Identification and characterization of monocyte prostaglandin synthase 2 activity as a risk factor for and a component o...

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Identification and characterization of monocyte prostaglandin synthase 2 activity as a risk factor for and a component of human autoimmune disease
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Litherland, Sally Ann, 1960-
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Monocytes   ( mesh )
Prostaglandin Endoperoxide Synthase   ( mesh )
Risk Factors   ( mesh )
Dinoprostone   ( mesh )
Autoimmune Diseases -- etiology   ( mesh )
Diabetes Mellitus -- etiology   ( mesh )
Diabetes Mellitus, Type I -- etiology   ( mesh )
Gene Expression Regulation   ( mesh )
Signal Transduction   ( mesh )
Research   ( mesh )
Department of Pathology and Laboratory Medicine thesis Ph.D   ( mesh )
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Thesis:
Thesis (Ph.D.)--University of Florida, 1997.
Bibliography:
Bibliography: leaves 66-80.
Statement of Responsibility:
by Sally A. Litherland.
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Typescript.
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Vita.

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IDENTIFICATION AND CHARACTERIZATION OF MONOCYTE
PROSTAGLANDIN SYNTHASE 2 ACTIVITY AS A RISK FACTOR FOR AND
COMPONENT OF HUMAN AUTOIMMUNE DISEASE












By


Sally A. Litherland


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

UNIVERSITY OF FLORIDA


1997

























Copyright 1997

by

Sally A. Litherland




























For my parents and family

and in loving memory of

Mrs. Nellie Morgan and Dr. Les Morgan,

my guardian angels and my inspiration.














ACKNOWLEDGMENTS


When a project of this magnitude is undertaken even under normal

circumstances, it represents the cooperative effort of many hard working

individuals. When done on an accelerated timeline as this one, it would be

impossible without a coordinated team effort of experienced experts,

dedicated trainees, and talented support personnel. The author wishes to

express her heartfelt gratitude to all who contributed to the successful

completion of this work.

First and foremost, the author wishes to thank her mentor, Dr. Michael J.

Clare-Salzler, who took a chance on the unknown commodity that was this

very determined individual, and allowed her to try her own ideas as well as

freely share his own with her. Mike is not only a gifted doctor and scientist,

but also a truly good human being. The author is honored to call him mentor

and friend.

This work would not have been possible without the generous

participation of the many men, women, and children who volunteered to

donate their time and blood for analysis. The author wishes to express her

greatest appreciation and admiration for such selflessness in the name of

helping others who may be affected by autoimmune disease.

Next, the author thanks the other members of her committee, Dr.

Ammon Peck, Dr. Edward Wakeland, and Dr. Charles Allen, for their

consistent support of this doctoral work, their friendship, and sage advice.









Special thanks go to the Flow Cytometry laboratory personnel, Melissa

Chen, Neal Benson, and Kristie Grebe, for all the kind help and advice in

development of the methodology, data collection, and analysis for the flow

cytometric data. Thanks too to the author's laboratory personnel, especially

Dr. Tony Xie, whose seminal work in autoimmune mice was the basis and

rationale behind this work in humans.

The author also thanks her friends and colleagues for their support and

friendship, especially Scott Reno, Kryzsztof Witek, Win Noren, Robbin

Chapman and the rest of her SLSTP students and colleagues, Mimi Shao and

the Mission Operations Crew, Dr. Lyle Moldawer and his lab group, Rose

Mills, Bertha and Javier Sanchez, Donna Whittaker, Ramelle Ruff, Joanne

Johnson-Tardieu, Clive Wasserfall, Janet Cornellis, Dr. Jeff Anderson, Roberta

Cook, Mary Alice Dennis, and Karen Fuller.

Finally, but most importantly, the author wishes to thank her parents,

Allyn and Regina Litherland, for their faith in her, their love, and their

unfailing support of her educational endeavors.














TABLE OF CONTENTS


ACKNOWLEDGEMENTS........................................................ iv

ABSTRAC T.......................................................................... viii

CHAPTERS

1 EXPERIMENTAL BASIS AND RATIONALE...............................

Autoim m unity............................................................. 1
Autoimmune Insulin Dependent Diabetes....................... 1
Antigen Presentation in Autoimmunity............................. 3
Activation Induced Cell Death in Autoimmunity................ 5
Prostaglandins and Their Roles in Immune Responsiveness. 10
Prostaglandin E2 and T cell Responses........................... 16
PGE2 Role in Diabetes in the NOD................................. 18
IL10 Role in NOD Diabetes............................................. 21
PGE2 in Human Autoimmune Disease............................ 21

2 MATERIALS AND METHODS................................................ 24

Human Subject Populations........................................... 24
Sam ple Preparation....................................................... 25
Ex vivo Analysis of PGS2 Expression by Intracellular Flow
Cytom etry........................................................ 29
Monocyte Activation Analysis by Flow Cytometry ............. 33
In situ Immunohistochemical Analysis of PGS2 and Cellular
M arkers............................................................ 34
RT-PCR Analysis for PGS2, FAS, and Cytokine Profiles........ 35
Ceram ide Analysis........................................................ 35
Passive Cell Death, Cell Cycle Effects, and AICD Induction
A nalyses........................................................... 37
PGS2 Activation and Inhibition Assays............................ 39
Measurement of T cell Activation in PBMC Cultures ........... 40
Analysis of Hormonal Influence and Menstrual Cycle
Effects on PGS2 Expression in PBMC..................... 42
Statistical Methods for Data Analysis.............................. 43










3 RESULTS ........................................................................... 44

Identification of Aberrant PGS2 Expression in Humans with
Autoimmune Disease and Individuals at Risk for
Autoimmune Diabetes........................................ 44
Autoimmune CD 14+ Monocytes Express PGS2 without Co-
Expression of Other Activation Markers................. 46
PGS2 Expression Correlates with Clinical and Genetic
Markers of IMD High Risk in the Pre-diabetic
Population Tested............................................... 49
Age and Gender Affects the Expression of PGS2 by
Autoimmune PBMC............................................. 51
Insensitivity of PGS2 Expression to Suppression by IL10..... 52
PGE2 Inhibition of CD25 Expression on Activated T cells..... 53
CD8+ T cell Subpopulation Bias in CD25 Expression in
Autoimmune PBMC.............................................. 55
PGE2 Mediated CD25 Suppression Contributes to its Block
of A IC D .............................................................. 56
Decreased Spontaneous Cell Death and an Increase of
Cells in GO/G 1 and in Cycle in Autoimmune PBMC... 58
Ceramide Levels are Elevated in Autoimmune PBMC ......... 58

4 DISC USSIO N ..................................................................... 61

REFERENCES CITED............................................................. 66

BIOGRAPHICAL SKETCH....................................................... 81















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

IDENTIFICATION AND CHARACTERIZATION OF MONOCYTE
PROSTAGLANDIN SYNTHASE 2 ACTIVITY AS A RISK FACTOR FOR AND A
COMPONENT OF HUMAN AUTOIMMUNE DISEASE

By

Sally A. Litherland

August, 1997



Chairman: Michael J. Clare-Salzler, MD
Major Department: Pathology, Immunology, and Laboratory Medicine
Recent work in the nonobese diabetic mouse has demonstrated that

macrophage produced prostaglandin E2 (PGE2) contributes to antigen

presenting cell dysfunction and the development of diabetes. The

overproduction of PGE2 was the result of constitutive expression of the

normally inducible cyclooxygenase, prostaglandin synthase 2 (PGS2).

In this study, PGS2 expression was examined in over 200 samples of

human peripheral blood monocytes from normal controls, relatives of

autoimmune patients, and members of a test subject group consisting of

autoimmune individuals, diabetics, and individuals at low, moderate and high

risk for immune mediated diabetes (IMD). Aberrant PGS2 expression in

unactivated peripheral blood monocytes was found in 37% of the test subjects

as compared with 4% of normal controls. PGS2 expression correlated
viii









inversely with insulin secretary capacity, a diagnostic measure of risk for

diabetes; indicating that aberrant PGS2 expression may predispose

individuals to high risk of IMD.

Six of twelve subjects tested had lost sensitivity to interleukin 10

suppression, a normal regulatory control of PGS2 expression. Further

investigation is needed to determine if this type of regulatory dysfunction is

an underlying mechanism for the defect in vivo.

When PGS2 expression was inhibited in vitro, test subject T cells

increased IL2 receptor expression (CD25). The PGE2 suppression of CD25

correlates positively with the IMD susceptibility major histocompatibility

locus alleles DR4 and DQ00302. Through its effect on CD25 PGE2 inhibits

interleukin 2(IL2) signal transduction in T cells, a critical signal for T cell

activation, proliferation, and activation induced cell death (AICD).

Subject cells were resistant to AICD induced by anti-FAS antibody and

had an accumulation of ceramide, a second messenger in cell death signal

transduction. Ceramide accumulation was directly correlated with the

increase in PGS2 expression found in these individuals. Passive cell death,

defined as spontaneous death of cells ex vivo, was significantly lower in test

subject cells than controls. Concurrently, the percentage of cells remaining in

GO/G 1 and entering cycle was significantly higher.

This study confirms the identification of aberrant PGS2 expression as a

risk factor for human IMD and presents evidence for its role in the

immunopathogensis of multiple autoimmune diseases.














CHAPTER 1
EXPERIMENTAL BASIS AND RATIONALE

Autoimmunity

In autoimmune disease, normal regulatory controls for self-tolerance

are superseded and the body's immune defenses are turned against its own

tissues (Cohen and Young, 1992; Mountz et al., 1994;Ucker et al., 1994).

Tolerance of self antigens by peripheral blood mononuclear cells (PBMC) is

maintained by regulatory mechanisms including energy, active suppression

by regulatory cells and factors, and elimination of self-reactive cells by

Activation Induced Cell Death (AICD)(Squier et al., 1995; Podack, 1995).

Though normal healthy individuals can have autoreactive immune cells, these

are controlled either by elimination of the cells themselves or functional

regulation through a complex system of cell and cytokine interactions. If the

control of these cells is compromised, immune mediated pathology can result,

leading to overt autoimmune disease (Cohen and Young, 1992; Mountz et al.,

1994;Ucker et al., 1994).

Autoimmune Insulin Dependent Diabetes

One of the most common of autoimmune diseases in humans is immune

mediated diabetes (IMD), also referred to as insulin dependent diabetes

mellitus type (IDDM1) or juvenile onset diabetes. This debilitating disease

afflicts 1 out of every 5000 Americans and leads to major health complications

that are often life threatening. IMD is metabolically characterized as insulin

deficiency manifesting as severe blood hyperglycemia and cellular

hypoglycemia. Patients diagnosed with IMD must modify dietary intake, self-









monitor blood glucose, and take subcutaneous insulin injections in order to

prevent the development of diabetes-related complications including

ketoacidosis, retinal disease, kidney disease, neuropathology, and

atherosclerosis. Individuals with IMD have a reduced life expectancy by 10 to

20 years as a result of such complications (Winter et al., 1993).

The only confirmed genetic susceptibility locus for IMD is the HLA,

major histocompatibility locus (MHC). HLA DR and DQO loci are linked to

both IDDM susceptibility and IDDM resistance. Alleles DR 03 and DR 04 and

DQO 0302 and DQP 0201 have been described as susceptibility loci; whereas,

DR 02 and DQP 0602 have been defined as protective alleles for IMD (Winter

et al., 1993; Faustman, 1993). In addition, approximately 14 non-

histocompatibility Iddloci linked with IMD susceptibility have been

described, though the genes within these loci are largely undefined (Winter

et al., 1993).

Individuals genetically predisposed to IMD are further classified with

regard to risk by the presence of autoimmune antibodies in their serum.

Insulin autoantibodies (IAA+), anti-GAD like domain antibodies (GAD+), and

anti- islet cell antigens (ICA+) antibodies can be found in these individuals

prior to the manifestation of clinical symptoms. None of these autoantibodies

are thought to play a role in the pathology of IMD, but their presence in serum

is considered indicative of autoimmunity. Of these autoantibodies, ICA+

antibodies are considered the most predictive for increased risk for IMD

(Maclaren et al., 1975).

Unlike many autoimmune diseases, human IMD does not have a clear

gender bias in its incidence. However, there is an age effect seen in IMD

onset. Higher incidence of disease occurs in pre-pubescent children and

adolescents who are at genetic risk for IMD. The peak onset of IMD is found









during puberty and maybe linked to hormonal effects that occur with growth

and development(Winter et al., 1993, Leslie and Dubey, 1994).

The Nonobese Diabetic Mouse Model for IMD

Great advances in the understanding of human IMD have come from

the genetically inbred mouse model, the nonobese diabetic (NOD) mouse,

and congenic strains derived from this line (Serreze et al., 1989; Serreze et al.,

1993; Serreze and Leiter, 1988; Serreze and Leiter, 1994; Wicker et al., 1995;

Yui et al., 1996). When the mice are housed in a specific pathogen free

environment, NOD female mice have an 80% incidence of spontaneous

diabetes, while only 20% of males develop diabetes (Serreze and Leiter,

1994). The disease onset is at approximately 5-8 weeks of age. The pathology

of the disease in the NOD mice has features in common with IMD in humans,

including infiltration of the islets of Langerhans (insulitis), loss of insulin

producing islet (3 cells, MHC involvement, and the development of

autoantibodies. The condition is lethal in mice, as in humans, without direct

intervention with insulin replacement therapy (Serreze et al., 1989; Serreze et

al., 1993; Serreze and Leiter, 1988; Serreze and Leiter, 1994; Wicker et al.,

1995; Yui et al., 1996). The NOD mouse strain also develops autoimmune

destruction of salivary and lacrimal gland function similar to Sjogren's

Disease, and autoimmune thyroid disease (Faveeuw et al., 1994).

Antigen Presentation in Autoimmunity

Antigen presenting cell (APC) activation of T cells is impaired in both

animal models and humans with a genetic predisposition for immune

mediated diabetes (Lee et al., 1988; Ihm and Yoon, 1990; Clare-Salzler and

Mullen, 1992; Clare-Salzler et al., 1992; Clare-Salzler, 1994 and 1995). Defects

found in the MHC locus of both NOD mice and humans contribute to APC









impairment (Winter et al., 1993; Serreze et al., 1993; Serreze and Leiter, 1994;

Clare-Salzler et al., 1992).

T cell receptor (TCR) stimulation is the primary signal for activation of

specific T cells. Antigen presentation by self MHC on APC provide the TCR

with a critical stimulus for T cell activation, as can anti-CD3 antibody binding

and superantigen crosslinkage of TCR to MHC (Groux et al., 1993, Kawabe and

Ochi, 1991). APC transfer experiments in NOD mice have shown a protective

effect of dendritic cells against the development of autoimmune diabetes

(Clare-Salzler et al., 1992). These findings give evidence that highly

stimulatory APC-induced T cell stimulation is essential in the regulation of

self- tolerance (Clare-Salzler et al., 1992; Clare-Salzler and Mullen, 1992;

Jansen et al., 1994).

Interactions of APC and T cells are crucial to the regulation of the

immune response. During maturation in the thymus, stem cell derived

precursors of T lymphocytes encounter MHC presented self antigens in its

ontogeny. These cells must run a gauntlet of interactions with APC: testing for

both negative and positive responsiveness to self. Failure to present the

correct level of self-recognition at each stage of this process, that is, MHC

recognition for positive selection and low self antigen recognition for negative

selection, results in the elimination of such cells before they can be released

into the periphery (Jones et al., 1990; Webb et al., 1990).

Not all self antigen-recognizing lymphocytes are eliminated during

ontogeny. Autoreactive cells are found in the peripheral blood of both

nonautoimmune and autoimmune individuals. In the periphery, qualitative

and quantitative differences in the antigen presentation and the

microenvironment of the APC-T cell interaction control the outcome of T cell









activation. APC presentation of self-antigens to mature T cells leads to the

induction of tolerance; whereas, presentation of foreign peptides can start the

T cell activation process necessary for mounting a specific immune response

(Ucker et al., 1993, Clare-Salzler, 1995).

Quantitative differences in interaction with antigens during T cell-APC

encounters have been implicated as a major component for the production of

regulatory T cells as opposed to the production of effector T cells (Ucker et

al., 1993). High levels of antigen presentation are thought to lead to

production of suppressor/regulatory functional T cells; whereas, lower levels

of T cell activation would lead to the development of effector cells (Ucker et

al., 1993).

The presence or absence of co-stimulation is critical to how a T cell

interprets the primary antigen signal from the APC. Secondary signaling

involving B7:CD28 surface molecule interactions allows for full activation of

responsive T cells, so that they proliferate and differentiate, and mount an

immune response to the antigen. Modulation of the second signal, such as

delaying or blocking of the CD28:B7 signal, induces energy. Cytokine

mediated signals, such as ILl, IL4, IFNy, and IL2, promote T cell

differentiation. Variations in the quality and quantity of activation signals also

contribute to positive regulation of FAS and FASL surface molecules, priming

the system to eliminate cells and to control or terminate an immune response

(Cook and McCormick, 1995).

Activation Induced Cell Death in Autoimmunity

In normal, healthy individuals, selective cell death is used to control the

immune response of T cells recognizing self antigens (Martin et al., 1994;

Majno and Joris, 1995; Squier, 1995; M. Chen et al., 1995). Selective cell death









is an effective means to maintain control over potentially damaging activation

events that could turn the immune system's defenses against self (Squier et al.,

1995). This cell death is thought to be mediated by activation induced cell

death (AICD). Only cells undergoing high levels of activation are sensitive to

AICD, yielding selectivity in the process. Impairment of AICD may be a

critical event in the immunopathogensis of autoimmune diseases as it may

allow for the accumulation and inappropriate activation of autoreactive T cells

(Sneller et al., 1992; Mountz et al., 1994; Thompson, 1995).

When the AICD pathway of a cell is triggered by biochemical signals

and events, it leads to the orderly disassembly of the cell's structure into

membrane enclosed particles termed apoptotic bodies (Steller, 1995). These

particles allow for phagocytic removal the dying cell by surrounding cells or

professional phagocytes without concurrent release of intracellular

components. The packaging of the cell remnants prevents an inflammatory

response from being triggered by release of intracellular antigens and

biochemically reactive components; thereby, decreasing the chances of cell

damage by a self-directed immune response (Mountz et al., 1994; Steller,

1995).

Elimination of autoimmune T cells through AICD is a mechanism used in

the development of peripheral tolerance (Liu and Janeway, 1990). This

elimination of self-responsive T cells has been found to be specific for antigen

or superantigen activated cells. This specificity of AICD is mediated by the

TCR-mediated recognition of the antigen-MHC complex (Webb et al., 1990;

Groux et al., 1993).

AICD acts in concert with cell proliferation to control the overall

immune response. Like proliferation, AICD of T cells occurs in the setting of









cell activation mediated through the TCR. Superantigen induced activation of

the TCR in vivo and in vitro has been used as a model system in which to

define the progress of an immune response from induction to termination. In

this model system, superantigens such as Staphylococcus aureus enterotoxin

B (SEB) and retroviral MTV Mis- 1 a protein, specifically activate a VP subclass

of T cells (Kawabe and Ochi, 1991;Huang and Crispe, 1993; Gonzalo et al.,

1994;Weber et al., 1995; Nishimura et al., 1995). These superantigens first

stimulate the T cells of the specific TCR subclass to rapidly proliferate. Fully

activated T cells undergo an array of changes including phospholipase A2

(PLA2) induction, reactive oxygen intermediate generation, down regulation

of the TCR and other changes in their cell surface markers including

upregulation of FASand FASL expression (Huang and Crispe, 1993;Weber et

al., 1995;Nishimura et al., 1995). The T cell response is rapidly diminished by

elimination of the activated T cell population. The expression of FASand FASL

is a critical for the induction of apoptosis in responding T cells by AICD

(Kawabe and Ochi, 1991; Dhein et al, 1995). These experiments together

suggest that the progression of cells from activation to death is a normal

regulatory mechanism for control of the lymphocyte population.

Signaling for Induction of AICD in the Immune System

AICD is an active process, requiring protein synthesis and signal

transduction to initiates events within the nucleus of the cell (Steller, 1995). It

is thought that all cells are capable of undergoing a preprogrammed 'suicide'

process (Ucker et al., 1993; Nagata and Golstein, 1995).

In the immune system, the FAS receptor/FASLligand binding system is

known to induce AICD in the FASbearing cell(Alderson et al, 1993). FAS/FASL

receptor-ligand proteins have been cloned and studied in humans and mice









(Nagata and Golstein, 1995; Suda et al., 1993; Alderson et al., 1993; Cheng et

al., 1995). These proteins are members of the TNFca-TNFaR families of

regulatory proteins (Nagata and Golstein, 1995; Owen-Schaub et al., 1992).

FAShas been found to be homologous to APO-1 (Cifone et al., 1993; Krammer

et al., 1994). FASis a receptor protein expressed on almost all cells of the

body, but is either in an inactive state or in low abundance so that it is not

readily recognized by the FASL bearing cells of the immune system under

normal conditions (Nagata and Golstein, 1995). FASL, its ligand, is inducible

and found on few cell types including monocytes and both CD4+ and CD8+

subpopulations. FASL binding of FASis a primary mechanism of cytotoxic T

lymphocytes for killing of targeted cells by apoptosis (Owen-Schaub et al.,

1992; Podack, 1995). AICD mediated by FAS/FASL interactions first requires

recognition of antigen through the TCR, activation of the T cell, and

cooperation of the target cell for cell death to occur. Triggers for FASL

induction include TCR-MHC engagement either by antigen or TCR-directed

antibody, starvation for growth factors, cell maturation, energy, and

stimulation by TNFa, IFNy ILl ,antigen, superantigen, or GM-CSF and CSF-

l(Groux et al., 1993; Huang and Crispe, 1993;Wu et al., 1994; Kim et al., 1991;

Liu and Janeway, 1990; Kawabe and Ochi, 1991; Brunner et al., 1995). Many of

these AICD triggering molecules are also known to stimulate T cell

proliferation. The variation in the response to the same factor may be

dependent on quantitative variations and factors influencing the cell and its

microenvironment (Alderson et al., 1993; Gulbins et al., 1995).

The discovery of two mutant strains of mice, lpr/lprmice and gid/gld

mice, has suggested the importance of AICD in the immunopathogensis of

autoimmune diseases (Sneller et al., 1992; Chervonsky et al., 1997). Lpr/lpr









mice are defective in their production of FAS receptor; whereas, gld/g1dmice

are deficient in FASL (Watanabe-Fukunaga et al., 1992). The FAS-FASL signal

transduction pathway is considered the primary mechanism for AICD in

immune cells. These mice are viable but have high levels of nonmalignant

lymphocyte proliferation as well as develop lymphomas and leukemias at an

unusually high rate. This increase in both nonmalignant and cancerous

immune cell overgrowth in these mice, suggests that the loss of AICD

dramatically affects the normal clonal deletion of lymphocytes. In the

periphery, proliferating 'undying' self-reactive cells found in these mice

increase their potential risk for the development of autoimmunity (Howie et

al., 1994). These mouse strains eventually develop characteristic autoimmune

responses and disease that parallel those seen in human systemic lupus

erthematosus (SLE)(Sneller et al., 1992; Emlen et al., 1994).

Signal Transduction of AICD

Once a cell receives the AICD signal through FAS-FASL or TNFa-

TNFaR binding, the next steps in the process are to transmit the signal via

secondary and tertiary messengers to the nucleus of the cell, prompting

activation of the death program genes and deactivation of cell proliferation

genes(Mountz et al., 1994).

When a cell is signaled to undergo AICD by FAS/FASL binding, PLA2

activity is induced within the cell (Jayadev et al., 1994; Schutze et al., 1994;

Hannun, 1994; Hannun and Obeid, 1995). The induction of PLA2 in turn

catalyzes the turnover of fatty acids and phospholipids in the membrane

(Jayadev et al., 1994; Wu et al., 1994; Obeid et al., 1993). As the catabolism of

the membrane lipids continues, the cell flips portions of the lipid bilayer of its

outer membrane so that phosphoserine containing regions are now visible on









the outer leaf of the membrane (Steller, 1995). This abnormal lipid

arrangement is recognized by receptors on phagocytic cells and causes the

removal of the dying cell even before it completes the AICD process (Hannun

and Obeid, 1995). Activation of lipid metabolism in the membrane also

triggers activation of the enzyme sphingomyelinase within the cell which in

turn cleaves sphingomyelin from the cell membrane into fatty acids and

ceramide (Cifone et al., 1993). The actions of ceramide effectively turn off

expression of genes that drives the cell through its cycle and induce others

that are involved in the progression of the AICD (Kinoshita et al., 1995;

Hannun, 1997; Gill et al., 1994). Ceramide acts a signal transduction molecule

in AICD as well as blocking cell cycle and cell survival functions through its

effects on BCL2 and Rb gene product function (Hannun, 1994; Hannun and

Obeid, 1995;Bose et al., 1995; Hannun, 1997). Ceramide-activated kinases and

phosphatases are involved in the activation of chromosomal degradation

enzymes that digest the chromatin of the cell into discrete, nucleosomal size

fragments (approx. 180 bp)(McConkey et al., 1994; Martin et al., 1994; Tian et

al., 1995). This uniform or 'ladder' of chromatin fragmentation is considered

diagnostic for differentiating apoptotic cell death from oncotic processes of

necrosis (Majno and Joris, 1995). In cell death triggered by daunorubicin, a

chemotherapeutic agent, it has been shown that even sphingomyelinase-

independent production of ceramide allows for the propagation of the AICD

signal (Bose et al., 1995).
Prostaglandins and Their Roles in Immune Responsiveness

Prostaglandins (PG) are lipid metabolytes derived from the free fatty

acid, arachidonic acid (AA)(Hyslop and Nucci, 1993; DeWitt and Smith, 1995).

The prostaglandin family of molecules is important in regulation of









inflammatory responses. Prostaglandin E2 (PGE2), the predominant species

produced by blood monocytes and macrophages, is an immunomodulating

molecule with potent effects on T lymphocyte activation and function (Figure

1; DeWitt and Smith, 1995).

Figure 1. Biosynthesis Pathways for Prostaglandins. Adapted from Dewitt and Smith, 1995.


PHOSPHOUPIDS, TRIGLYCERIDES, CHOLESTEROL ESTERS

SUPASES





ARACHIDONIC ACID

CYCLOXKYGENASE PGH SYNTHASE: PGS1 OR PGS2

PGG2
PEROXIDASE

PGH2



PGD2 PCG2
SYNIITHASES



PGFa TX


o>-- /- / COON


OH

PGE2








Synthesis of Prostaglandins

PG synthesis begins with the release of AA from cell membrane lipids

through the actions of phospholipases (PL)(Hyslop and Nucci, 1993). AA is

converted to PGH2 through the cyclooxygenase and peroxidase activities of

prostaglandin synthase, PGS. This enzyme is found in 2 isoforms, PGSl(COXl)

or PGS2(COX2). Further conversion of PGH2 to the other PG species,

including PGE2, requires additional enzymes that are cell specific in their

expression. The prostaglandin synthase step of the pathway is rate-limiting

and the focus of regulatory mechanisms controlling prostaglandin production

(DeWitt and Smith, 1995).

PGS 1 is a developmentally regulated enzyme that promotes and

mediates so-called 'housekeeping' functions of circulating hormones in a wide

variety of cell types. Its expression is constitutive and dependent on the

availability of AA substrate from the actions of cytoplasmic membrane bound

PLA2. PGS1 is located in the endoplasmic reticulum membrane and its

product acts in both autocrine and paracrine fashions (Morita et al., 1995:

DeWitt and Smith, 1995). Homozygous transgenic PGS 1 knockout mice are

viable and have no apparent pathology if born from heterozygote mating,

suggesting that the functions of this enzyme after parturition are not unique

and can be replaced by other activities (Langenbach et al., 1995).

PGS2, on the other hand, appears to have essential functions in kidney

development, regulation of inflammation and immune response (Morham et

al., 1995). PGS2 is bound to the nuclear and nearby endoplasmic reticulum








membranes(Morita et al., 1995). It derives its substrate pool from the

induction of cPLA2, a cytoplasmic enzyme that releases AA from cellular lipids

in response to activation stimuli (Hyslop and Nucci, 1993). With the cloning

and sequencing of both genes, it was found that PGS2 is structurally related,

but unique, when compared to PGS 1 (Goppelt-Struebe, 1995). The PGS2 gene

resides on the distal arm of chromosome 1 in both the human and murine

genome (Kosaka et al., 1994). This enzyme is expressed in only a limited

number of tissues; mostly as an induced activity during immune and

inflammatory responses (Riese et al., 1994). A low basal level of PGS2

expression is found in the immunoprivileged sites, brain and testes, as well as

a unique and essential expression found in the macula densa of the kidney

(DeWitt and Smith, 1995). Transgenic PGS2 knockout mice develop poorly,

and die within 6 weeks after birth, usually due to kidney malfunction and

nephritis. These mice also exhibit suppressed immune responsiveness

including poor macrophage responsiveness to endotoxin (LPS) stimulus

(Sneller et al., 1992; Morham et al., 1995; Tsujii and DuBois, 1995;Chervonsky

et al., 1997).

Monocyte/Macrophage Prostaglandin Synthase 2 Expression

Peripheral blood monocytes and tissue macrophages are the major

sources of PGE2 within the human immune system. Its expression is

considered a hallmark of monocyte and macrophage activation (Sweet and

Hume, 1996). During an immune response, PGE2 production is enhanced by

the induction of PGS2 expression. Monocyte activation by ILl a & (3, TNF a, or








LPS promotes PGS2 expression within the 6 hours of stimulation (Sweet and

Hume, 1996; Ristimaki et al., 1994; Reddy and Herschmann, 1994). When PGS2

expression is accompanied by a cPLA2 catalyzed release of AA, its activity

promotes the production of PGE2 (Dewitt and Smith, 1995).

Regulation of PGS2 expression is done through a complex series of

signal cascades involving multiple points of feedback regulation by its

product, PGE2. TNFa and ILl a & 0 stimulation of monocytes induces PGS2

expression as well as IL12 production. IL12 in turn promotes even higher

PGS2 expression. IL12 also promotes IL10 production from monocytes and

other immune cells (Segal et al., 1997; Gerosa et al., 1996; Daftarian et

al.,1996; Ludviksson et al., 1997; Ehrhardt et al., 1997). IL10 acts as a major

regulatory suppressive signal for immune response effects including IL12,

TNFa, IL1 a & 0, and PGS2 expression as well as the induction of cell surface

receptors for secondary activation signals. (de Waal Malefyt et al., 1991;

Isomaki et al., 1996; Mertz et al., 1994; Spittler et al., 1995; Meisel et al., 1996;

Dai et al., 1997; Takenaka et al., 1997). IL10 suppresses PGS2 expression

approximately 16-18 hours after activation (D'Andrea et al., 1993; Strassmann

et al., 1994; Niiro et al.,1995; Berger et al., 1996). Therefore, PGS2 expression

is self-limiting; in that its product, PGE2, downregulates IL12, TNFa, and IL-1,

promoters of PGS2 expression, and upregulates IL10, a suppressor of PGS2

(van der Pouw Kraan et al., 1995; Ludviksson et al., 1997).








Prostaglandin Effects on Immune Microenvironment

PGE2 is a potent modulator of the inflammatory response and is present

in abundance during the early response phase of inflammation.

Prostaglandins have a physiological role in pain, fever, vasodilatation leading

to localized swelling and heat, and phagocytic activity enhancement for

activated macrophages and natural killer cells present at wound sites (Lu et

al., 1995; DeWitt and Smith, 1995).

Some of same signals that promote PGS2 expression and PGE2

production also promote AICD. These include ILl a & 0, TNFa, IFNy, and AA

(Watson and Wijelath, 1990; Kim et al., 1991; Cifone et al., 1993; Obeid et al.,

1993; Jayadev et al., 1994; Ristimaki et al., 1994; Wu et al., 1994). Ceramide,

along with its role as a signal transduction molecule in AICD, also promotes

the production of PGE2 through induction of PGS2. In turn, PGE2 suppresses

the signal transduction activities of ceramide (Ballou et al., 1992; Hannun,

1997). The mechanisms involved in this feedback regulation intertwining

these two signal pathways are still poorly understood (Ballou et al., 1992;

Jarvis et al., 1994a and 1994b; Hannun, 1994; Hannun and Obeid, 1995).

Many of PGE2 effects on cells are mediated through cAMP (Foegh,

1988;Holter et al., 1991; Snijdewint et al., 1993). Changes in cellular levels of

cAMP affects protein kinases involved in cell activation and proliferation

control (Anastasia et al., 1992; Paliogianni et al., 1993; Riese et al., 1994; van

der Pouw Kraan et al., 1995). PGE2 elevated cAMP levels affects RAS








dependent gene expression including Rafand rasp21 expression (Pastor et

al., 1995; Cook and McCormick, 1993; Gulbins et al., 1995). These genes are

involved in the activation of MAP kinases which are crucial to the activation of

cell proliferation gene expression as well as signal transcription in AICD

(Baixeras et al., 1994; Goetzl et al., 1996).

Prostaglandin E2 and T cell Responses

Prostaglandins are potent modulators of T cell activation. PGE2 effects

on T cell regulation appear to be two-fold: 1) modulation T cell function and 2)

inhibition of AICD signal transduction (Lenardo, 1991; Lu et al., 1995). PGE2

role in cell death shares mechanistic components with its role in the control of

cell activation.

Prostaglandin E2 effects on IL2 signal transduction

PGE2 induced cAMP inhibits T cell expression of IL2 and its receptor

(IL2R) (Minakuchi et al., 1990; Anastassiou et al., 1992; Paliogianni et al., 1993;

D. Chen et al., 1994). By blocking IL2 production and IL2 signal transduction,

prostaglandins effectively inhibits T cell proliferation and block their

progression to AICD.

PGE2 specifically inhibits the upregulation of the alpha subunit (a,

CD25) of IL2R (Antonaci et al., 1991; Giordano et al., 1993). This subunit is

expressed on T cells after antigen stimulus and is considered a marker for T

cell activation (Schorle et al., 1991; Antonaci et al., 1991; Giordano et al.,

1993). The 1 and y subunits of the IL2R are constitutively expressed as a low








affinity receptor on mature resting T cells and promote IL2 signal transduction

leading to cell proliferation. The signal from this low affinity receptor

promotes BCL2 and c-myc driven cell functions that preserve cell viability and

drive cell cycling (Ahmed et al., 1997). The a subunit of IL2R is upregulated

by T cell activation and complexes with the 3y chains to create a high affinity

receptor for IL2. The new level of signal transduction promoted by the a

chain containing receptor promotes AICD of T cells. In vitro studies using

differential inhibitors of IL2 and non-IL2 dependent cell proliferation showed

that this effect is independent of its augmentation of T cell proliferation

(Lenardo, 1991; Groux et al., 1993; Miethke et al., 1994; Kishimoto et al., 1995;

Sakaguchi et al., 1995; Taguchi and Takahashi, 1996; Kneitz et al., 1995;

Fournel et al., 1996; Wang et al. 1996; Parijs et al., 1997; Ahmed et al. 1997;

Sharfe et al., 1997; Zhu and Anasetti, 1995). Studies of lymphoid cells from a

patient with a novel human immune disorder suggest that loss of IL2R a chain

function can also block normal activation induced downregulation of the cell

viability factor BCL-,, thereby, enhancing the sensitivity of activated T cells to

AICD (Sharfe et al., 1997).

Prostaglandin E2 and Metalloproteinase Activity

Another possible mechanism by which PGE2 affects T cell activation

and AICD is through prostanoid activation of metalloproteinases, which can

enzymatically process surface regulatory molecules such as FASL, FAS, and

TNFaR from cells (Mariani et al., 1995; Kayagaki et al., 1995).

Metalloproteinases secreted by monocytes and macrophages during








extracellular matrix remodeling are readily induced by the autocrine activity

of PGE2 (Lang and Bishop, 1993; Sunderkotter et al., 1994; Mertz et al., 1994;

Clare-Salzler, 1994).

Metalloproteinase release of FASL and TNFx has been postulated to

upregulate FAS-FASL mediated AICD and TNFac mediated cell activation,

respectively. However, similar metalloproteinase cleavage of FAS,

TNFa receptor, and other surface receptors could prohibit signal transduction

through these molecules; thereby, leaving T cells resistant to cell death

(Goetzl et al., 1996).

PGE2 Role in Diabetes in the NOD Mouse

Recent work in the NOD mouse has revealed that an excess of

macrophage produced PGE2 contributes to APC dysfunction(Prescott and

White, 1996). The overexpression of PGE2 was found to be the result of a

defect in cyclooxygenase expression. Messenger RNA (mRNA) for

prostaglandin synthase 2 (PGS2), normally inducible, is expressed

constitutively in all NOD macrophages; and PGS2 protein is elevated in the

estrus phase of mature females, the most susceptible NOD group for diabetes

(Xie, dissertation, 1997).

High levels of PGS2 mRNA have been found in the unactivated

macrophages of pre-diabetic NOD mice beginning at 4 weeks of age. NOD

macrophages produce elevated levels of PGE2 when cultured alone, when

compared with control mouse strains. In addition, in vitro studies have shown








that macrophages from NOD mice have marked elevation in their PGE2

production when co-cultured with NOD T cells (Xie, dissertation, 1997).

The aberrant expression of PGS2 and PGE2 production are corrected

by the congenic replacement of the chromosomal locus containing the NOD

PGS2 gene with the normal gene locus from a nonautoimmune strain, C57B 10.

This genetic change is related to a 50% reduction in disease incidence

(Wicker et al., 1995; Xie, dissertation, 1997). The reverse congenic; that is, a

normal, nonautoimmune mouse (C57B6 background) containing only the

chromosome 1 locus (including the NOD PGS2 gene) from the NOD,

expresses PGS-2 constitutively and shows lymphocyte infiltration into the

pancreas (Yui et al., 1996; Xie ,dissertation, 1997; Garchon et al. 1994).

Confirmation of PGS2 enzymatic activity as the cause of the PGE2

elevated levels seen in the NOD was established by in vitro and in vivo testing

of the effects on PGE2 production by drugs that specifically block PGS 1 or

PGS2 (i.e., indomethacin, for both PGS1 and PGS2, and NS398, specific for

PGS2). When applied to T cell-macrophage-coculture systems, these enzyme

inhibitors reversed PGE2 production to baseline levels (Futaki et al., 1994;

Xie, dissertation, 1997).

Production of high levels of PGE2 by NOD mouse macrophages

interferes with AICD induction in V08+ T cells stimulated in vivo and in vitro

by SEB superantigen. In vivo and in vitro treatment of NOD monocytes with

PGS2 inhibitory drugs reversed this interference and allowed for elimination








of the superantigen stimulated subpopulation of V~8+ T cells (Webb et al.,

1990; Kawabe and Ochi, 1991; Xie, dissertation, 1997). These data indicate

that the PGS2 defect seen in these autoimmune mice has a direct effect on

AICD.

Levels of both FAS, and its ligand, FASL expression are abnormally

high in NOD spleen and lymph node cells as compared to nonautoimmune

control strains, B6 and BALB/c, in the resting state and with stimulation by

anti-CD3 and SEB stimulation TCR activation. These results suggest that

elimination of activated T cells by AICD is impaired in NOD mice at a point

distal in the signal cascade to these receptors.

Lipid analysis of NOD lymph node cells and the NOD monocyte

derived cell line, ZK7, showed higher levels of ceramide, sphingomyelin, and

PGE2 in these cells than in lymph node cells of control B6 mice. These high

levels of lipids in lymph node cells and monocytes derived from NOD mice

are apparent in the unmodified state as well as with TNFa stimulation, as

compared with the low but detectable levels of these lipids in the lymph node

cells of B6 nonautoimmune mice. The accumulation of ceramide in NOD cells

suggests that there is a block in the AICD process distal to ceramide

production in the signal transduction pathway.

These data suggest that signal induction for AICD in the NOD is

activated, but that the process is blocked later in the signal transduction

pathway. One hypothesis is that NOD cells are primed and ready for AICD,








but are somehow restrained from completing the process. Knowing that PGE2

production is aberrantly high in these cells, it is possible that ceramide signal

transduction may be blocked at the level of RASactivation by the effects of

cAMP generated by PGE2 (Gulbins et al., 1995).

IL 10 Role in NOD Diabetes

The role of IL10 in NOD diabetes has been the focus of recent studies

using NOD transgenic mice. IL10 accelerates onset of autoimmune diabetes in

these transgenic autoimmune mouse models. Overexpression of IL10 in the

pancreas can replace all Idd susceptibility loci except the NOD MHC in

promotion of diabetes. IL10 immunosuppression of PGS2, IL12, TNFc, and ILl

in normal immune responses may be impaired in this transgenic model;

allowing autoimmune responsiveness to be enhanced rather than suppressed

(Lee et al., 1996). The mechanism of this IL10 effect has yet to be elucidated.

PGE2 in Human Autoimmune Disease

Macrophage derived PGE2 has been implicated in the dysregulation of

T cell activation characteristic of many autoimmune diseases (Clare-Salzler,

1994 and 1995). PGE2 may influence the generation of effector T cells as

opposed to regulatory T cells by altering the quantitative signal the T cell

receives via IL2/IL2R binding, IFNy generation, kinase activation, and RAS

dependent gene activation (Anastasia et al., 1992; Cook and McCormick,

1993; Paliogianni et al., 1993; Ucker et al., 1993; Riese et al., 1994; van der

Pouw Kraan et al., 1995; Mauel et al., 1995). PGE2 roles in blocking AICD are








less well defined. PGE2 effects on cell activation and cell death are most likely

not separate and independent mechanisms, as they both appear to be

dependent on specific activation signaling between monocytes/ macrophages

and T cells as well as overlap mechanistically (i.e., through cAMP elevation

and effects on signal transduction).

Preliminary Data on PGS2 Expression in Human Autoimmune Diseases

Elevated PGE2 production and concurrent elevation of PGS2 mRNA

have been found in cultured peripheral blood monocytes of pre-diabetic

individuals, patients with IMD (Figure 2), new onset SLE patients (5/7 tested),

and autoimmune thyroiditis patients (4/6 tested). In contrast, PGE2 levels and

inducible PGS2 activity were significantly lower in normal controls tested

under the same conditions (Clare-Salzler, 1994). These data suggest that the

defect in PGS2 expression may be a factor common to a number of

autoimmune diseases.

This study focused the potential of spontaneous PGS2 expression and

aberrant PGE2 production to have a role in the immunopathogensis of human

IMD and other autoimmune diseases. Mechanisms by which a defect in PGS2

expression could manifest, affect cell activation and/or AICD, and correlate

with onset of clinical diabetes were characterized.










Figure 2. a. PGE2 Production from Cultured Human Monocytes. b. RT-PCR amplification
products from reaction with PGS2 specific primers. Lanes represent gene expression after 24
hr culturing under the following conditions: lane 1) induction with LPS; 2) no induction.
G3PDH RT-PCR run in parallel as internal control. c. Summary of PGS-2 expression in
autoimmune patients and normal controls. PGS2 mRNA measured by RT-PCR amplification
from 100,000 purified monocytes cultured for 16hr. *significant difference (p<0.0001)
**(p<0.0008) by Fisher T Test (from Clare-Salzler, 1994).


A.


p = 0.0007
n=25


24000-

22000-

20000-

18000-

16000-

14000-

12000-

10000-

8000-

6000-

4000-

2000-

0-


ICA+ SUBJECTS


ICA+ Normal
Subject C control
0 LPS 0 LPS


PGS2



P-ACTIN


0


0 n= 14




NORMAL CONTROLS


Subject Group PGS2 % PGS2
m RNA+/total mRNA+
Healthy Controls 2/23 8.6%

ICA+/pre-IDD 29/35 83%

C A-/Established IDD 6/7 85.7 %















CHAPTER 2
MATERIALS AND METHODS

Human Subject Populations

Sixty test subjects (aged 3 to 75, 41 females and 26 males) were drawn

from volunteers participating in the Clinical Research Center Diabetes

Prevention Trial and associated clinical research protocols. All subjects

included in the test group were considered autoimmune by virtue of testing

positive for autoantibodies or by other clinical criteria. Subject volunteers

were drawn from two protocol groups within the test population:

1) Natural History Group (NH) and 2) Subcutaneous Group (SQ).

The NH group contained individuals who were relatives of diabetics

and currently not on any trial treatment protocol. These individuals were

being monitored their progression toward possible diabetes. These

individuals were classified as at lower risk for onset of diabetes by virtue of

their intravenous glucose tolerance test (IVGTT) values above the established

threshold for their age group; i.e., < 100 for > 1 lyrs of age; <75 for children

younger than 11, and/or lack of specific islet cell antigen (ICA) antibodies. As

individuals in the NH group developed ICA autoantibodies, they were

reclassified as being at moderate risk for IMD.









The SQ group consisted of persons considered at high risk for early

onset of IMD by virtue of clinical criteria (i.e., IVGTT values below the

established threshold for their age group, the presence of antibodies to islet

cell antigens (ICA+), and genetic profile (Iddrisk groups)). These individuals

were on a prophylactic protocol of daily subcutaneous insulin injections and

blood glucose monitoring. These subjects would stop their treatments at least

3 days prior to clinic visits; and therefore, were not on insulin treatment at the

time of sampling.

Subjects with active autoimmune disease (AI) including Hashimoto's

thyroiditis, Addison's disease, Graves' disease, vitiligo, ulcerative colitis, and

rheumatoid arthritis were found in both NH and SQ groups. In addition, some

subjects from both groups developed IMD over the course of the trial and

were re-classified as diabetic (D). Subjects were sampled twice on average,

with at least 3 months time between samplings.

Ninety control samples were drawn from 30 healthy laboratory or clinic

personnel (age 18 to 55, 13 female, 12 male) and from volunteer relatives of

autoimmune patients that had with no clinical history of autoimmune disease

(age 35 to 45, 1 female and 4 male).

Sample Preparation

Over 200 blood cell samples were drawn following informed consent

from autoimmune subjects or their legal guardians and normal controls

according to protocols approved for the University of Florida Shands

Teaching Hospital IRB. All human blood samples used in these studies were

obtained by trained clinical staff and with informed consent as prescribed by

the Clinical Research Center. Proper biohazard handling and disposal

procedures were used in all work with these samples. Samples were received

for analysis within 1 hour of collection and were placed into sodium azide









containing buffer within 90 minute after receipt. A minimum of volume Iml of

whole blood was sufficient for the analysis.

Materials

Endotoxin-free Ficoll-Hypaque was purchased from Sigma and used in

a 2/3 ratio to isolate PBMC from whole blood. Phosphate buffered saline

(lxPBS) stock was made from endotoxin-free lOx solution (Sigma, Gibco,

Whittaker Biochemical) and pH adjusted to 7.4 with sodium hydroxide

(Sigma). Gibco RPMI 1640 plus glutamine powdered medium was

reconstituted in milli-Q water and supplemented with 2g/L sodium

bicarbonate (Baker reagent grade), 10%(v/v) heat-inactivated endotoxin-free

fetal bovine serum (HyClone certified grade), and 1%(v/v) PSN antibiotic mix

(Sigma penicillin, streptomycin, neomycin mix for tissue culture), pH adjusted

to 7.4, then filter sterilized. A solution of 0.01 %(w/v) trypan blue (Sigma) in

lxPBS was used for counting viable cells. DAPI chromatin stain (used at

lug/ml) was a gift from Dr. Michael Paddy of the Center for Structural Biology,

University of Florida. Propidium iodide (PI), Lipopolysaccharide (LPS,

endotoxin; working concentration of lug/ml) and Phytohemagglutanin (PHA,

lectin; working concentration of 5-10 Oug/ml), were purchased from Sigma. The

metalloproteinase inhibitor, Galardin, TNFa, and TNFa binding protein were

gifts from Dr. Lyle L. Moldawer, Department of Surgery, College of Medicine,

University of Florida. Annexin-PI labeling kits were purchased from R-D

Research and used according to manufacturer's suggestions.

PCR primers for cell markers and cytokines were the generous gifts of

Dr. Ammon Peck and Dr. Jeff Anderson, Department of Pathology, College of

Medicine, University of Florida.

ELISA kits for PGE2 were purchased from Cayman Chemical Company

and used according to manufacturer's directions. ELISA kits for T cell









cytokines IL2, IL10, and IFNy from Genzyme and run according to

manufacturer's protocols. IL10 ELISA were either purchased from Genzyme

or were run as a collaborative effort in the laboratory of Dr. Lyle Moldawer,

Department of Surgery, College of Medicine, University of Florida.

Antibodies

Fluorescein (FITC) labeled anti-human PGS2 mouse monoclonal

antibody (IgG ) was derived from ascites fluid was a gift from or purchased

from the Cayman Chemical Company. This monoclonal antibody was

originally developed by Cremion and coworkers (1995) of France and is

currently available as a FITC conjugated ascites isolate IgG 1 from Cayman

Chemical Company. Earlier runs of the flow cytometric (FACS) analysis were

performed using an unlabeled rabbit polyclonal anti-PGS2 sera and

unlabeled mouse monoclonal antibody, both purchased from Cayman

Chemical Company. Mouse monoclonal antibody conjugates (PE or FITC)

raised against human monocyte markers, CD 14 (IgG2a), CD69 (IgG 1), DR

(IgG2b), CD80 (IgG ), CD86 (IgG ), TNFa (IgG ), IL10 (IgG2a), CD25 (IgG ),

CD4 (IgG 1), CD8 (IgG 1), and FAS (IgG 1) were purchased from Pharmingen.

Anti-human CD 105 direct FITC label mouse monoclonal antibody (IgM) was a

gift of Dr. M. Schieder of Germany, originally purchased from Serotec.

Human blood antigen absorbed mouse isotype control antibodies for each of

the above antibodies were purchased from Sigma and Pharmingen to serve as

nonspecific binding controls for background set point. Non-immune rabbit

serum from Cayman Chemical Company was used as the control for

polyclonal anti-PGS2 antibody studies. Unlabeled rabbit polyclonal or mouse

monoclonal antibodies were detected by goat (Fab)2 FITC-conjugated

fragments, specific for rabbit (Sigma) or mouse IgG (Cappel).
Reaaents for Label Preparation









Lyophilized mixed mouse serum (Sigma) was reconstituted in

endotoxin-free water (Gibco) to a concentration of Img protein/ml and used

as a blocking agent at a working concentration of 20ug/million cells. Blocking

was supplemented with 10ul/100ul cell suspension of autologous human

plasma when available. FACS buffer consisted of l%(w/v) RIA grade

BSA(Sigma) and 0.1%(w/v) sodium azide (Sigma) dissolved in IxPBS and

bought to pH 7.4 before filter sterilization. The cells were fixed in a solution

of 4% formaldehyde in IxPBS. Saponin buffer was made by dissolving

0.5%(w/v) saponin permeabilizing agent(Sigma) in FACS Buffer and

readjusting the pH to 7.4 prior to filter sterilization. All FACS solutions were

stored refrigerated until use.

Cell Preparation

One to twenty milliliters(ml) of whole blood were collected into

heparinized vacutainers at each sample drawing. Peripheral blood

mononuclear cells (PBMC) were isolated by centrifugation (500xg, 30min,

20C) on Ficoll gradients. After the serum layer was removed and sampled,

the PBMC were collected from the top of the gradient, washed with lxPBS,

and resuspended in RPMI + 10% FCS. The PBMC were then counted,

viability assessed, and diluted to 0.5million cells/200ul with azide containing

FACS buffer. To generate a positive controls for PGS2 expression and

monocyte activation, aliquots of control cells were transferred to

polypropylene culture tubes prior to the dilution in FACS buffer and cultured

with 1-10ug/ml LPS known to readily induces PGS2, (Sweet and Hume, 1996)

or with 5-10ug/ml PHA for nonspecific cell activation, for16-24hr at

370C/5%CO2 before analysis.









Ex vivo Analysis of PGS2 Expression by Intracellular Flow Cytometry

Previous work on human monocyte PGS2 expression was done with

adherence purified monocytes. Macrophage adherence to a surface can

induce early response genes such as c-fos (Sweet and Hume, 1996). Since

PGS2 is considered a marker for monocyte/macrophage activation, a

sensitive method for detecting PGS2 without monocyte isolation from freshly

isolated PBMC was devised. This method allowed for differentiation of the

amount of PGS2 protein expression inherent in subject and control PBMC from

that induced by the adherence. In addition, use of the intracellular flow

cytometric (FACS) analysis method to detect intracellular PGS2 protein

allowed for concurrent detection of defined markers of monocyte activation.

The human PGS2 peptide used to raised the anti-PGS2 monoclonal

antibody was kindly provided Cayman Chemical for specificity testing. A

FASL peptide purchased from Calbiochem was used as a nonspecific antigen

control. Monocyte activators, LPS (working concentration of lug/ml) and PHA

(working concentration of 10 Oug/ml) were purchased from Sigma.

Fluorescent Antibody Labeling of Cells for FACS Analysis

All antibodies were used at an optimal working concentration of 0.5-

lug/million cells. Ficoll-isolated PBMC were aliquoted into Falcon 5042

polystyrene tubes at 0.5million cells/tube as dilutions in FACS buffer. The

cells were then incubated with 10ul of mouse serum and 10ul of autologous

human plasma for 20min at room temperature. After 20min, anti-surface

marker antibodies or their isotype controls were added to the appropriate

tubes and the cell incubated for an additional 20min at room temperature.

The cells were then washed (500xg centrifugation, 15C, 5min) with 1ml of

FACS Buffer and the supernatants discarded. 500ul of cell fixation solution

was then added to each tube and the cells allowed to fix for 20min. After trial









runs with alternative fixation methods (i.e., paraformaldehyde, ethanol), 4%

formaldehyde was found to be the most reproducible and stable fixative

which allows retention of surface antigens and stable cell for

permeabilization. Fixed cells were washed twice with 500ul of the

permeabilizing saponin buffer and the supernatant poured off. Saponin

permeabilization was found to be superior to ethanol or Triton/Tween 20 in

that it allowed for good surface antigen retention as well as optimal access of

antibodies to the target protein. This procedure requires saponin to remain in

all solutions post fixation to maintain cell permeability. After the final washes

of the labeled cells, the samples are returned to the non-saponin containing

buffer to close the pores formed in the membrane. In the residual volume

(approximately 200ul), 0.Sug of anti-PGS2-FITC antibody or isotype control

was added into the appropriate tubes and allowed to incubate with the

permeabilized cells for 1 hour. Following incubation, all tubes were washed 3

times with S00ul of saponin buffer and finally resuspended in 200ul of FACS

Buffer. Sample analysis of 10,000 events were used each sample on a Becton

Dickinson FACSort instrument with an argon laser excitation of 488nm,

15millivolt for FITC and PE, and detected by fluorescence at 530 +/-15 and

580 +/-21 nm, respectively.

For each sample, aliquots of unlabeled cells, and isotype antibodies

were run in parallel. In the majority of experiments, sets of subject and

control PBMC samples were processed and analyzed in parallel. Analysis of

the FACS data was performed using the Becton Dickinson PC LYSYS II

program or WinMDI freeware. Positivity for a given antigen was determined

as the percentage of cells with a fluorescence intensity above the maximum

level of the non-specific (isotype) antibody control as well as by mean

fluorescence intensity.









PGS2 specificity testing was done by FACS analysis with antibody

preparations that were incubated refrigerated overnight in a 10:1 peptide to

antibody ratio of either PGS2 peptide or a nonspecific peptide from FAS-L

protein as the intracellular labeling material.

This FACS method is highly reliable even with high variance human

peripheral blood samples (PBMC) when run with a full complement of

controls and standardized to the nonspecific isotype control for antibody

background binding. Reproducible and well separated responses to the

intracellular labeling of PGS2 in human peripheral blood were obtained by

this method (Figure 3). Unactivated normal control PBMC CD 14+ cells exhibit

low labeling for PGS2 often equal to or lower than the fluorescent labeling

seen in the control isotype matched antibody (Figure 3, panel A). Overnight

culture in RPMI + 10% FCS yields a low level expression in normal controls

(Figure 3,panel B). With 10ug/ml LPS activation, the induction of PGS2

expression was clearly seen as a 1-2 log shift in the fluorescent labeling by

PGS2 (Figure 3, panel C).

Specificity/accuracy of the PGS2 expression detection method was

tested by pre-absorbing the anti-PGS2 specific monoclonal against either the

peptide used in the production of anti-PGS2 antibodies or a nonspecific

peptide from FASL protein. Over 90% of the anti-PGS2 activity was retained

after preincubation with the FAS-Lpeptide; whereas, less than 2% of the

activity above the isotype control background remained after incubation with

the specific PGS2 peptide (Figure 4).

Multiple, independent samplings from healthy control individuals have

low within-sample variation as compared to the population variance (SD=5 to









Figure 3. Contour Plot of Intracellular FACS Analysis of Human PBMC. A.) cells ex vivo
without stimulation or culture; B.) cells from the same sample cultured 24hours without
stimulation; and C.) cells from the sample cultured 24hours in the presence of LPS.


FL 4imts (3) vY FSC4-Hi (1) FL1 -H4it (3) vs FSC41Heig (1) FL1 -Hei (3) vs FSC- oeH (1)

A B C .








10 10 10 10' 0' 10' 10 10 C 0 10
PGS2 Specific FITC Fluorescence Intensity _-_





Figure 4. Specificity of Antibodies for Detection of PGS2. LPS stimulated control PBMC (left
four dot plots) and unstimulated test subject PBMC (right three dot plots) were subjected to
FACS analysis for PGS2 expression. In the upper left corner plot, the samples were labeled
with nonspecific isotype antibodies (background fluorescence); the upper right plot cells
were labeled with the specific anti-PGS2 antibodies, the lower left plot cells labeled with the
anti-PGS2 antibody that had been previously incubated with a nonspecific peptide and the
lower right/center plot cells labeled with the anti-PGS2 antibody that had been previously
incubated with the specific PGS2 peptide.


PGS2
- peptide




10o AI i


PGS2-FITC Fluorescence--+*


LPS Stimulated Control
PBMCs


Unstimulated Subject
PBMCs









7% within sampling; 5% standard derivation for population). Comparison of

the sensitivity of this method with that of Western blot analysis using the same

antibody for detection of PGS2 shows the FACS method is many fold more

sensitive and requires 10-100 fold fewer cells to obtain a detectable signal

(Towbin et al., 1979). FACS based analysis is theoretically capable of

detecting specific antigen expression down to the single cell level. Dual

labeling with the intracellular protein specific antibody in conjunction with

cell surface markers can enhance the assay by also defining the population

expressing this intracellular antigen. By comparison, Western blot analysis is

limited to the level of protein detectable on PAGE gels (at best lOng by silver

staining methods), which requires 100-1000 fold more cells for extraction

(Towbin et al, 1979). This would put the quantitative potential of FACS

analysis at a level similar to RT-PCR quantitation of mRNA levels in a cell.

Monocyte Activation Analysis by Flow Cytometry

Co-labeling cells for surface markers and intracellular PGS2 can

expand the FACS method described above expanded to help identify the cell

populations expressing the intracellular PGS2, and help define their activation

state. Co-localization studies of two intracellular proteins in the same cell, that

is, intracellular TNFa, intracellular IL10 in conjunction with intracellular PGS2

labeling, were also possible and used as further indicators of PBMC activation

status (Sweet and Hume, 1996).

Fluorescent Antibody Labeling of Cells for FACS Analysis

All antibodies were used at an optimal working concentration of

lug/million cells. The cells were then incubated in FACS Buffer with lOul of

mouse serum and lOul of autologous human plasma for 20 minutes at room

temperature(rt). After 20 minutes, anti-surface marker antibodies or their

isotype controls were added to the appropriate tubes and the cell incubated









for an additional 20 minutes at rt. The cells were then washed (500xg

centrifugation, 15C, 5min) with 1ml of FACS Buffer and the supernatants

discarded. Five hundred microliters of Cell Fixation Solution was then added

to each tube and the cells fixed for 20 minutes, rt. Fixed cells were washed

twice with 500ul of the permeabilizing Saponin Buffer. An 0.5ug aliquot of anti-

PGS2-FITC antibody or isotype control was added into the appropriate tubes

of permeabilized cells and incubated for 1 hour, rt. Following incubation, all

tubes were washed 3 times with S00ul of Saponin Buffer and finally

resuspended in 200ul of FACS Buffer. FACS analysis was performed as

described above for intracellular FACS. Positivity for a given antigen was

determined as the percentage of cells with a fluorescence intensity above the

maximum level of the non-specific (isotype) antibody control as well as by

mean fluorescence per cell.

In situ Immunohistochemical Analysis of PGS2 and Cellular Markers

Fluorescent labeling of cells was confirmed by microscopic inspection

of cytospin preparations made from the FACS cell preparations. DAPI was

used to stain chromatin for ease in cell localization under the fluorescent

microscope. The same staining method was applied to chamber-well slides of

adherent cell cultures for in situ immunohistochemical detection of

CD 14+/PGS2+ monocytes. For in situ and cytospin labeled cells, post-fixation

and permeabilization staining of cellular chromatin with DAPI was used to

help locate and quantitate the percent positive cells per field.

Photomicroscopy was done on an Olympus IMT-2 Fluorescent Inverted

microscopy with the kind permission of Dr. Michael Paddy, Center for

Structural Biology, College of Medicine, University of Florida.









RT-PCR Analysis for PGS2, FAS, and Cytokine Profiles

Freshly isolated PBMC samples were collected and aliquots of 1-8

million cells were frozen under 100% ethanol and stored at -80C until

analyzed. Frozen cell samples were thawed, centrifuged out of ethanol, and

the cell pellet air or vacuum-dried. mRNA was extracted from the cell pellet

(approximately 1,000,000 cells) using the QIAGEN rapid mRNA kit method

and immediately converted into cDNA by reverse transcription (RT) using

Perkin-Elmer reagents and kit protocols. The resultant cDNA was then used

as template for PCR amplification using primers specific for human PGS2 gene

and either Perkin-Elmer or Qiagen PCR reagent kits. Sample were routinely

processed without a hot start for 35 cycles in a GeneMachine II thermocycler

using the temperature protocol of 900C, 105sec, 35 cycles of (720C 45sec,

65C 30sec) with a 7-10min extension period at 750C at the end of the run.

Samples were loaded onto 1.0% Agarose gels containing 0.02% ethidium

bromide (EtBr, Sigma) and run on IBI electrophoresis system for lhr at 80V-

100V, then visualized and photodocumented using a Stragagene EagleEye

transillumination system. Similar analyses were carried out using primers for

IL10, IL2, IL4, TNFa, TGFP, IL12, ILla0 & P, FAS, FASL, CD4, CD8, CD25,and

CD3.

Ceramide Analysis

Cells freshly collected from ficoll separated peripheral blood were

extracted for lipids by the method of Bligh and Dyer, 1959. These lipid

extracts were stored as dried pellets under nitrogen gas at -80C until analysis.

The lipid profile shown in Figure 5 was obtained by separation using High

Performance Thin Layer Chromatography (HPTLC) using lipid extracts

obtained from million cells solubilized in 10-20ul of chloroform just prior to

application. The mobile phase used for the majority of analysis was









Chloroform: Methanol: Water, in a volume ratio of 65:25:4. All reagents were

HPLC grade and all glassware was alkali and alcohol washed between runs.

Lipids were detected by exposure to iodine vapors and analysis was by

densitometry scanning (Perkin Elmer MD 1 software) relative to known

concentration single standards for ceramide, sphingomyelin, and

prostaglandin run on the same plate (Figure 5). All data points represent the

average of two replicates of each sample as compared to 4 replicates of each

standard.

Confirmation of ceramide identification and quantitation was

established by a series of HPTLC runs of replicates of the same sample sets

(control and subject) in multiple solvent mixes to show comigration of the

standard and sample spots. In addition, cell samples were spiked with a

known quantity of standards) prior to extraction and followed through HPTLC

analysis to calculate recovery percentages.

Figure 5. High Performance Thin Layer Chromatography of PBMC Lipids. Example HPTLC
plate showing ceramide, PGE2, and sphingomyelin standards along with human PBMC
samples from a normal control and an autoimmune subject. Mobile phase is Chloroform:
Methanol: Water in a 65:25:4 volume ratio. Sample spots contain lipid extract from million
cells. Standards, g samples


b 0.1 1.0 3.0 3.0 3:0 blk co trol sub ct

CERAMIDE
PGE2

SPHINGOMYELIN
SPHINGOMYELIN > -> *









Passive Cell Death, Cell Cycle Effects, and AICD Induction Analyses

To measure the effect of aberrant PGS2 activity in subjects on

susceptibility to cell death, passive cell death, progress through cell cycle,

and sensitivity to AICD, samples were analyzed using specialized FACS

labeling that detected changes in the cell chromatin content and

morphological studies for characteristic cellular changes of apoptotic death.

Propidium Iodide Intercalation for Chromatin Content and Cell Cycle Analysis

Propidium Iodide (PI) intercalation is detectable by FACS and is

proportional to the length and topological structure of cellular chromatin.

From one PI intercalation measurement of chromatin content, cell cycle status

can be determined. Cells with a full complement of chromatin (2n

chromosome number) will define the peak of cells in GO/G 1 phase. Those

replicating (>2n to 4n) will fluoresce at a greater intensity depicting the S, M,

and G2 cycling cells. Cells with less than complete chromatin content (<2n)

are in the process of chromatin breakdown and a portion represent cells

undergoing apoptotic death (Figure 6).

PBMC were prepared for intracellular FACS as described above with

and without surface identification marker labels. At least 30 minutes prior to

FACS analysis, 100mg/ml PI in FACS Buffer was added to an aliquot of fixed

permeabilized cells and analyzed as is.

Annexin/PI Analysis for Induction of AICD

In an attempt to measure the early events of AICD, freshly isolated

PBMC were placed in FACS buffer and lOul of Annexin and lOul of PI added to

each nonpermeabilized aliquot. These samples are read by FACS analysis

within 15minutes to detect cells that can take up the PI (i.e., membrane








Figure 6. Example of Propidium Iodide Intercalation FACS Analysis for Cell Chromatin
Content. Histogram depiction of PI intercalation fluorescence shows peaks at less than 2n, 2n,
and greater than 2n levels of chromatin indicating cells that have broken chromatin (dead or
apoptotically dying), cells with a full compliment of chromatin (2n, cells in GO and G 1 phases
of the cell cycle), and cells with greater than 2n chromatin (cells with replicating DNA and
dividing cells:G2, S, M phases of the cell cycle).


event number
(0
N 00/01 (2nPI)
M2

Cells in Cycle (>2n PI)
M3


<2n PI intercalation: Apoptotic & Dead Cells


1u- 1u,


Intercalated PI Fluorescence- >




compromised, dead cells) and surface label with Annexin which binds

phosphoserine residues that have flipped to the outer leaf of the cell

membrane; an early event in apoptosis.

TUNEL Analysis for AICD

Uniform nucleosome sized cleavage of cellular DNA is considered one

of the characteristics of cells undergoing AICD (Mountz et al., 1994; Gold et

al., 1994). To analyze this property both qualitatively and quantitatively,


I


104









Terminal Transferase tailing with FITC-conjugated UTP was used both in FACS

analysis and in situ immunohistochenmical protocols. Boeringer-Mannheim In

Situ Death Detection Kit reagents were used in cell suspensions or on

adherence cell cultures prepared in chamberwell slides. Duplicate cell

samples were fixed and permeabilized as described above for intracellular

FACS analysis and then mixed with a reaction cocktail containing FITC-

conjugated UTP. One sample was left as is and Terminal Transferase (TdT)

enzyme was added to the other. The samples were incubated for 1 hour at

37C and then cytospun on to slides for in situ analysis. The samples without

TdT acted as background controls for the enzymatically labeled duplicate.

TUNEL labeled cells were also run as colabeled samples with intracellular

labels for chromatin (DAPI) and surface cell identification markers. FACS

analysis for TUNEL labeling was done using the procedures described above

for intracellular FACS analysis.

Morphological Analysis for AICD

Cytospins of cells were prepared and stained by Difquik staining for

morphology and photodocumentation of the cellular changes of the cells

undergoing AICD or PCD: membrane blebbing and nuclear fragmentation

with condensed chromatin (Martin et al, 1994).

PGS2 Activation and Inhibition Assays

PGS2 activity was both stimulated and suppressed in vitro to test its

effects on monocyte and T cell activation. PBMC isolated on ficoll gradients

were aliquoted into polypropylene tubes and supplemented with PHA or LPS

for nonspecific activation stimulation of T cells and monocytes, respectively.

In addition to the stimulatory compounds, cultures were supplemented with

inhibitory substances: 10g/ml IL10 or the PGS2 specific inhibitor, 5PM NS398

or 10lg/ml Galardin, a broad spectrum metalloproteinase inhibitor. IL10 was









used as a physiologically important and appropriate regulator of PGS2

activity; whereas, NS398 was used as a pharmaceutical agent specific for

inhibition of PGS2 activity which does not affect gene or protein expression.

Galardin was used to counteract the effect of PGE2 induction of

metalloproteinases; a potential mechanism of action for PGE2 effects on cell

activation and inhibition of AICD. After incubation at 37C/5%CO2 for up to 24

hours, both cell and supernatant samples were taken from these cultures for

FACS analysis for PGS2, T cell surface markers, and monocyte activation

markers, ELISA analysis of PGE2 and T cell cytokines, and lipid analysis by

HPTLC.

Measurement of T cell Activation in PBMC Cultures

Loss of T cell tolerance regulation is central to the current theories for

the underlining cellular dysfunction that precipitates autoimmune disease

(Katz et al, 1995). To investigate the effect of aberrant PGS2 activity on T cell

function and APC-T cell interactions, control and AI PBMC cultures were

subjected to T cell activation stimuli. These polypropylene tube cultures were

made from ficoll separated PBMC in medium alone or supplemented with 5-

10ug/ml PHA or 1-10ug/ml LPS; and with or without the specific PGS2 inhibitor

5pM NS398. The cultures were maintained in 37C/5%CO2 for up to 72hrs

before collection and analysis.

T cell Activation Analysis

T cell activation was assessed by FACS analysis for T cell markers CD4,

CD8, CD3 and CD25 as well as the monocyte markers described above.

Supernatants from these cultures were collected for ELISA determination of T

cell cytokines IL2, IL4, IFNy, IL10, and for PGE2. The fold change in CD25

expression and cytokine production between PHA or LPS alone and PHA or









LPS plus NS398 was used to determine the change in T cell activation with and

without PGS2 activity.

T cell Proliferation Analysis

PBMC cultures were established in triplicate in 96 well dishes after

ficoll preparation. These cultures were supplemented with Sug/ml PHA for

nonspecific T cell activation with or without concurrent supplementation with

SpM the PGS2 specific inhibitor, NS398. The cultures were allowed to incubate

for 3 days at 37C/5% C02 before 1pCi/well of tritiated thymidine was added

to each well. The cells were incubated for 24hrs more and then the

suspension cells harvested by suction onto glass fiber filters and extensively

washed. After drying, the filters were read by dry count beta counter and

analyzed for 3H thymidine incorporation as a measure of proliferation.

T cell AICD Induction Analysis

In order to determine the susceptibility of the PBMC T cell population to

AICD induced by PHA, cultures of control and subject PBMC were established

in polypropylene tubes as above, in the presence or absence of PHA and with

or without concurrent supplementation with NS398 to block PGS2 activity.

These cultures were maintained for 1 to 5 days at 37C/5%CO2 then applied to

a second ficoll gradient to remove dead cells. The ficoll-repurified cells were

plated in 24-well dishes that had been previously coated with and without

2ug/well anti-FAS, low endotoxin, azide-free monoclonal antibody. The cells

were allowed to incubate 18-22 hours with the antibody at 37C, 5%CO2. The

cells were harvested and labeled as described above for T cell markers,

TUNEL, and PI intercalation.









Analysis of Hormonal Influence and Menstrual Cycle Effects on PGS2

Expression in PBMC

Previous studies on human and animal uterus and vagina tissues have

shown a cyclic expression of PGS2 linked to the female estrus cycle (Leslie

and Dubai, 1994). PGS2 expression is elevated in the preluteal phase of the

cycle and appears to be involved in control of AICD in uterine lining prior to

exfoliation and menstruation.

PGS2 expression in PBMC was tested on blood samples from two

healthy female controls, one non-autoimmune and one a relative of

autoimmune, both of which were not on estrogen/progesterone therapy.

These volunteers reported their cycle day as days from the first day of their

last period and were drawn at least once a week from that date through to

their next period. These cells from samples prepared for PGS2 FACS analysis

and serum samples taken for hormone analysis. It was not possible to collect

weekly samples from the test subject individuals since they were available for

sampling only once every 3-6 months. However, female subjects and controls

were asked to estimate their cycle day at the time of each draw and asked

whether or not they were on any hormone therapy at the time of their visits.

This information was used to compare cycle timeframe with their PGS2

expression.

In addition, PBMC from both male and female subjects and controls

were cultured in polypropylene tubes with and without 1-1 Oug/ml

progesterone or estrogen. These cultures were allowed to incubate 24hr at

37C/5%CO2 and then collected for analysis by FACS.

FACS analysis for PGS2 expression was carried out on the two types of

samples asdescribed above. Estradiol RIA analysis on serum samples was





43


performed by the Department of Pharmacology/Medical Chemistry, College

of Pharmacy, University of Florida laboratories.

Statistical Methods for Data Analysis

Data from the above analyses was analyzed for statistical significance

using Student T test for pair wise comparisons, ANOVA for multifactoral

correlative analysis and nonparametric analysis for high variance

comparisons. Microsoft Excel, Instat, GraphPad Prizm, Cricket Graph III

software was utilized in these analyses.















CHAPTER 3
RESULTS
Identification of Aberrant PGS2 Expression in Humans with Autoimmune

Disease and Individuals at Risk for Autoimmune Diabetes

FACS analysis of intracellular immunofluorescent staining with

monoclonal and polyclonal antibodies specific for PGS2 protein showed both

an increase in mean fluorescent intensity (a measure of protein/cell level) and

percentage of cells expressing PGS2 in the CD 14+ monocytes in autoimmune

PBMC sample as compared to those of normal healthy controls (Figure 7).

The increase in percentage of cells positive for PGS2 expression was

found to be significant by one-way ANOVA and modified Student t test for

sample populations with unequal variances (p=0.0003 ANOVA; post t test

p<0.0001; n controls= 25, n relatives= 5,n total subjects=60;Figure 8).

These findings were confirmed by in situ immunohistochemical analysis that

showed an increased number of CD 14+/PGS2+ cells in autoimmune adherent

cell cultures over that in normal control cultures(Figures 9a and 9b). These

data indicate the previously detected increase in PGS2 mRNA leads to a

quantifiable difference in protein expression in autoimmune cells. Analysis of

mRNA from the ex vivo PBMC cells did not yield consistent results due to the

low percentage of monocytes in most samples (i.e., visible actin control bands

in 5/15 tested). When mRNA was qualitatively detectable, it followed the

same pattern as the previous work with adherence isolated monocytes.












45
U,








A



0

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r "0 wa




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4 W 4
oo U3 o.
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A sample diabetic PBMC gave a visible cDNA product from PGS2 specific
primers in the absence of any added monocyte activation factors; whereas, 2
control and 2 relative samples did not.

Figure 8. Percentage of Cells Expressing PGS2 ex vivo is Significantly Higher in Subjects as
Compared to Controls. Data from over 200 blood samples taken from test group(60
individuals), relatives (5 individuals), and controls (25 individuals). % PGS2/CD 14+ values
for relatives, all subjects group, moderate and high risk subjects, diabetics and AI subgroups
were found significantly different from the normal control group by one way ANOVA
(p=0.0003) and by pairwise posttest for populations with unequal variance; p values as shown
below graph. Solid bars indicate mean of group values; dotted line represents control group
mean plus 2 standard deviations; level used to define positivity for PGS2 expression.


ANOVA P=0.0003


+ x
++ X


CONTROL
MEAN + 2SD

0


CONTROLS
P P<0.0001 """
P=0.0003 *
P=0.0004 **
P= 0.003 "*
P=0.01


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...

lip,


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000
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....... .... AA A
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+++
+
+
++
-+-
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I Al


RELATIVES SUBJECTS LOW RISK MOD. RISK HIGH RISK
*****


SAMPLE GROUP


CONTROLS RELATIVES ALLSUBJECTS LOW RISK MOD. RISK HIGH RIK IMD Al

SAMPLINGS 54 32 125 18 30 50 19 8
INDIVIDUALS 25 5 60 18 29 15 6 6
# POSITIVE 1 3 23 3 7 12 3 5
% POSITIVE 4% 60% 37% 17% 24% 80% 50% 83%




Autoimmune CD 14+ Monocytes Express PGS2 without Co-Expression of


Other Activation Markers


Induction of PGS2 expression is considered a marker for macrophage

development, with expression occurring later in development (Sweet and

Hume, 1996). By co-labeling cells for surface markers and intracellular PGS2,


****


"**








47
Figure 9 a and b. Fluorescent Photomicroscopic Visualization of Co-labeling of Freshly
Isolated AI PBMC with anti-PGS2-FITC, anti-CD 14-PE, and DAPI. Colocalization of all stains on
the same cells seen and indicate the intracellular location of the PGS2 protein detected to be
surrounding the nuclear contained chromatin stained with DAPI(panel B and A). In contrast,
the surface CD 14 label is peripheral to these staining.

A. B.





CD14+
PGS2+ __.
monocyte


DAPI stained anti-PGS2-FITC
ICA+ PBMCs co-stain on same
PBMCs
FACS analysis was used to identify the cell populations expressing the
intracellular PGS2, and help define their activation state (Figure 10).
Samples from a total of 42 subjects (ICA+ moderate risk, high risk,
diabetic, and autoimmune subject subgroups) and 23 control individuals
(CTL) were analyzed. The proportion of CD 14+ monocytes expressing
intracellular PGS2 was significantly higher in ICA+ subjects as compared to
the control group (subjects meanl3.4% 2.9, n=42;58 samplings, controls
mean 2.7% .65, n=23;45 samplings; p=0.0006). The mean percentage of
monocytes (ICA+ vs CTL) expressing DR (21.1 4.5 vs 18.8 5.6), CD69
(23.34.6 vs 28.3 7.5), intracellular TNFa (3.4 0.79 vs 3.8 1.9),
intracellular ILl0(1.5 1.3 vs 1.6 0.61) and CDI05 (5.91.9 vs 3.32.1) was
not significantly different in ICA+/PGS2+ subject cells relative to unstimulated
ICA-/PGS2- control cells (ICA+ n=22 vs Ctl n= 19).















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Six out of seventy-four samplings of thirty individuals in the control and

relative groups were found to have a spontaneous expression of PGS2 that

was greater than 2 standard deviations (SD) above the mean of all control

samples. Three of these samples and cultures of controls cells activated with

PHA, LPS, and TNFa, were tested for monocyte activation markers. The PGS2

expression of these cells was accompanied by elevated expression of CD69,

CD 105, DR, intracellular IL10 and intracellular TNFc (Figure 10). In contrast,

the spontaneous expression of PGS2 seen in subjects was not accompanied by

elevation of any other marker expression above the levels seen in control cell

cultures (Figure 10, last row). The reduction of intracellular TNFax and IL10

seen in some of the activated controls maybe a result of activation induced

secretion. The lack of activation markers other than PGS2 in the unstimulated

controls and subject samples suggested that monocytes are not activated by

the isolation processing and the PGS2 levels seen in these samples indicate

some other form of expression alteration inherent to the subject cells. These

data suggest that the increase in PGE2 expression is occurring out of

sequence in the monocyte activation cascade; possibly altering the further

activation of the monocyte.

PGS2 Expression Correlates with Clinical and Genetic Markers of IMD High

Risk in the Pre-diabetic Population Tested

The 1 + 3 minute insulin levels from IVGTT clinical data reflect the

insulin responsiveness of the pre-diabetic individual tested. Values less than

100 for adults and children over 11 years of age and 75 for children under 11









years of age are used as criteria for evaluating risk for diabetes; as are the

expression of certain autoantibodies, and HLA genetic subgroups (DR and

DQf3). A comparison of PGS2 expression levels with these clinical and genetic

markers for IMD risk in a pre-diabetic population shows a marked

segregation for the trait with high risk factors for progression to diabetes

(Figure 11). PGS2 expression is predominant in high risk ICA+ individuals,

especially those carrying the HLA alleles DR 04 or 01 and DQ3 0302, 0201, or

05. The expression of PGS2 inversely correlates with IVGTT 1 +3min insulin

levels (p=0.0201; correlation of highest PGS2 values of 46 subjects,

hyperbolic curve regression analysis for best fit; r squared = 1.78).

Figure 11. Subpopulation of Pre-diabetic Subjects at High Risk for progression IDDM are
Found to be Expressing Uninduced PGS2 and High Levels of PGE2 Production. Low risk (NH
group ICA-), moderate risk (NH group, ICA+), high risk (SQ group and ICA+, low IVGTT NH
group members) subjects, diabetics (IMD), and individuals with other autoimmune diseases
were analyzed as to their IVGTT 1 + 3 minute insulin levels correlation with PGS2 expression.
The correlation was found to be significant (p=0.0201, n= 46 total subjects) and to best fit a
hyperbolic curve (r squared value= 1.78).
CONTROL MEAN
350- +2 SD
325- 0 0 LOW
S300- A A MODERATE
S275 HIGH
J 275-
< IMD
250-
Z CORRELATION P=0.0201 O Al
2251
HYPERBOLIC CURVE R = 1.78
S200-
Z 1751
150- A
+125 A
S100 ----- --------------------------------- IVGTT
V THRESHOLDS
> : \7 v o X

25 V
V
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80
% PGS2/CD 14+ CELLS








Age and Gender Affects the Expression of PGS2 by Autoimmune PBMC

The test subject group had a sampling bias of females (41 individuals)

to males (26 individuals). The age range was not random among the sexes,

with a bias toward older females (>30 years of age) and younger males (< 20

years of age). These sex and age bias did not show any significant differences

in PGS2 expression when tested by Student t test or ANOVA (gender and age

subgroups; 108 samplings of subjects). Control females had PGS2 expression

significantly lower than that seen in subject females (n controls = 29, mean

2.35%, SD=4.5; n subjects=62, mean 11.56%, SD=21.5; p= 0.012, Student t

test). Based on the finding that the highest expression of macrophage PGS2 is

found in the estrus phase of the female NOD mouse, PBMC from human

females were tested to determine if a similar variation in PGS2 expression was

linked to their menstrual cycle. Analysis of one control female and one

control relative was done over a period of 30 days, testing PBMC for PGS2

expression and serum for estradiol levels (Figure 12).


The observation drawn from this and collaborative interviews with

female subjects and other controls (concerning estrogen and progesterone

therapies and cycle history) is that PGS2 expression appears to vary with the

menstrual cycle. These data suggest an increase in PGS2 expression occurs

prior to the luteal phase of the cycle. More data on a larger sample population

is needed to accurately determine the exact peak of PGS2 expression.

In vivo esterdiol or progesterone treatment of PBMC from both males and

females in control and test subject groups gave variable PGS2 expression

results by FACS, suggesting that there is individual variation in










responsiveness to the hormones. Progesterone concentration may play a role

in this responsiveness, with sensitivity being dependent on estrogen induced

progesterone receptor expression or other unknown factors.


Figure 12. Analysis of Monocyte PGS2 Expression During the Menstrual Cycle. Two control
female volunteers, one normal healthy control and one nonautoimmune relative control, were
monitored over a 30 day period for PGS2 Expression in PBMC and esterdiol level in serum.
Cycle day was recorded from the individual's report of the first day of their period designated
as day zero.

-o- PGS2/CD14 control --a-- estradiol control
-o- PGS2/CD14 relative ----- estradiol relative

20- 150
19 -140
18-
17- -130
16- -120
+ 15- -110
13- -100
S12- -90 .
11-o 5
10-7
9 -70
s 7-
-50 2
5. 1: 2. .---- 0
4- -30
3- 20
2 10
0-------------..--------------------------------"<" /-----
-5-4-3-2-1 0 1 2 3 4 5 6 7 8 9101112131415161718192021222324252627282930
Cycle Day



Insensitivity of PGS2 Expression to Suppression by IL10

ELISA analysis of IL10 production by PBMC in culture suggested that

subject cells produce the immunosuppressive cytokine at levels equal to or

greater than normal controls and nonautoimmune relatives (n controls = 37;

mean 92. lpg/million cells, SD=94.1; n subjects = 43; mean 98.5pg/million

cells, SD= 120.2).

In vitro studies of PBMC cultured in the presence of 500ng/ml IL10

alone or with LPS activation stimulus showed that PGS2 expression in 6 of 12

subject samples assayed were insensitive to IL10 suppression. Of 12 subjects

and 8 controls samples cultured, the controls on average decreased their









PGS2 expression down to 45% of the baseline levels (SD= 12%); whereas,

subject samples on average increased their PGS2 expression to 2.64 times the

level seen in their baseline cultures (SD= 1.7). When stimulated by LPS, the

differential PGS2 expression maintained the same insensitivity pattern; with

controls decreasing their expression ( mean 0.52 fold, SD=0.26) and subjects

remaining at the elevated levels induced in them by LPS (92% of LPS

stimulated level, SD=68%). The high variability in the data suggests that

factors other than IL10 insensitivity are involved in PGS2 expression.

PGE2 Inhibition of CD25 Expression on Activated Subject T cells

PBMC from controls, nonautoimmune relatives, and subjects were

tested in vitro for the effect of the PGS2 inhibition on IL2 signal transduction in

T cell AICD and proliferation. These cultures were stimulated with the T cell

mitogen, PHA, at levels that would induce both proliferation and AICD (5-

lOug/ml). The samples were set with or without the specific PGS2 inhibitor,

NS398 (5pM) and then assayed by FACS and ELISA for T cell activation,

proliferation and cell death. Tritiated thymidine uptake was lower overall in

the subject samples (n=7, mean uptake 702 l1cpm) than in controls (n=3, mean

uptake 10618 cpm) but no large changes were seen with NS398 treatment

(subjects 1.2 fold increase, controls 0.95 fold change). ELISA for PGE2

indicated that the PGE2 was present in unactivated and activated subjects and

activated controls, but were lowered or totally inhibited in NS398 treated

cultures. IL4 levels were undetectable or at the limit of detection by ELISA in

all samples(1/4 controls;0/8 subjects). IFNy was not detected in most cultures;

however, in samples with expression, NS398 treatment caused a increase in

IFNy (5/8 subjects mean increase 42.3 fold; 3/4 controls mean change 0.94

fold). Basal IL2 expression as measured by ELISA was low to undetectable in

most of the samples tested; however, in subject samples with IL2 detectable in









PHA activated culture, NS398 treatment caused an increase in its expression

(11/16 subjects, mean increase 77 fold). In contrast, control samples had little

change in IL2 expression in the presence of NS398 (2/4 controls mean change

1.2 fold).

FACS analysis for IL2 receptor a protein, CD25, showed a significant

increase in CD25 expression on subject CD3+ cells with NS398 treatment

during PHA stimulation (Figure 13;p=0.04, Student t test). Subject CD3+ cells

increased their CD25 expression by an average of 2.0 times (SD=3. 1, n=30);

whereas, controls decreased their expression to 0.45 fold (SD=0.37, n= 10)

and relatives decreased theirs to 0.49 fold (SD= 1.4, n=8) compared to

expression with PHA stimulation alone. The increase in CD25 expression was

especially strong in ICA+ SQ group individuals with the HLA alleles of DR4,

DQ00302 (p=0.01).


Figure 13. CD25 Expression Induction on CD3+ T Cells with PHA Stimulation and Treatment
with NS398. Cells were cultured in the presence of PHA and with or without NS398. The
Fold Increase reported in this graph was calculated as the % of cells with CD25/CD3+
phenotype in NS398 and PHA relative to culture with PHA alone. 'Controls' in this graph
represent normal controls and nonautoimmune relative. 'ICA+ Subjects' represents ICA+
diabetics, autoimmune subjects, moderate and high risk individuals.
10-



Z 7-






S3: It .04.

CONTROL ICA+ SUBJECTS
4P=0.04

SAMPLE GROUP






55



CD8+ T cell Subpopulation Bias in CD25 Expression in Autoimmune PBMC

FACS analysis was used to detect CD25 expression on T cell subtypes

in PBMC mixed cell cultures grown under conditions identical to those used

for the CD3+ analysis. These data show that the significant increase in

CD25/CD3+ expression is due to an increase in CD25 expression on CD8+ T

cells (p=0.01,Figure 14). In contrast, though the CD4 cells increase as well,

this increase was not significantly different from the changes seen in control

and relative samples under the same conditions(p=0.29, Figure 14).



Figure 14. CD25 Expression Increase with NS398 Treatment with PHA Stimulation on CD8+
and CD4+ T cells. Twenty-four hour cultures of PBMC with 5ug/ml PHA and with or without
5uM NS398. Data presented as fold increase with the addition of NS398 to the PHA stimulated
cultures as compared with PHA alone. Dotted lines represent the mean of the control samples
tested. The p values listed below the graph are from Student t test analysis of the control and
subject sample pairs (CD8+ and CD4+). A significant difference was seen in the increased
%CD25 expression on CD8+ in the test group relative to controls (p=0.04). CD4+ cell
expression of CD25 was also increased with NS398 present; however, this was not
significantly different from controls.


20-
V
r 19-
18-
17-
JI 16-
Wi 15-
S" 14-
+0 13-
N I- 12-
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f 8 1.0= NO CHANGE

z 6-
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13
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1- V---E--- -4-
o- ----ri-----------------------'----
0v V
0 300
CONTROL CD8+ SUBJECT CD8+ CONTROL CD4+ SUBJECT CD4+

P=0.04 P=0.29
SAMPLE GROUP









PGE2 Mediated CD25 Suppression Contributes to its Block of AICD

To test whether the influence of PGE2 on CD25 was linked to a

suppression of AICD, PBMC cultures were set up as before but maintained

24hr or 5 days in the presence of 5ug/ml PHA with or without 5SiM NS398

treatment. After ficoll removal of dead cells, the cultures were transferred to

cell dishes coated with anti-FAS antibody, a known apoptosis inducer of

activated T cells. Wells without antibody were seeded in parallel and after

24hr incubation, the cells were again analyzed for CD25 expression and for

induction of cell death. Plate bound anti-FAS antibody substituted for FAS-L

crosslinking of FASreceptors; thereby, providing a standardized AICD

stimulus. The anti-FAStreatment allowed for greater cell death of CD25+

cells when NS398 was present in subject cell cultures than when cultured with

PHA alone prior to the exposure to antibody (Figure 15; control % PI<2n

CD25+ cells fold index mean=0.94, SD 0.16; subject mean=2.4, SD 2.76). FAS

expression on subject cells was not found to be significantly different from

that of control cells, regardless if grown under unstimulated or PHA

stimulated conditions. Measurement of FASL expression was attempted but

the results were ambiguous due to poor antibody binding. CD25 cells in many

of the subject samples were also high in FAS expression, but unlike CD25+

cells in controls, were not susceptible to AICD induction by PHA or anti-FAS

without NS398 present.

When galardin treatment used in place of NS398, increased FAS and

CD25 expression on both controls and subjects but not enhancement anti-FAS

mediated AICD induction in subjects. Metalloproteinase activity was not

measured in these cultures.





























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Decreased Spontaneous Cell Death and Increase of Cells in G0/G 1 and in

Cycle in Autoimmune PBMC

A significant decrease in spontaneous PCD was noted in the subject

group compared with controls or with relatives by FACS analysis of

propidium iodide intercalation(Figure 16, panel A: ANOVA p=0.04;sample n

of controls=47, mean=66% SD=23; n relatives= 24, mean 68% SD=19; n

subjects= 87, mean=54% SD=25). A concurrent significant increase in both

the cell populations remaining in GO/G 1 (Figure 16, panel B; 2n chromatin;

ANOVA p=0.03) and in cells entering cycle (Figure 16, panel C; PI

intercalation >2n level;ANOVA p=0.02). These data were collaborated by

TUNEL FACS analysis and morphological staining of cells, suggesting that the

decrease in PCD seen in these assays is due to a decrease in apoptotic death.

Ceramide Levels are Elevated in Autoimmune PBMC

NOD mice were found to have increased levels of the AICD

intermediate, ceramide, as compared to BALB/c and B6 control strains. It is

possible that the changes in PGE2 expression could be a part of a larger

problem with lipid metabolism and AICD induction. To examine this

possibility in humans, lipid analysis was done on the subject and control

PBMC in parallel to the PGS2 expression FACS analysis. Lipid extracts from

fresh PBMC of controls and subjects were analyzed by HPTLC. Lipid spots that

comigrated with ceramide standards were found to be increased in subjects

as compared to controls (n controls=7, mean 1.034pg/million cells, SD= 1.27; n











A. PASSIVE CELL DEATH


Doo


oO
00
000 0


ooo ooit'bo
0o 0%o




o 8


CONTROLS RELATIVES
***


0 CONTROLS
0 RELATIVES
o SUBJECTS


CONTROL MEAN

ANOVA P=0.04


SUBJECTS
... P=0.0 1
....* P=0.007


SAMPLE GROUP


B. GO/G 1 STATIONARY
PHASE CELLS


00
00 0 0'
00 9 0

0oo 0
a00 0 9-V
.....-.- .... .......... ..... ....... -.- 0
0n0 000


o CONTROLS
0 RELATIVES
0 SUBJECTS








CONTROL MEAN
AN OVA P=0.03


CONTROLS RELATIVES SUBJECTS
P=0.03
P=0.04
SAMPLE GROUP


C. M/S/G2 CYCLING CELLS


0
00
oo


0 CONTROLS
0 RELATIVES
0 SUBJECTS



ANOVA P=0.02





CONTROL MEAN


CONTROLS


RELATIVES SUBJECTS


P=0.01
SAMPLE GROUP


,IS
Z Z
_J-
U

wZ
U-
E


oU..


Figure 13. Propidium Iodide
Analysis of Cell Cycle
Stages. A. Passive Cell Death
in Controls, Relatives, and
Subjects. B. Stationary Phase
(GO/G1) Cells. C. Cells in
Cycle.(M, S, & G2).


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ZZ
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U 2


2 Z
CM 0

I-J

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SD










subjects= 11, mean 5.391 pg/million cells, SD= 8.29). The change in ceramide


levels was different between genders, with females being higher than males


in both groups (control females mean 1.571 pg/ml; control males mean 0.317;


subject females mean 7.755; subject males mean 2.554). Linear regression


analysis depicted in Figure 17 shows this increase was positively correlated


with the increase in PGS2 expression observed in these subjects.


Figure 17. Correlation of Ceramide Levels with PGS2 Expression. A. in Controls. and B. in
Subjects. Data represents average of duplicate samples run on the same HPTLC plate.
Linear regression correlation analysis of 10 subject and 7 control samples is depicted. Dotted
curves represent 95% confidence intervals.


CONTROL CELLS
N=7; P=0.545; R2=0.06
NO CORRELATION

0 MALE
5- 0 FEMALE
Z
0 4-


" --- ................. .... ... .......-



0 o

0 3 6 9 12 15 18 21 24 27 30
% PGS2/CD14+ CELLS


z
0
U)
,-I

w
a9.

L)
U


SUBJECT CELLS
N=10; P=0.0018; R2=0.68

O MALE
FEMALE ---
O FEMALE .-"


0 10 20 30 40 50 60 70 80 90 100
% PGS2/CD1 4+ CELLS















CHAPTER 4
DISCUSSION

The aberrant PGS2 mRNA expression and associated PGE2

overproduction was recently described by Xie (1997) in the NOD mouse and

by Clare-Salzler(1995) in human peripheral blood monocytes of individuals at

risk of IMD, those with overt IMD, and with other autoimmune diseases

including rheumatoid arthritis, vitiligo, ulcerative colitis, Addison's Disease,

Graves' Disease, Hashimoto's thyroiditis, and SLE. This study gives a

quantitative definition to the PGS2 expression defect in humans and provides

insight into possible mechanisms of its role in immunopathogensis.


Intracellular FACS analysis of freshly isolated human PBMCs allowed

for detection of ex vivo quantitation of PGS2 expression without the

background activation caused by adherence purification of monocytes or the

transient increase in expression that accompanies in vitro culturing. Aberrant

PGS2 protein expression in unactivated peripheral blood monocytes was

found in 37% of the test subjects as compared with 4% of normal healthy

controls (p<0.0001). The presence of the enzyme without indicators of

monocyte activation gives support to the idea that the monocyte/macrophage

population has been altered in autoimmune individuals, yielding a defect in

APC function which affects T cell activation. The presence of this aberrant

PGS2 expression correlated inversely with insulin responsiveness as

measured by IVGTT (p=0.0201), indicating a link between the defect and









immunopathogensis of IMD. This link presents the opportunity for use of

PGS2 expression as a barometer for disease activity; both as a diagnostic

indicator of risk for IMD and a potential target for therapeutic intervention.

Nonsteroidal anti-inflammatory drugs (NSAID) such as the PGS2 specific

inhibitor NS398 and related compounds, may constitute a new

pharmacological avenue for the treatment and prevention of autoimmune

disease.

In many autoimmune diseases there is a gender bias and/or an age of

onset effect. IMD does not have a defined gender bias but does have an age

of onset prevalence that coincidences with puberty (Winter et al., 1993). The

sample population tested in this study had a bias in the population toward

sexually mature females and pre-pubescent males. This may represent a

sampling bias, not the incidence level in the total at-risk population. Adult

control females were found to have cyclic variations in their PGS2 expression

that coincided with their menstrual cycle. Estrogen and progesterone

therapies, such as birth control use and estrogen replacement therapy, are

postulated to affect PGS2 expression. These influences on PGS2 expression

contribute to the sample variance seen in the study population. It is interesting

to note that even with this potential signal interference in the data, female

subjects were found to have a significantly higher level of PGS2 expression

than female controls (p=0.01). Though no gender or age bias for IMD could

be defined from this study, the observations suggest a more in-depth study is

needed into the effects of sexual maturation and the use of hormone-based

therapies in the risk assessment for IMD and other autoimmune diseases.

The underlying cause of the PGS2 expression defect is still unknown.

The immunosuppressive cytokine IL10 has been the object of recent

transgenic and congenic analysis in the NOD mouse (Lee et al., 1996). The









PGS2 expression of some test group samples in vitro showed insensitivity to

interleukinlO suppression, a normal regulatory control of PGS2 expression.

Further investigation is needed to determine if such a regulatory dysfunction

is an underlying mechanism for the defect in vivo.

When PGS2 expression was inhibited in vitro, test subject T cells

increased IL2 receptor expression (CD25) and their susceptibility to AICD

(p=0.04). This effect is especially prevalent in cells from subjects with the

IMD susceptibility major histocompatibility locus alleles DR4 and DQj0302

(p<0.001). This prevalence suggests a linked effect between PGE2

interference with IL2 signaling and the high risk HLA allele on T cell

activation. Moreover, the greatest change in CD25 expression attributable to

PGS2 expression was seen in the subject CD8+ T cells (p=0.01). CD8+ T cells

are thought to be the primary subpopulation involved in suppression

regulation of T cell functions, including T helper cell differentiation to Thl or

Th2 cells (Fukuse et al., 1992; Eimasry et al., 1986; Eimasry et al., 1987;

Balashov et al., 1995; Goetzl et al., 1995). A selective influence on the

activation and elimination potential of CD8+ regulatory T cells would have

dramatic effects on the regulatory control of peripheral tolerance, T helper

cell functional bias (Thl verses Th2), as well as appropriate conclusion of

immune responses and memory cell development. Interference with these

types of immune regulation could promote autoimmunity and

immunopathogensis.

Passive cell death, defined as spontaneous death of cells ex vivo, was

significantly lower in test subject cells than nonautoimmune relatives and

normal controls(p=0.04). Concurrently, the percentage of subject cells

remaining in GO/G 1 and entering cycle were significantly higher(p=0.03 and

0.02, respectively). Preliminary data suggests that subject cells in culture









were also resistant to AICD induced by anti-FAS antibody. These data

suggest that autoimmune PBMC are unusually resistant to cell killing. The

precedence for lack of cell death leading to autoimmune disease has been

shown in the 1prand gid mouse models and more recently in humans with SLE

and SLE-like lymphoproliferative disease (Howie et al., 1994; Sneller et al.,

1992; Emlen et al., 1994). Resistance to AICD in vitro was reversible in this

study by inhibition of PGS2 expression. PGE2 effects on IL2, CD25,

metalloproteinase cleavage of surface ligands, and receptors all can

contribute to its effects on AICD signaling. However, surface signal effects

alone did not account for all of the PGE2 effects on AICD. Galardin treatment

prevented loss of surface molecules including FAS and CD25 from subject

cells, but did not promote greater susceptibility to AICD induction. Though

Galardin is a broad spectrum inhibitor of metalloproteinase, its anti-

proteolytic effects may not affect the molecules involved in the resistance to

AICD seen in these cells. Another possible mechanism for the effectiveness of

NS398 inhibition of PGS2 expression in promoting AICD susceptibility is that

PGE2 may be affecting AICD signal transduction at a point beyond the initial

surface receptor-ligand interaction. The accumulation of ceramide, a second

messenger in AICD signal transduction, directly correlated with the increase

in PGS2 expression found in the test subject individuals(p=0.002). Ceramide

functions in promotion of kinases and dephosphorylases important for AICD

and in arresting cell cycle progression (Hannun and Obeid, 1995). Recent

data show that PGS2 expression is promoted by ceramide; whereas, the action

of ceramide as a nuclear signal transduction molecule are somehow blocked

by PGE2 (Ballou et al., 1992; Hannun, 1997). This interplay of lipid

metabolytes of AA represents another level of signal transduction/cell

activation regulation that is just now being uncovered. Ceramide has been









implicated in control of ICE, MAP kinases, and RAS/RAFrelated proteins

(Hunnan, 1997). PGE2, through its actions on cAMP, also affects the same

metabolic pathways. The complex interplay and balance of these lipid

components may be similar to the BCL2-BAX/BCL-xprotein level balances

needed to maintain a cell in a viable, functional state. The mechanism of how

PGE2 is promoting resistance to cell death and; thereby, setting the stage for

development of autoimmune dysfunction lies in its role in signal transduction.

Further studies into the lipid metabolism of cells is needed to elucidate this

role.

This study confirms the identification of aberrant monocyte PGS2

expression as a risk factor for IMD and presents evidence for its role as a

component of the immunopathogensis of multiple autoimmune diseases.













REFERENCES CITED


Ahmed, N. N., H. L. Grimes, A. Bellacosa, T. 0. Chan, and P. N. Tsichilis. 1997.
Transduction of Interleukin-2 Antiapoptotic and Proliferative Signals
via Akt Protein Kinase." Proceedings of the National Academy of
Sciences USA. 94: 3627 3632.

Alderson, M. R., R. J. Armitage, E. Maraskovsky, T. W. Tough, E. Roux, K.
Schooley, T. Ramsdell, and D. H. Lynch. 1993. "Fas Transduces
Activation Signals in Normal Human T Lymphocytes." Journal of
Experimental Medicine. 178: 2231-2235.

Anastasia, E. D., F. Paliogianni, J. P. Balow, H. Yamada, and D. T. Boumpas.
1992. "Prostaglandin E2 and Other Cyclic AMP-Elevating Agents
Modulate IL-2 and IL-2Ra Gene Expression At Multiple Levels." Journal
of Immunology. 148(9): 2845-2852.

Anastassiou, E. D., F. Paliogianni, J. P. Balow, H. Yamada, and D. T. Boumpas.
1992. "Prostaglandin E2 and Other Cyclic AMP-Elevating Agents
Modulate IL-2 and IL-2Rot Gene Expression at Multiple Levels." Journal
of Immunology. 148: 2845-2952.

Antonaci, S., C. Tortorella, A. Poignano, A. Ottolenghi, E. Jirillo, and L.
Bonomo. 1991. "Modulating Effects on CD25 and CD71 Antigen
Expression by Lectin Stimulated T Lymphocytes in the Elderly."
Inmunopharmacology and Immunotoxicology. 13(1&2): 87-100.

Baixeras, E., L. Bosca, C.Staiber. A. Gonzalez, A.C. Carrera, J.A. Gonzalo, and
C. Martinez-A. 1994. "From Apoptosis to Autoimmunity: Insights from
the Signaling Pathways Leading to Proliferation or to Programmed
Death. p.53-92. in "Apoptosis in Immunity"; dedicated volume of
Immunological Reviews-, G. Moller; ed. 142: December.

Balashov, K. E., S. J. Khoury, D. A. Hafler, and H. L. Weiner. 1995. "Inhibition
of T cell Responses by Activated Human CD8+ T Cells is Mediated by
Interferon-y and is Defective in Chronic Progressive Multiple Sclerosis."
Journal of Clinical Investigation. 95: 2711-2719.








Ballou, L. R., C. P. Chao, M. A. Holness, S. C. Barker, and R. Raghow. 1992.
"Interleukin-1-mediated PGE2 Production and Sphingomyelin
Metabolism." Journal ofBiological Chemistry. 267(28): 20044-20050.

Berger, S., H. Ballo, and H. J. Stutte. 1996. "Immune Complex-induced
Interleukin-6, Interleukin-10, and Prostaglandin Secretion by Human
Monocytes: a Network of Pro- and Anti-inflammatory Cytokines
Dependent on the Antigen: Antibody Ratio." European Journal of
Immunology. 26: 1297-1301.

Bligh, E. G. and W. J. Dyer. 1959. "A Rapid Method of Total Lipid Extraction
and Purification." Canadian Journal of Biochemistry and Physiology.
37(8): 911-917.

Bose, R., M. Verheij, A. Haimovitz-Friedman, K. Scotto, Z. Fuks, and R.
Kolesnick. 1995. "Ceramide Synthase Mediates Daunorubicin-Induced
Apoptosis: An Alternative Mechanism for Generating Death Signals."
Cell. 82: 406-414.

Brunner,T., R.J. Mogil, D. LaFace, N. J. Yoo, A. Mahboubl, F. Echeverri, S. J.
Martin, W. R. Force, D. H. Lynch, C. F. Ware, and D.R. Green. 1995.
"Cell-autonomous Fas (CD95)/Fas-ligand Interaction Mediates
Activation-induced Apoptosis in T-cell Hybridomas." Nature. 373:441-
448.

Chen, D. and E. V. Rothenberg. 1994. "Interleukin 2 Transcription Factors as
Molecular Targets of cAMP Inhibition: Delayed Inhibition Kinetics and
Combinatorial Transcription Roles." Journal of Experimental Medicine.
179: 931-942.

Chen, M., J. Quintans, Z. Fuks, C. Thompson, D. W. Kufe, and R. R.
Weichselbaum. 1995. "Suppression of Bcl-2Messenger RNA
Production May Mediate Apoptosis after Ionizing Radiation, Tumor
Necrosis Factor ac, and Ceramide." Cancer Research. 55:991-994.

Cheng, J., C. Lu, W. J. Koopman, and J. D. Mountz. 1995. "Characterization of
Human Fas Gene: Exon/Intron Organization and Promoter Region."
Journal of Immunology. 154: 1239-1245.

Chervonsky, A. V., Y. Wang, F. S. Wong, I. Visintin, R. A. Flavell, C. A.
Janeway, Jr., and L. A. Matis. 1997. "The Role of Fasin Autoimmune
Diabetes." Cell. 89: 17-24.

Cifone,M. G., R. De Maria, P. Roncaioli, M. R. Rippo, M. Azuma, L. L. Lanier, A.
Santoni, and R. Testi. 1993. "Apoptotic Signaling through CD95








(Fas/Apo-1) Activates an Acidic Sphingomyelinase." Journal of
Experimental Medicine. 177: 1547-1552.

Clare-Salzler, M. J. 1994. "Monocyte Dysfunction in Pre-IDDM; Spontaneous
Expression of Prostaglandin Synthase-2." manuscript in preparation.

Clare-Salzler, M. J. 1995. "The Role of NOD Macrophages in the
Dysregulation of Peripheral Tolerance." manuscript in preparation.

Clare-Salzler, M. J., J.Brooks, A. Chai, K. Van Herle, and C. Anderson. 1992.
"Prevention of Diabetes in Nonobese Diabetic Mice by Dendritic Cell
Transfer." Journal Clinical Investigation. 90: 741-748.

Clare-Salzler, M. J. and Y. Mullen. 1992. "Marked Dendritic Cell-T cell
Cluster Formation in the Pancreatic Lymph Node of the Non-Obese
Diabetic Mouse." Immunology. 76: 478-484.

Clare-Salzler, M. J., J.Brooks, A. Chai, K. Van Herle, and C. Anderson. 1992.
"Prevention of Diabetes in Nonobese Diabetic Mice by Dendritic Cell
Transfer." Journal Clinical Investigation. 90: 741-748.

Cohen, I. R. and D. B. Young. 1992. "Autoimmunity, Microbial Immunity and
the Immunological Homunculus." Immunology Today. 13: 490.

Cook, S. J. and F. McCormick. 1993. "Inhibition by cAMP of Ras-Dependent
Activation of Raf." Science. 262: 1069-1072.

Crenmion, C., A. Habib, J. Maclouf, P. Pradelles, J. Grassi, and Y. Frobert.
1995. Differential measurement of constitutive (COX-1) and inducible
(COX-2) cyclooxygenase expression in human umbilical endothelial
cells using specific immunometric enzyme immunoassays. Biochim
BiophysActa. 1264:341-348.

Daftarian, P. M., A. Kumar, M. Kryworuchko, and F. Diaz-Mitoma. 1996. "IL10
Production is Enhanced in Human T Cells by IL- 12 and IL-6 and in
Monocytes by Tumor Necrosis Factor-a." Journal of Immunology. 157:
12-20.

Dai, W. J., G. Kohler, and F. Brombacher. 1997. "Both Innate and Acquired
Immunity to Listeria monocytogenes Infection are Increased in IL- 10-
Dificient Mice." Journal of Immunology. 158: 2259-2267.

D'Andrea, A., M. Aste-Amezaga, N. M. Valiante, X. Ma, M. Kubin and G.
Trinchieri. 1993. "Interleukin 10 (IL-10) Inhibits Human Lymphocyte
Interferon y-Production by Suppressing Natural Killer Cell Stimulatory








Factor/IL-12 Synthesis in Accessory Cells." Journal of Experimental
Medicine. 178: 1041-1048.

de Waal Malefyt, R., J. Abrams, B. Bennett, C. G. Figdor, andJ. E. de Vries.
1991. "Interleukin 10 (IL-10) Inhibits Cytokine Synthesis by Human
Monocytes." Journal of Experimental Medicine. 174: 1209-1219.

DeWitt, D. and W. L. Smith. 1995. "Yes, but Do They Still Get Headaches?"
Cell. 83: 345-348.

Dhein, J., H. Walczak, C. Baumler, K.-M. Debatin, and P. H. Krammer. 1995.
"Autocrine T-cell Suicide Mediated by APO-1/(Fas/CD95)." Nature.
373: 438-440.

Ehrhardt, R. 0., B. R. Ludviksson, B. Gray, M. Neurath, and W. Strober. 1997.
"Induction and Prevention of Colonic Inflammation in IL-2-Deficient
Mice." Journal of Immunology. 258: 566-573.

Eimasry, M. N., E. J. Fox and R. R. Rich. 1986. "Opposing Immunoregulatory
Functions of CD8+ Lymphocytes: A Requirement for Monocytes in
Suppressor Cell Induction." Journal of Immunology. 137(8): 2468-
2477.

Eimasry, M. N., E. J. Fox and R. R. Rich. 1987. "Sequential Effects of
Prostaglandins and Interferon-y on Differentiation of CD8+ Suppressor
Cells." Journal of Immunology. 139(3): 688-694.

Emlen, W., J. Niebur, and R. Kadera. 1994. "Accelerated In Vitro Apoptosis
of Lymphocytes from Patients with Systemic Lupus Erythematosus."
Journal of Immunology. 152: 3685-3692.

Faustman, D. 1993. "Mechanisms of Autoimmunity in Type I Diabetes."
Journal of ClinicallImmunology. 13(1): 1-7.

Faveeuw, C., M.-C. Gagnerault, and F. Lepault. 1994. "Expression of Homing
and Adhesion Molecules in Infiltrated Islets of Langerhans and Salivary
Glands of Nonobese Diabetic Mice." Journal of Immunology. 152:
5969-5977.

Foegh, M. L. 1988. "Immune Regulation by Eicosanoids." Transplantation
Proceedings. 20(6): 1158-1161.

Fournel, S., L. Genestier, E. Robinet, M. Flacher, and J.-P. Revillard. 1996.
"Human T cells Require IL-2 but not G 1/S Transition to Acquire








Susceptibility to Fas-Mediated Apoptosis." Journal of Immunology.
157: 4309-4315.

Fukuse, S., C. Terajima, A. Koontz, M. Kelley, D. R. Webb, and B. H. Devens.
1992. "Interleukin-2 Stimulates the Development of Anergy via the
Activation of Nonspecific Suppressor T Cells." International Archives of
Allergy Immunology. 99: 411-415.

Futaki, N., S. Takahashi, M. Yokoyama, I. Arai, S. Higuchi, and S. Otomo. 1994.
"NS-398, A New Anti-inflammatory Agent, Selectively Inhibits
Prostaglandin G/H Synthase/Cyclooxygenase (COX-2) Activity in
vitro." Prostaglandins. 47: 55-59.

Garchon,H.-J., J.-J. Luan, L. Eloy, P. Bedossa, and J.-F. Bach. 1994. "Genetic
Analysis of Immune Dysfunction in Non-obese Diabetic (NOD) Mice:
Mapping a Susceptibility Locus Close to the Bc1-2 Gene Correlates with
Increased Resistance of the NOD T cells to Apoptosis Induction."
European Journal of Immunology. 24: 380-384.

Gerosa, F., C. Paganin, D. Peritt, F. Paiola, M. T. Scupoli, M. Aste-Amezaga, I.
Frank, and G. Trinchieri. 1996. "Interleukin-12 Primes Human CD4 and
CD8 T cell Clones for High Production of Both Interferon-y and
Interleukin-10." Journal of Experimental Medicine. 183: 2559-2569.

Gill, B.M., H. Nishikata, G. Chan, T. L. Delovitch, and A. Ochi. 1994. "Fas
Antigen and Sphingomyelin-Ceramide Turnover-Mediated Signaling:
Role in Life and Death of T Lymphocytes." p. 113-126. in "Apoptosis in
Immunity"; dedicated volume of ImmunologicalReviews; G. Moller; ed.
142: December.

Giordano, C., R. DeMaria, M. Todaro, G. Stassi, A. Mattina, P. Richiusa, G.
Galluzzo, F. Panto, and A. Galluzzo. 1993. "Study of T-Cell Activation in
Type I Diabetic Patients and Pre-Type I Diabetic Subjects by
Cytometric Analysis: Antigen Expression Defect in vitro." Journal of
Clinical Immunology. 13(1): 68-78.

Goetzl, E. J., S. An, and L. Zeng. 1995. "Specific Suppression by Prostaglandin
E2 of Activation-Induced Apoptosis of Human CD4+CD8+ T
Lymphoblasts." Journal of Immunology. 154: 1041-1047.

Goetzl, E. J., M. J. Banda, and D. Leppert. 1996. "Matrix Metalloproteinases in
Immunity." Journal of Immunology. 156: 1-4.

Gold, R., M. Schmied, G. Giegerich, H. Breitschopf, H. P. Hartung, K. V. Toyka,
and H. Lassmann. 1994. "Differentiation between Cellular Apoptosis








and Necrosis by the Combined Use of in situ Tailing and Nick
Translation Techniques." Laboratory Investigation. 71(2): 219-225.

Gonzalo, J.A., E. Baixeras, A. Gonzalez-Garcia, A. George-Chandy, N. Van
Rooijen, C. Martinez-A., and G. Kroemer. 1994. "Differential in vivo
Effects of a Superantigen and an Antibody Targeted to the Same T Cell
Receptor." Journal of Immunology. 152: 1597-1608.

Goppelt-Struebe, M. 1995. "Regulation of Prostaglandin Endoperoxide
Synthase (Cyclooxygenase) Isozyme Expression." Prostaglandins,
Leukotrienes, and Essential Fatty Acids. 52: 213-222.

Groux, H., G. Monte, B. Plouvier, A. Capron, and J. C. Ameisen. 1993. "CD3-
mediated Apoptosis of Human Medullary Thymocytes and Activated
Peripheral T cells: Respective Roles of Interleukin- 1, Interleukin-2,
Interferon-y, and Accessory Cells." European Journal of Immunology.
23: 1623-1629.

Gulbins, E., R. Bissonnette, A. Mahboubi, S. Martin, W. Nishioka, T. Brunner,
G. Baier, G. Baier-Bitterlich, C. Byrd, F. Lang, R. Kolesnick, A. Altman,
and D. Green. 1995. "FAS-Induced Apoptosis Is Mediated Via a
Ceramide-Initiated RAS Signaling Pathway." Immunity. 2:341-351.

Hannun, Y. A. 1994. "The Sphingomyelin Cycle and the Second Messenger
Function of Ceramide." Journal of Biological Chemistry. 269(5): 3125-
3128.

Hannun, Y. A. 1997. "Functions of Ceramide in Coordinating Cellular
Responses to Stress." Science. 274: 1855-1859.

Hannun, Y. A. and L. M. Obeid. 1995. "Ceramide: An Intracellular Signal for
Apoptosis." Trends in Biological Sciences. 20(2): 73-77.

Holter, W., A. M. Spiegel, B. H. Howard, S. Weber, and M. R. Brann. 1991.
"Expression of GTP-Binding Proteins and Prostaglandin E2 Receptors
During Human T Cell Activation." Cellular Immunology. 134: 287-295.

Howie, S. E. M., D. J. Harrison, and A.H. Wyllie. 1994. "Lymphocyte
Apoptosis--Mechanisms and Implications in Disease." p. 141-156. in
"Apoptosis in Immunity"; dedicated volume of Immunological Reviews;
G. Moller; ed. 142: December.

Huang, L. and I. N. Crispe. 1993. "Superantigen-Driven Peripheral Deletion
of T Cells." Journal of Immunology. 151(4): 1844-1851.








Hyslop, S. and G. De Nucci. 1993. "Prostaglandin Biosynthesis in the
Microcirculation: Regulation by Endothelial and Non-endothelial
Factors." Prostaglandins, Leukotrienes, and Essential Fatty Acids. 49:
723-760.

Ihm, S.-H. and J.-W. Yoon. 1990. "Studies on Autoimmunity for Initiation of
P-Cell-Specific Cytotoxic Effectors and Insulitis in NOD Mice."
Diabetes. 39: 1273-1278.

Isomaki, P., R. Luukkainen, R. Saario, P. Toivanen, and J. Punnonen. 1996.
"Interleukin- 10 Functions as an Antiinflammatory Cytokine in
Rheumatoid Synovium." Arthritis & Rheumatism. 39(3): 386-395.

Jansen, A., F. Homo-Delarche, H. Hooijkaas, P. J. Leenen, M. Dardenne, and
H. A. Drexhage. 1994. "Immunohistochemical Characterization of
Monocytes-Macrophages and Dendritic Cells Involved in the Initiation
of the Insulitis and 3-Cell Destruction in the NOD Mice." Diabetes. 43:
667-674.

Jarvis, W.D., F. A. Fornari, Jr., J. L. Browning, D. A. Gewirtz, R. N. Kolesnick,
and S. Grant. 1994a. "Attenuation of Ceramide-induced Apoptosis by
Diglyceride in Human Myeloid Leukemia Cells." Journal of Biological
Chemistry. 269(50): 31685-31692.

Jarvis, W.D., R. N. Kolesnick, R.A. Fornari, R. S. Traylor, D. A. Gewirtz, and S.
Grant. 1994b. "Induction of Apoptotic DNA Damage and Cell Death by
Activation of the Sphingomyelin Pathway." Proceedings of the National
Academy of Sciences USA. 91:73-77.

Jayadev, S. C. M. Linardic, and Y. A. Hannun. 1994. Identification of
Arachidonic Acid as a Mediator of Sphingomyelin Hydrolysis in
Response to Tumor Necrosis Factor a." Journal of Biological Chemistry.
269(8): 5757-5763.

Jones, L. A., L. T. Chin, D. L. Longo, and A. M. Kruisbeek. 1990. "Peripheral
Clonal Elimination of Functional T Cells." Science. 250: 1726-1729.

Katz, J. D., C. Benoist, and D. Mathis. 1995. "T Helper Cell Subsets in Insulin-
Dependent Diabetes." Science. 268: 1185-1188.

Kawabe, Y. and A. Ochi. 1991. "Programmed Cell Death and Extrathymic
Reduction of V08+ CD4+ T Cells in Mice Tolerant to Staphylococcus
aureus enterotoxin B." Nature. 349: 245-248.








Kayagaki, N., A. Kawasaki, T. Ebata, H. Ohmoto, S. Ikeda, S. Inoue, K. Yoshino,
K. Okumura, and H. Yagita. "Metalloproteinase-mediated Release of
Human Fas Ligand." Journal of Experimental Medicine. 182: 177-1783.

Kim, M-Y., C. Linardic, L. Obeid, andY. Hannun. 1991. "Identification of
Sphingomyelin Turnover as an Effector Mechanism for the Action of
Tumor Necrosis Factor a and y-Interferon." Journal of Biological
Chemistry. 266: 484-489.

Kinoshita, T., T. Yokata, K. Araj, and A. Miyajima. 1995. "Suppression of
Apoptotic Death in Hematopoietic Cells by Signaling through the IL-
3/GM-CSF Receptors." EMBOJournal. 14(2):256-275.

Kishimoto, H., C. D. Surh, and J. Sprent. 1995. "Upregulation of Surface
Markers on Dying Thymocytes." Journal of Experimental Medicine.
181: 649-655.

Kneitz, B., T. Herrmann, S. Yonehara, and A. Schimpl. 1995. "Normal Clonal
Expansion but Impaired Fas-Mediated Cell Death and Anergy Induction
in Interleukin-2-Deficient Mice." European Journal of Immunology.
25:2572-2577.

Kosaka, T., A. Miyata, H. Ihara, S. Hara, T. Sugimoto, 0. Takeda, E. Takahashi,
and T. Tanabe. 1994. "Characterization of the Human Gene (PGS2)
Encoding Prostaglandin-Endoperoxide Synthase 2." European Journal
of Biochemistry. 221: 889-897.

Krammer, P. H., J. Dhein, H. Walczak, I. Behrmann, S. Mariani, B. Matiba, M.
Fath, P.T. Daniel, E. Knipping, M. 0. Westendorp, K. Stricker, C.
Baumler, S. Hellbardt, M. Germer, M. E. Peter, and K.-M. Debatin.
1994. "The Role of APO-1 -Mediated Apoptosis in the Immune System."
in "Apoptosis in Immunity";dedicated volume of Immunological
Reviews, G. Moller; ed. 142: December.

Lang, R. A. and J. M. Bishop. 1993. "Macrophages Are Required for Cell
Death and Tissue Remodeling in the Developing Mouse Eye." Cell. 74:
453-462.

Langenbach, R., S. G. Morham, H. G. Tiano, C. D. Loftin, B. I Ghanayem, P. C.
Chulada, J.F. Mahler, C. A. Lee, E. H. Goulding, K. D. Kluckman, H.S.
Kim, and 0. Smithies. 1995. "Prostaglandin Synthase 1 Gene
Disruption in Mice Reduces Arachidonic Acid-Induced Inflammation
and Indomethacin-Induced Gastric Ulceration." Cell. 83: 483-492.








Lee, K.-U., K. Amano, and J.-W. Yoon. 1988. "Evidence for Initial Involvement
of Macrophage in Development of Insulitis in NOD Mice." Diabetes.
37: 989-991.

Lee, M.-S., R. Mueller, L. S. Wicker, L. B. Peterson, and N. Sarvetnick. 1996.
"IL-10 is Necessary and Sufficient for Autoimmune Diabetes in
Conjunction with NOD MHC Homozygosity." Journal of Experimental
Medicine. 183: 2663-2668.

Lenardo, M. J. 1991. "Interleukin-2 Programs Mouse ab T Lymphocytes for
Apoptosis." Nature. 353: 858-861.

Leslie, C. A. and D. P. Dubey. 1994. "Increased PGE2 from Human Monocytes
Isolated in the Luteal phase of the Menstrual Cycle. Implications for
Immunity?" Prostaglandins. 47:41-54.

Liu,Y. and C. A. Janeway,Jr. 1990. "Interferon y Plays a Critical Role in
Induced Cell Death of Effector T Cell: A possible Third Mechanism of
Self-tolerance." Journal of Experimental Medicine. 172: 1735-1739.

Lu, X., W. Xie, D. Reed, W. S. Bradshaw, and D. L. Simmons. 1995.
"Nonsteroid Antiinflammatory Drugs Cause Apoptosis and Induce
Cyclooxygenases in Chicken Embryo Fibroblasts." Proceedings of the
National Academy of Sciences. 92: 7961-7965.

Ludviksson, B. R., B. Gray, W. Strober, and R. 0. Ehrhardt. 1997.
"Dysregulated Intrathymic Development in the IL-2-Dificient Mouse
Leads to Colitis-Inducing Thymocytes." Journal of Immunology. 158:
104-111.

Maclaren, N. K., S. W. Huang and J. Fogh. 1975. "Antibody to cultured human
insulinoma cells in insulin-dependent diabetes." Lancet. 1: 997.

Majno, G. and I. Joris. 1995. "Apoptosis, Oncosis, and Necrosis." American
Journal of Pathology. 146(1): 3-15.

Mariani, S. M., B. Matiba, C. Baumler, and P. H. Krammer. 1995. "Regulation
of Cell Surface APO- 1/Fas (CD95) Ligand Expression by
Metalloproteases." European Journal oflImmunology. 25: 2303-2307.

Martin, S. J., D. R. Green, and T. G. Cotter. 1994. "Dicing with Death:
Dissecting the Components of the Apoptosis Machinery." Trends in
Biological Sciences. January: 26-30.








Mauel, J., A. Ransijn, S. B. Corradin, and Y. Buchmuller-Rouiller. 1995.
"Effects of PGE2 and of Agents that Raise cAMP Levels on Macrophage
Activation Induced by IFN-y and TNF-a." Journal ofLeukocyte Biology.
58:217-224.

McConkey, D. J., P. Nicotera, and S. Orrenius. 1994. Signaling and
Chromatin Fragmentation in Thymocyte Apoptosis." in "Apoptosis in
Immunity"; dedicated volume of InmmunologicalReviews, G. Moller; ed.
142: December.

Meisel, C., K. Vogt, C. Platzer, F. Randow, C. Liebenthal, and H.-D. Volk.
1996. "Differential Regulation of Monocytic Tumor Necrosis Factor-
a and Interleukin-10 Expression." European Journal of Immunology.
26: 1580-1586.

Mertz, P. M., D. L. DeWitt, W. G. Stetler-Stevenson, and L. M. Wahl. 1994.
"Interleukin 10 Suppression Monocyte Prostaglandin H Synthase-2."
Journal of Biological Chemistry. 269(33): 21322-21329.

Miethke, T., R. Vabulas, R. Bittlingmaier, K. Heeg, and H. Wagner. 1996.
"Mechanisms of Peripheral T Cell Deletion: Anergized T cells are Fas
Resistant but Undergo Proliferation-associated Apoptosis." European
Journal of Immunology. 26: 1459-1467.

Minakuchi, R., M.C. Wacholtz, L. S. Davis, and P. E. Lipsky. 1990.
"Delineation of the Mechanism of Inhibition of Human T Cell Activation
by PGE2." Journal of Immunology. 145(8): 2616-2625.

Morham, S. G., R. Langenbach, C. D. Loftin, H. F. Tiano, N. Vouloumanos, J. C.
Jennette, J. F. Mahler, K. D. Kluckman, A. Ledford, C. A. Lee, and 0.
Smithies. 1995. "Prostaglandin Synthase 2 Gene Disruption Causes
Sever Renal Pathology in the Mouse." Cell. 83: 473-482.

Morita, I., M. Schindler, M. K. Regier, J. C. Otto, T. Hori, D. L. DeWitt, and W. L.
Smith. 1995. "Different Intracellular Locations for Prostaglandin
Endoperoxide H Synthase-1 and -2." Journal of Biological Chemistry.
270(18): 10902-10908.

Mountz, J. D., J. Wu, J. Cheng, and T. Zhou. 1994. "Autoimmune Disease: A
Problem of Defective Apoptosis." Arthritis & Rheumatism. 37(10): 1415-
1420.

Nagata, S. and P. Golstein. 1995. "The FasDeath Factor." Science. 267: 1449-
1455.








Niiro, H., T. Otsuka, T. Tanabe, S. Hara, S. Kuga, Y. Nemoto, Y. Tanaka, H.
Nakashima, S. Kitajima, M. Abe, and Y. Niho. 1995. "Inhibition by
Interleukin- 10 of Inducible Cyclooxygenase Expression in
Lipopolysaccharide-Stimulated Monocytes: Its Underlying Mechanism
in Comparison with Interleukin-4." Blood. 85(12): 3736-3745.

Nishimura, Y., A. Ishii, Y. Kobayashi, Y. Yamasaki, and S. Yonehara. 1995.
"Expression and Function of Mouse Fas Antigen on Immature and
Mature T cells." Journal of Immunology. 154: 4395-4403.

Obeid, L. M., C. M. Linardic, L. A. Karolak, and Y. A. Hannun. 1993.
"Programmed Cell Death Induced by Ceramide." Science. 259:1769-
1771.

Owen-Schaub, L. B., S. Yonehara, W. L. Crump III, and E. A. Grimm. 1992.
"DNA Fragmentation and Cell Death Is Selectively Triggered in
Activated Human Lymphocytes by Fas Antigen Engagement." Cellular
Immunology. 140: 197-205.

Paliogianni, F., R. L. Kincaid, and D. T. Boumpas. 1993. "Prostaglandin E2
and Other Cyclic AMP Elevating Agents Inhibit Interleukin 2 Gene
Transcription by Counteracting Calcineurin-dependent Pathways."
Journal of Experimental Medicine. 178: 1813-1817.

Parijs, L. V., A. Biuckians, A. Ibragimov, F. W. Alt, D. M. Willerford, and A. K.
Abbas. 1997. "Functional Responses and Apoptosis of CD25(IL2Ra)-
Deficient T cells Expressing a Transgenic Antigen Receptor." Journal of
Immunology. 158: 3738-3745.

Pastor, M. I., K. Reif, D. Cantrell. 1995. "The Regulation and Function of
p21ras during T-cell Activation and growth." Immunology Today.
16(3):158-164.

Podack, E.R. 1995. "Functional Significance of Two Cytolytic Pathways of
Cytotoxic T Lymphocytes." Journal of Leukocyte Biology. 57: 548-552.

Prescott, S. M. and R. L. White. 1996. "Self-Promotion? Intimate Connections
Between APC and Prostaglandin H Synthase-2." Cell. 87:783-786.

Reddy, S. T., and H. R. Herschman. 1994. "Ligand-induced Prostaglandin
Synthesis Requires Expression of the TIS 10/PGS-2 Prostaglandin
Synthase Gene in Murine Fibroblasts and Macrophages." Journal of
Biological Chemistry. 269(22): 15473-15480

Riese, J., T. Hoff, A. Nordhoff, D.L. DeWitt, K. Resch, and W. Kaever. 1994.
"Transient Expression of Prostaglandin Endoperoxide Synthase-2








during Mouse Macrophage Activation." Journal ofLeukocyte Biology.
55: 476-482.

Ristimaki, A. S. Garfinkel, J. Wessendorf, T. Maciag, and T. Hla. 1994.
"Induction of Cyclooxygenase-2 by Interleukin 1-a." Journal of
Biological Chemistry. 269(16): 11769-11775.

Sakaguchi, S., N. Sakaguchi, M. Asano, M. Itoh, and M. Toda. 1995.
"Immunologic Self-Tolerance Maintained by Activated T Cells
Expressing IL-2 Receptor a-Chains (CD25)." Journal of Immunology.
155: 1151-1164.

Segal, B. M., D. M. Klinman, and E. M. Shevach. 1997. "Microbial Products
Induce Autoimmune Disease by an IL- 12-Dependent Pathway." Journal
of Immunology. 158: 5087-5090.

Schorle, H., T. Holtschke, T. Hunig, A. Schimpl, and I. Horak. 1991.
"Development and Function of T cells in Mice Rendered Interleukin-2
Deficient by Gene Targeting." Nature. 352: 621-624.

Schutze, S., T. Machleidt, and M. Kronke. 1994. The Role of Diacylglycerol
and Ceramide in Tumor Necrosis Factor and Interleukin- 1 signal
Transduction." Journal ofLeukocyte Biology. 56: 533-541.

Serreze, D. V., K. Hamaguchi, and E. H. Leiter. 1989. "Immunostimulation
Circumvents Diabetes in NOD/Lt Mice." Journal ofAutoimmunity. 2:
759-776.

Serreze, D. V. and E. H. Leiter. 1988. "Defective Activation of T Suppressor
Cell Function in Nonobese Diabetic Mice." Journal of Immunology.
140(11): 3801-3807.

Serreze, D.V., J. W. Graedeke, and E. H. Leiter. 1993. "Hematopoietic Stem-
cell Defects Underlying Abnormal Macrophage Development and
Maturation in NOD/Lt Mice: Defective Regulation of Cytokine Receptors
and Protein Kinase C." Proceedings of the National Academy of
Sciences. 90: 9625-9629.

Serreze, D. V. and E. H. Leiter. 1994. "Genetic and Pathogenic Basis of
Autoimmune Diabetes in NOD Mice." Current Opinion in Immunology.
6: 900-906.

Sharfe, N., H. K. Dadi, M. Shahar, and C. M. Roifman. 1997. "Human Immune
Disorder Arising from Mutation of the a chain of the Interleukin-2








Receptor." Proceedings of the National Academy of Sciences. 94:
3168-3171.

Sneller, M. C., S. E. Straus, E. S. Jaffe, J. S. Jaffe, T. A. Fleisher, M. Stetler-
Stevenson, and W. Strober. 1992. A Novel Lymphoproliferative/
Autoimmune Syndrome Resembling Murine lpr/gld Disease." Journal
of Clinical Investigation. 90: 334-341.

Snijdewint, F. G. M., P. Kalinski, E. A. Wierenga, J. D. Bos, and M. L.
Kapsenberg. 1993. "Prostaglandin E2 Differentially Modulates
Cytokine Secretion Profiles of Human T Helper Lymphocytes." Journal
of Immunology. 150: 5321-5329.

Spittler, A., C. Schiller, M. Willheim, C. Tempfer, S. Winkler and G. Boltz-
Nitulescu. 1995. "IL-10 Augments CD23 Expression on U937 Cells and
Down-regulates IL-4-driven CD23 Expression on Cultured Human
Blood Monocytes: Effects of IL- 10 and Other Cytokines on Cell
Phenotype and Phagocytosis." Immunology. 85: 311-317.

Squier, M. K. T., A. J. Sehnert, and J. J. Cohen. 1995. "Apoptosis in
leukocytes." Journal of Leukocyte Biology. 57: 2-10.

Steller, H. 1995. "Mechanisms and Genes of Cellular Suicide." Science. 267:
1445-1449.

Strassmann, G., V. Patil-Koota, F. Finkelman, M. Fong, and T. Kambayashi.
1994. "Evidence for the Involvement of Interleukin 10 in the
Macrophages by Prostaglandin E2." Journal of Experimental Medicine.
180: 2365-2370.

Suda, T., T. Takahashi, P. Golstein, and S. Nagata. 1993. Molecular Cloning
and Expression of the Fas Ligand, a Novel Member of the Tumor
Necrosis Factor Family." Cell. 75: 1169-1178.

Sunderkotter, C., K. Steinbrink, M. Goebeler, R. Bhardwaj, C. Sorg. 1994.
"Macrophages and Angiogenesis." Journal of Leukocyte Biology. 55:
410-422. 1994.

Sweet, M. J., and D. A. Hume. 1996. Endotoxin signal transduction in
macrophages. JLeukocyte Biology. 60: 8-26.

Taguchi, 0. and T. Takahashi. 1996. "Administration of Anti-interleukin-2
receptor ax antibody in vivo induces localized autoimmune disease."
European Journal of Immunology. 26: 1608-1612.








Takenaka, H. S. Maruo, N. Yamamoto, M. Wysocka, S. Onon, M. Kobayashi, H.
Yagita, K. Okumura, T. Hamaoka, G. Trinchieri, and H. Fujiwara. 1997.
"Regulation of T cell dependent and -independent IL-12 production by
the three Th2-type cytokines IL-10, IL-6, and IL-4." Journal of Leukocyte
Biology. 61: 80-87.

Thompson, C. B. 1995. "Apoptosis in the Pathogenesis and Treatment of
Disease." Science. 267: 1456-1462.

Tian,Q., J.-L. Taupin, S. Elledge, M. Robertson, and P. Anderson. 1995. "Fas-
activated Serine/Threonine Kinase (FAST) Phosphorylates TIA-1 during
Fas-mediated Apoptosis." Journal of Experimental Medicine. 182: 865-
874.

Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of
proteins from polyacrylamide gels to nitrocellulose sheets: procedure
and some applications. Proceedings of the National Academy Sciences.
76(9):4350-4354.

Tsujii, M. and R.N. DuBois. 1995. "Alterations in Cellular Adhesion and
Apoptosis in Epithelial Cells Overexpressing Prostaglandin
Endoperoxide Synthase 2." Cell. 83: 493-501.

Ucker, D.S., J. Meyers, and P. S. Obermiller. 1993. "Activation-Driven T Cell
Death; II. Quantitative Differences Alone Distinguish Stimuli Triggering
Nontransformed T Cell Proliferation or Death." Journal of Immunology.
149: 1583-1592.

Ucker, D.S., L. D. Hebshi, J. F. Blomquist, and B. E. Torbett. 1994.
"Physiological T-Cell Death: Susceptibility is Modulated by Activation,
Aging and Transformation, but the Mechanism is Constant." in
"Apoptosis in Immunity"; dedicated volume of Immunological Reviews,
G. Moller; ed. 142: December.

van der Pouw Kraan, T. C. T. M., L. C. M. Boeije, R. J.T. Smeek, J. Wijdenes,
and L. A. Aarden. 1995. "Prostaglandin E2 is a Potent Inhibitor of
Human Interleukin 12 Production." Journal of Experimental Medicine.
181: 775-779.

Wang, R., A. M. Rogers, B.J. Rush, and J. H. Russell. 1996. "Induction of
Sensitivity to Activation-induced Death in Primary CD4+ Cells: a Role
for Interleukin-2 in the Negative Regulation of Responses by Mature
CD4+ T Cells." European Journal of Immunology. 26: 2263-2270.








Watanabe-Fukunaga, R., C. I. Brannan, N. G. Copeland, N. A. Jenkins, and S.
Nagata. 1992. "Lymphoproliferation Disorder in Mice Explained by
Defects in Fas Antigen that Mediates Apoptosis." Nature. 356:314-317.

Watson, J. and E. S. Wijelath. 1990. "Interleukin-one Induced Arachidonic
Acid Turnover in Macrophages." Autoimmunity. 8:71-76.

Webb, S., C. Morris, and J. Sprent. 1990. "Extrathymic Tolerance of Mature T
Cells: Clonal Elimination as a Consequence of Immunity." Cell. 63:
1249-1256.

Weber, G. F., S. Abromson-Leeman, and H. Cantor. 1995. "A Signaling
Pathway Coupled to T Cell Receptor Ligation by MMTV Superantigen
Leading to Transient Activation and Programmed Cell Death."
Immunity. 2: 363-372.

Wicker, L. S., J.A. Todd, and L. B. Peterson. 1995. "Genetic Control of
Autoimmune Diabetes in the NOD Mouse." AnnualReviews in
Immunology. 13: 179-200.

Winter, W. E., T. Chihara, and D. Schatz. 1993. "The Genetics of Autoimmune
Diabetes." American Journal of Diabetes Consultants. 147: 1282-1290.

Wu, T., S. J. Levine, M. G. Lawrence, C. Logun, C. W. Angus, andJ. H.
Shelhamer. 1994. "Interferon-g Induces the Synthesis and Activation of
Cytosolic Phospholipase A2." Journal of Clinical Investigation. 93: 571-
577.

Xie, T. 1997. "The Role of Prostaglandin Synthase 2 (PGS2) in the
Immunopathogensis of NOD Diabetes". Doctoral Dissertation.
University of Florida. College of Medicine.

Yui, M., K. Muralidharan, B. Moreno-Altamirano, G. Perrin, K. Chestnut, and E.
K. Wakeland. 1996. "Production of Congenic Mouse Strains Carrying
NOD-derived Diabetogenic Genetic Intervals: An Approach for the
Genetic Dissection of Complex Traits." Mammalian Genome. 7(5):
331-334.

Zhu, L. and C. Anasetti. 1995. "Cell Cycle Control of Apoptosis in Human
Leukemic T Cells." Journal of Immunology. 154: 192-200.















BIOGRAPHICAL SKETCH


Sally A. Litherland was born in Melbourne, Florida. She received her

Bachelor of Science in Food and Consumer Protection/Food Science from the

University of Florida in 1981, a Masters of Science in Nutritional Biochemistry

from University of Florida in 1983, and a Masters of Science in Molecular

Biology from Cornell University Medical College in 1990. She worked as the

Senior Mission Support Scientist and Deputy Manager of Mission Operations

for the Kennedy Space Center NASA Life Sciences contractor from 1990 to

1994. She has been a Science Consultant and Educational Public Affairs

Lecturer for NASA from 1990 to the present. Upon completion of her doctoral

studies, she will return to Florida's Space Coast area to pursue a career in

education and academic research.






I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of Doctor of Philosophy.



Micha V. Clare-S~ r,Chair
Assoc'a e Professor of Pathology,
Immunology, an oratory
Medicine

I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of Doctor of Philosophy.



AmmonL-Peek
..Prfessor of Pathology, Immunology,
And Laboratory Medicine

I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of Doctor of Philosophy.



Edward Wakeland
Professor of Pathology, Immunology,
And Laboratory Medicine

I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope
and quality, as a dissertation for the degree of Doctor of Philosophy.



Charles Allen
Professor of Biochemistry and
Molecular Biology









This dissertation was submitted to the Graduate Faculty of the
Department of Pathology, Immunology,and Laboratory Medicine, College of
Medicine, and to the Graduate School and was accepted as partial fulfillment
of the requirements for the degree of Doctor of Philosophy.


August, 1997


Dean, Graduate School






































UNIVERSITY OF FLORIDA
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