receptor takes place more slowly. TGF-pi is known to bind with a relatively high
affinity to the type III receptor. The type III receptor is a proteoglycan, which is
suggested to be involved in the modulation of type I receptor activity. Certain
proteoglycans have been known to act as reservoir of growth factors. Hence,
extracellular type III receptors might modulate the response of cells by acting as a
nonsignaling binding protein capable of concentrating stores of TGF-P near the cell
surface (Boyd and Massague, 1989). Intracellularly, activation of the type III
receptor might initiate rate-limiting steps in the TGF-P signaling pathway.
Our hypothesis mentioned earlier, that the peptides might be weak inhibitors
because of the time involved in the signaling assay was not valid. We found that
even when the cells were pulsed with the peptide for a short period of time TGF-P
induced signaling was not inhibited. However, the kinetic studies might help us shed
some light into the early molecular events involved in the TGF-P signaling pathway.
The kinetic studies could be a starting point for the analysis of the ligand-receptor
assembly kinetics at the cell surface.
119
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TGF-P signaling was inhibited by almost 70% with the soluble rTGF-{$ type
II receptor. Furthermore, we also observed a remarkable difference in the inhibitory
effect when the cells were pulsed as opposed to continuous presence of the soluble
receptor. When the cells were pulsed, the inhibition was 70% as opposed to a 25%
when they were not. This suggests that the pulsing the cells with the peptide might
be a good model system for studying their effects on TGF-P induced signaling
pathways. Furthermore, peptides that span the regions covered by the soluble
receptor are being designed in our laboratory to determine the contact sites between
the receptor subunits and the ligand.
An understanding of the ligand-receptor interactions will facilitate in better
understanding the TGF-P mediated effects in both CD4+ and CD8+T cells. TGF-Pi
is a powerful immunosuppressive agent and is known for its therapeutic potential in
autoimmune diseases including T1DM (Piccirillo et al., 1998). A better
understanding of the assembly of the TGF-P signaling complex and the initial events
that lead to this assembly would provide us with a tool with which therapeutic
strategies can be developed.
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CHAPTER6
Summary and perspectives
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6.1. Summary
Type 1 Diabetes Mellitus or T1DM is an autoimmune disease characterized
by T cell mediated destruction of islet P-cells. The Non-Obese Diabetic (NOD)
Mouse is an excellent animal model for the study of pathogenesis of T1DM because
it shares many features with the human disease. Over time, the disease progresses
from a benign form to a malignant form leading to the destruction of p-cells (Bach
and Mathis, 1997). The complex interactions between the disease-promoting and
disease-protective factors determine the delayed or accelerated progression of the
disease.
Data obtained with animal models such as the NOD mouse suggest a disease
promoting role for Thl cytokines such as IFN-y and IL-2 and a disease-protective
role for Th2 cytokines such as IL-4 and IL-10 (Rabinovitch, 1998). Existing
evidence suggests that CD8+ T cells initiate the disease process (Wang et al., 1996),
CD4+ T cells are predominant during the early stages of the disease and both CD8+
and CD4+ T cells are required for maximum destruction of P cells. CD8+ T cells are
one of the major sources of IFN-y, during the disease initiation process and IFN-y
has been implicated to be a key cytokine in priming islet P-cells for T cell mediated
destruction (von Herrath and Oldstone, 1997).
As discussed in great detail in Chapter 1, PGEi, by virtue of its ability to
inhibit cellular immune responses is a potential protective factor in T1DM. The
model proposed in Chapter 1 suggests a feedback inhibitory role for PGEi in T1DM.
122
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The effect of PGE 2 on IFN-y release from CD 8 + T cells was investigated in Chapter
2. PGEi exerts its diverse effects on cells by binding to four subtypes of receptors
(EP receptors) (Coleman et al., 1994). In Chapter 3, the involvement o f the receptor
subtypes and the associated signaling pathway in the PGEi-mediated inhibition was
determined. Signal integration in T cells as discussed in Chapter 1 is determined by
positive and negative regulatory factors that act on T cells. The positive and negative
regulatory signals act in complex ways and can counteract each other. In CD4+ T
cells, it has been shown that EL-2 can counteract the inhibitory effects of PGE2 (Miao
et al., 1996). Furthermore, IL-12 has been shown to have no effect on the modulatory
effect o f PGE2 (Hilkens et al., 1995). The role o f IL-2 and IL-12 on PGE2-mediated
inhibition was investigated in Chapter 4. The findings are as described below:
1. PGE2 inhibits TCR-dependent IFN-y release from both NOD-derived clonal and
polyclonal CD 8 + T cells. Based on studies that show a disease-promoting role of
IFN-y, this suggests a potential disease-protective role for PGE2 in T1DM.
2. EP2 receptors mediate the inhibitory effect of PGE 2 on IFN-y release. This
conclusion is based on the following findings: Misoprostol, an EP2/EP 3/EP 4
receptor selective agonist inhibited TCR-dependent IFN-y release suggesting that
either EP 2 or EP 3 or EP4 receptors mediate the inhibitory effect. 19-OH PGE 2, an
EP2 receptor selective agonist also inhibited the TCR-dependent IFN-y release
showing that EP 2 receptors are definitely involved in the PGE2 mediated
inhibition of IFN-y release.
123
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3. The inhibitory effect of PGE2 is not mediated by EP1/EP3 receptors. This was
confirmed by using an EP1/EP3 selective agonist, sulprostone, which had no
inhibitory effect on TCR-dependent IFN-y release from the CD8 + T cell clone,
8 D8 . The efficacy of sulprostone was tested and confirmed by measuring
intracellular calcium in a cell line, P815, known to express EP3 receptors. In
addition, since sulprostone has been shown to induce uterine contractions,
sulprostone was added to NOD-derived uterine tissue. We found that sulprostone
induced contractions when added both freshly and after incubation to mimic
experimental conditions, confirming that sulprostone was indeed active even
after the prolonged culture periods used in the experiments with CD8 + T cells.
4. From the results obtained a this point during this thesis research it was clear that
EP2 receptors were involved in mediating the inhibitory effect and there was no
involvement of EP| and EP3 receptors. However, EP4 receptors could also
contribute to the PGE2 -mediated inhibition of IFN-y, since misoprostol inhibited
IFN-y release over a broader range than 19-OH PGE2 . Using curve-fitting
analysis we determined the involvement of both EP2 and EP4 receptors in the
PGE2 -mediated inhibition of IFN-y release.
5. EP2 and EP4 receptors signal by increasing intracellular cAMP. By using the
cAMP analogs 8 -Br-cAMP and Sp-cAMPS, we were able to mimic the
inhibitory effect of PGE2 on IFN-y release from both CD8 + clonal and polyclonal
T cells. These data, together with the above results, confirm the role of EP2 /EP4
receptors on the inhibitory effect by PGE2.
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6. The sensitivity of T cells to PGEi depended on their activation state. Recently
expanded cells were insensitive to the effects of PGE2 than resting cells or cells
expanded two weeks before stimulation though the TCR. Sensitivity of CD8+ T
cells to PGEi is also determined by the TCR signal strength.
7. PGEi-mediated inhibition of IFN-y release is partially reversed by IL-2. This
suggests that accessory signals such as IL-2 could counteract the inhibitory effect
of PGE2 and the precise combination of these factors determines the benign or
malignant nature of the islet-inflammatory infiltrate.
8. However, IL-12, an accessory cell derived cytokine had little or no effect on the
inhibitory effect of PGE2. Both factors appeared to act independently from each
other. This further suggests that in autoimmune diseases such as IDDM a delicate
balance between the disease-promoting and protective factors determine the
progression of disease.
TGF-P is an immunomodulatory agent known to inhibit CD8+ T cell
mediated immune responses. Since previous studies have extensively discussed the
effect of TGF-P in T-cells, the emphasis of this research was to understand the
ligand-receptor interactions that lead to the TGF-p mediated effects in T-cells. In
order to address this issue, we performed structural studies using peptides derived
from the TGF-P 1 sequence and kinetic analysis that facilitated our studies using
peptides (Chapter 5). Our findings are as discussed below:
1. Peptides derived from the TGF-Pi ligand sequence had no inhibitory effect on
TGF-p induced signaling.
125
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2. A kinetic analysis revealed that the time required for treating the cells with the
ligand is rather short and also depended on the ligand concentration. These
"pulsing" experiments suggested that the limiting step in signaling might not be
ligand binding but rather ligand diffusion. The kinetic analysis provides us with a
model to study the ligand-receptor assembly.
3. TGF-P induced signaling was inhibited by the soluble form of the type II
receptor. This confirmed that the protocol in which the cells were pulsed with the
ligand for a short period was a useful system to study ligand-receptor assembly.
6.2. Future directions
In the light of the above findings, we can extend our research to the following
areas:
1. Studies have shown that in macrophages exogenous PGEi and endogenous PGEi
have contrasting effects on macrophage function. Hence, it would be interesting
to investigate what effect, if any, endogenous PGEi would have on CD8+ T cell
function.
2. Alterations, if any, in EP receptor expression between NOD and control
mice-derived CD8+ T cells can be explored by characterization of EP receptor
expression in T cells from different strains.
3. Quantitation of soluble factors such as IL-2 and IL-12 in the islet
microenvironment in addition to that of PGEi can be done to understand the
balance between both promoting and protective factors.
126
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4. The soluble form of the type II receptor was effective in inhibiting the TGF-P
induced signaling. Hence, peptides derived from the exoplasmic domain of the
type II receptor could be designed and their effect on inhibition of TGF-P
signaling studied.
5. The short ligand-binding time observed in our kinetic studies could be due to the
binding of the ligand to the type III receptor and subsequent presentation of the
ligand to the type II receptor. The involvement of the type III receptor in
ligand-receptor interactions could be tested by pulsing the cells with the ligand
and then subsequently blocking the type II receptor binding sites with the soluble
form of the type II receptor.
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Asset Metadata

Creator Ganapathy, Vidyalakshmi A. (author)

Core Title Endogenous regulatory factors in the inhibition and down-regulation of immune responses

School Graduate School

Degree Doctor of Philosophy

Degree Program Pharmaceutical Sciences

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biology, molecular,health sciences, immunology,Health Sciences, Pharmacy,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Hamm-Alvarez, Sarah (committee chair), [illegible] (committee member), Okamoto, Curtis T. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-151081

Unique identifier UC11328305

Identifier 3054736.pdf (filename),usctheses-c16-151081 (legacy record id)

Legacy Identifier 3054736-0.pdf

Dmrecord 151081

Document Type Dissertation

Rights Ganapathy, Vidyalakshmi A.

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus, Los Angeles, California 90089, USA