IDENTIFICATION AND CHARACTERIZATION OF MONOCYTE
PROSTAGLANDIN SYNTHASE 2 ACTIVITY AS A RISK FACTOR FOR AND
COMPONENT OF HUMAN AUTOIMMUNE DISEASE
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
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.
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
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
ABSTRAC T.......................................................................... viii
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
Sally A. Litherland
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
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.
EXPERIMENTAL BASIS AND RATIONALE
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
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,
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
CYCLOXKYGENASE PGH SYNTHASE: PGS1 OR PGS2
o>-- /- / COON
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
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;
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
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
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).
p = 0.0007
Subject C control
0 LPS 0 LPS
0 n= 14
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 %
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).
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.
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.
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.
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
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.
10o AI i
LPS Stimulated Control
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
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
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
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).
N 00/01 (2nPI)
Cells in Cycle (>2n PI)
<2n PI intercalation: Apoptotic & Dead Cells
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,
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
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
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
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.
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.
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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.
MEAN + 2SD
P= 0.003 "*
....... .... AA A
RELATIVES SUBJECTS LOW RISK MOD. RISK HIGH RISK
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,
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.
DAPI stained anti-PGS2-FITC
ICA+ PBMCs co-stain on same
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).
350- +2 SD
325- 0 0 LOW
S300- A A MODERATE
Z CORRELATION P=0.0201 O Al
HYPERBOLIC CURVE R = 1.78
S100 ----- --------------------------------- IVGTT
> : \7 v o X
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
+ 15- -110
S12- -90 .
5. 1: 2. .---- 0
-5-4-3-2-1 0 1 2 3 4 5 6 7 8 9101112131415161718192021222324252627282930
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
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,
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.
S3: It .04.
CONTROL ICA+ SUBJECTS
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.
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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
... P=0.0 1
B. GO/G 1 STATIONARY
00 0 0'
00 9 0
a00 0 9-V
.....-.- .... .......... ..... ....... -.- 0
AN OVA P=0.03
CONTROLS RELATIVES SUBJECTS
C. M/S/G2 CYCLING CELLS
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).
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.
N=7; P=0.545; R2=0.06
5- 0 FEMALE
" --- ................. .... ... .......-
0 3 6 9 12 15 18 21 24 27 30
% PGS2/CD14+ CELLS
N=10; P=0.0018; R2=0.68
O FEMALE .-"
0 10 20 30 40 50 60 70 80 90 100
% PGS2/CD1 4+ CELLS
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
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
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.
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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
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.
..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.
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.
Professor of Biochemistry and
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.
Dean, Graduate School
UNIVERSITY OF FLORIDA
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