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Anti-exocrine autoimmunity in the NOD mouse model of Sjogren's Syndrome

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Title:
Anti-exocrine autoimmunity in the NOD mouse model of Sjogren's Syndrome
Creator:
Robinson, Christopher Paul, 1970-
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English
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x, 150 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Antibodies ( jstor )
Cells ( jstor )
Cytokines ( jstor )
Diabetes ( jstor )
Gels ( jstor )
Lacrimal apparatus ( jstor )
Saliva ( jstor )
Salivary glands ( jstor )
Sjogrens syndrome ( jstor )
Type 1 diabetes mellitus ( jstor )
Autoimmunity ( mesh )
Cytokines ( mesh )
Department of Pathology and Laboratory Medicine thesis Ph.D ( mesh )
Dissertations, Academic -- College of Medicine -- Department of Pathology and Laboratory Medicine -- UF ( mesh )
Exocrine Glands -- chemistry ( mesh )
Exocrine Glands -- physiopathology ( mesh )
Gene Expression ( mesh )
Lacrimal Apparatus ( mesh )
Mice, Inbred, NOD ( mesh )
Mice, SCID ( mesh )
Models, Immunological ( mesh )
Research ( mesh )
Salivary Glands ( mesh )
Salivary Proteins ( mesh )
Sjogren's Syndrome -- etiology ( mesh )
Sjogren's Syndrome -- physiopathology ( mesh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 1997.
Bibliography:
Bibliography: leaves 136-149.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Christopher Paul Robinson.

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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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002287013 ( ALEPH )
49346360 ( OCLC )
ALP0164 ( NOTIS )

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ANTI-EXOCRINE AUTOIMMUNITY IN THE NOD MOUSE MODEL OF
SJOGREN'S SYNDROME













By



CHRISTOPHER P. ROBINSON


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








ACKNOWLEDGEMENTS


No usual thanks are sufficient to acknowledge my debt and sincere appreciation to

both of my co-mentors, Dr. Michael G. Humphreys-Beher and Dr. Ammon B. Peck.

Their insight has influenced both my personal and professional progression through this

graduate program. In addition, I would like to thank Dr. Linda Brinkley, Dr. Michael

Clare-Salzler, and Dr. Joel Schiffenbauer for their advice and contributions toward this

project.

1 am deeply grateful to Janet Cornelius, Micah Kerr, Jeff Anderson, Elizabeth

Bowen, Jason Brayer, and Kim Nguyen for providing technical assistance on this project.

Special thanks to all of my friends in the Peck, Humphreys-Beher, Clare-Salzler, and

Hillman laboratories who have made these past four years a genuine pleasure.

I would like express my sincere gratitude to my parents, Paul and Wesley

Robinson, without whose love, encouragement and support I would not be the person that

I am today. Lastly, I wish to thank my wife, Meryl, for her love, understanding, and

confidence in me.















TABLE OF CONTENTS


Page

ACKNOWLEDGEMENTS...................................................... ii

LIST OF TABLES... ............... .. .... ................... ........................ v

LIST OF FIGURES ....................................... ... .................... .. vi

ABSTRACT......................... ................... ...................... ix

CHAPTERS

1 INTRODUCTION ............................ ........ ...... ........ 1


2 CHARACTERIZATION OF THE CHANGING LYMPHO-
CYTE POPULATIONS AND CYTOKINE EXPRESSION
IN THE EXOCRINE TISSUES OF AUTOIMMUNE
NOD M ICE ................. .......... ........ ... ................... 30

Introduction ............ ............ .......... .. .......... 30
Materials and Methods.............. ..... ......... ... .. 32
Results .................. ............................. 37
Discussion .................................... .. ..... .. 50

3 GENETICALLY PROGRAMMED DEVELOPMENT OF
SALIVARY GLAND ABNORMALITIES IN THE NOD
(NON-OBESE DIABETIC)-SCID MOUSE IN THE
ABSENCE OF DETECTABLE LYMPHOCYTIC INFIL-
TRATION: A POTENTIAL TRIGGER FOR SIAL-
OADENITIS OF NOD MICE ................ ... .......... 56

Introduction ................. .. .............. ......... 56
M materials and M ethods ........................................... 57
R results .................. ..... .................................. 6 1
Discussion ............ ... ............. ......... ......... .... 74









4 EXPRESSION OF PAROTID SECRETARY PROTEIN IN
MARINE LACRIMAL GLANDS AND ITS POSSIBLE
FUNCTION AS A BACTERIAL BINDING PROTEIN ........... 80

Introduction .............. .......................... .......... 80
Materials and Methods ............................... ....... 81
Results............... .. .................... ....... 87
D discussion ............. .. ...................... .............. 10 1


5 EVALUATION OF NOVEL PROTEOLYTIC ACTIVITY
DETECTED IN THE SALIVA OF AGING NOD MICE .......... 105

Introduction ............... .. ... ..... ......... 105
M materials and M ethods ................. .......... ............ 107
Results ...................... .......... .......... ........... 116
Discussion ................. .......... ................. 124

6 CONCLUSION AND FUTURE DIRECTION ...................... 129

RE FEREN CE S ............................ .... .. ................................. .. 136

BIOGRAPHICAL SKETCH .................................................. ......... 150














LIST OF TABLES


Table Page

1. Murine Primer and Probe Sequences ..... ....... ..... ............. 34

2. Percentage of CD3 and B220 in the Lymphocytic Infiltrates of
Submandibular (SMG) and Lacrimal (LAC) Glands of NOD
M ice ................................................... .............. ........ 43

3. CD45RB Expression in Lymphocytic Infiltrating Cells of
Submandibular (SMG) and Lacrimal (LAC) Glands of NOD
Mice...................... ...................... .......... 44

4. Analysis of NOD-scid Saliva................. ................... ....... ............ 62

5. Cysteine Protease Activity in Saliva...................... ............ 115














LIST OF FIGURES


Figure Page

1. Histological Profile of Tissues Showing Lymphocytic Infiltrates of the
Exocrine Tissues and Insulitis in the NOD Mouse............................ 38

2. Flow Cytometric Analysis ofCD4* and CD8* T Cell Populations in
Lymphocytic Infiltrates in Tissues From 12 wk NOD Mice................ 39

3. Histogram of the Temporal Expression ofCD4' and CD8' T cells in
Lymphocytic Infiltrates of Submandibular (SMG), Lacrimal (LAC),
Parotid Glands (PAR) and Spleen (SPL) from NOD Mice................... 40

4. Histogram of Selective TCR Vp Distribution in Infiltrating Lymphocytes
Isolated From Spleen, Pancreatic Islet, Submandibular, and Lacrimal
Glands of NOD M ice................................................ .......... 45

5. Interleukin mRNA Expression of Lacrimal and Submandibular Glands as
Determined By RT-PCR and Southern Blotting.................................. 48

6. Proinflammatory mRNA Expression of Lacrimal and Submandibular
Glands as Determined by RT-PCR and Southern Blotting................... 49

7. Cytoplasmic Amylase Activity of Parotid and Submandibular Glands............ 63

8. Temporal Changes in the Protein Profiles of Saliva From NOD-scid
M ice..................................... .... ........................... 64

9. N-Terminal Amino Acid Residue Sequences of the 32 kDa, 27 kDa,
and 20 kDa Protein Bands (Shown on Fig 8) and Their Alignment
With the Published Sequence of Murine Parotid Secretory
Protein (PSP)....................... ...... ........ ..... ................... 65

10. Identification of Parotid Secretory Protein in Saliva Using Polyclonal
Anti-PSP Antibody on Western Blot.................... ................ 69

11. Western Blot Analysis of Cytoplasmic Fractions of Submandibular and
Parotid Cell Lysates Using Anti-PSP Polyclonal Antibody..................... 70








12. Western Blot Analysis of Saliva and Cytoplasmic Fractions of Parotid
and Submandibular Cell Lysates Using Anti-Proline-Rich Protein
Polyclonal Antibody...... ........ ................. ............ .............. 71

13. Morphological Changes in the Salivary Glands of NOD-scid Mice................ 72

14. RT-PCR and Southern Blot Detection ofPSP mRNA Isolated From
M urine Lacrimal Glands............. ..... ..... ...... .......... .......... 88

15. Western Blot Detection of Parotid Secretory Protein in Murine Lacrimal
Glands.................................... .................. ... .. ........... 89

16. Purification of Parotid Secretory Protein ............................................. 90

17. Amylase Enzyme Activity in the Presence of Saliva Proteins ....................... 91

18. Bacterial Binding of PSP................ ................................ ............ 93

19. Autoradiograph of PSP Binding to Bacterial Membrane Proteins ................. 94

20. RT-PCR and Southern Blot Detection of PSP Transcripts in NOD
and C3H/HeJ Tissues .......................................................... 96

21. Western Blot Detection of Parotid Secretory Protein in 8 wk NOD
and C3H/HeJ Tissue Lysates........................... ......... ............ 97

22. Sequence of Parotid Secretory Protein cDNA Derived From the Parotid
Gland mRNA From the NOD Mouse Strain ............... .................. 98

23. Autoradiogram of Differential PSP Migration Following Incubation
With Saliva or Salivary Gland Lysates........................................ Ill

24. Western Blot of Differential PSP Migration Following Incubation of
NOD and BALB/c Saliva.................................... ................... 112

25. Western Blot Depicting EDTA Inhibition of the Proteolytic Cleavage
ofPSP.................. ....... .. ...... ................. 113

26. Western Blot Depicting Proteolytic Cleavage of PSP in the Presence
ofEGTA..................... ..... ..... ...... .................... ..... ..... ......... .. 114

27. Histogram of Cysteine Protease Activity in Salivary Gland Lysates................ 119

28. Western Blot Analysis of Saliva for the Presence of Apoptotic
Proteases.. ............................ ... ............... ............... 120










29. Western Blot Analysis of Saliva for the Presence of the Cysteine
Protease Inhibitor; Cystatin.................. ... ............................. 121

30. Zymogram Gel Showing Distinct MMP Activities in NOD and BALB/c
Saliva... ........................... ..... ................ .. 122














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


CHARACTERIZATION OF ANTI-EXOCRINE AUTOlMMUNITY IN THE NOD
MOUSE MODEL OF SJOGREN'S SYNDROME


By

Christopher P. Robinson

May, 1997



Chairman: Dr. Ammon B. Peck
Cochairman: Dr. Michael G. Humphreys-Beher
Major Department: Pathology and Laboratory Medicine


The primary goal of this dissertation is to elucidate key features of the

pathogenesis of anti-exocrine autoimmunity in the NOD (non-obese diabetic) mouse. In

addition to autoimmune islet cell destruction, the NOD mouse develops chronic

lymphocytic infiltration of the salivary and lacrimal tissues, leading to dramatic declines in

exocrine gland secretion. The finding that secretary dysfunction in the NOD mouse

correlates to the presence of leukocyte infiltration of the exocrine tissues prompted the

study of this strain as a novel model of secondary Sjbgren's Syndrome. To further

develop this model, the studies presented in this dissertation investigate the anti-exocrine

infiltrating cell populations, physiological and biochemical alterations in NOD exocrine

tissues, and the contribution of the NOD genetic background to the development of anti-









exocrine autoimmunity. By flow cytometric analysis, the infiltrating lymphocyte

populations are shown to be quantitatively and qualitatively similar to that observed in

exocrine biopsies from Sjogren's patients and other animal models of this disease.

Similarly, RT-PCR detection of cytokine gene expression demonstrates similar cytokine

profiles in the NOD exocrine tissues as that seen in human patients and other disease

models. The investigation of the NOD-scid strain shows that morphological and

biochemical alterations of the salivary glands of NOD mice occur in the absence of

lymphocytic infiltration, suggesting that intrinsic alterations of the salivary glands may

underlie autoimmune invasion. Furthermore, aberrant gene expression and proteolytic

processing of the salivary protein, PSP, were detected in both NOD and NOD-scid

animals. This protein, previously reported as salivary gland specific, is further found to be

expressed in the lacrimal glands of mice. The novel cleavage of PSP is the result of a

proteolytic activity uniquely present in the saliva of aging NOD and NOD-scid animals.

Both cysteine protease and matrix metalloproteinase activities were detected in older

NOD and NOD-scid samples, potentially indicating increased apoptotic activity and

glandular restructuring, respectively. Novel proteolytic activity may explain the loss of

submandibular acini in NOD-scid mice, as well as potential candidate enzymes for PSP

cleavage. As such, the findings presented in this dissertation lay the foundation for future

genetic and molecular studies investigating the underlying pathogenesis of autoimmune

Sicca syndrome.













CHAPTER I
INTRODUCTION


Sjogren's Syndrome is a progressive, debilitating disorder in which the body's

immune system destroys the mucinous secreting exocrine tissues resulting in the hallmark

features of dry mouth and dry eyes (sicca syndrome) (1). While classified as an orphaned

autoimmune disease, the estimated number of cases of Sjogren's Syndrome in the United

States is believed to range between 200,000 to 4 million (2). This wide range in

estimated case numbers is attributed to the difficulty in properly diagnosing the seemingly

ubiquitous nature of many of the patient complaints. In Europe, where the criteria for

diagnosis is less stringent than in the United States, the prevalence is estimated at 0.1 to

1.0% of the population. As typical of many autoimmune diseases, over 90% of Sjogren's

Syndrome patients are women, most of which are post-menopausal (2). Common

complaints include "trouble chewing or parched mouth," "burning throat," "grit or glass in

the eyes," "blurred vision," "itching skin," or "debilitating fatigue"(3). Indeed, Sjogren's

has been termed the "great mimicker" since many symptoms, often considered minor or

vague, resemble those seen in other disease states such as hepatitis C infection, autonomic

neuropathy, or drug treatment (1). Since symptoms are often reported to medical

specialists such as dentists or optometrists, Sjbgren's Syndrome is often misdiagnosed and

symptoms treated as individual entities instead of systemic disorders. These complications

in diagnosis have led to the consensus described by the National Organization for Rare








Disorders (NORD) president Abbey Meyers that "Sjogren's Syndrome isn't a rare disease;

it's just massively under-diagnosed". The etiology of Sjogren's is unknown, and there are

no known cures. It was not until 1933, however, that sufficient interest led to the

landmark studies presented to the medical community by Swedish physician, Henrik

Sjogren (4).



Henrik Sjogren



Individual case studies of dry mouth or dry eye patients were presented as early as

the late 1800's (5). By 1927, Gougerot made the connection between salivary and

lacrimal sicca symptoms, and the linkage of these symptoms to arthritic disease was first

presented (6,7). Tying these findings together, Henrik Sjogren studied a patient group

predominately consisting of women over 40 years of age and displaying

keratoconjunctivitis sicca (4). Sjogren noted the appearance of lymphocytic infiltrates in

both the salivary and lacrimal tissues, as well as salivary gland swelling. Over 50% of the

patients had a history of arthritis. For the next 20 years, little advancement was made

beyond these descriptive observations. In 1951, after publishing numerous supplemental

studies, Sjogren concluded that the major criteria for diagnosis were keratoconjunctivitis

sicca, xerostomia, and polyarthritis (8). While the etiology of disease remained unknown,

Sjogren's microscopic and descriptive findings laid the foundation for the diagnostic

criteria which is still in use today.











Clinical Presentation and Complications of Siogren's Syndrome


The protein and mucin-rich secretions derived from the salivary, lacrimal, and

other minor exocrine tissues, i.e. labial and hardarian glands, are essential for maintaining

the health and integrity of the oral and ocular surfaces. These secretions provide not only

the fluid and electrolytes necessary for tissue homeostasis, but also contain several

additional classes of protein constituents (9). These constituents include important anti-

microbial defense mechanisms against pathogens such as immunoglobulins, iron chelators,

and proteases; growth factors important for mucosal tissue maintenance and regeneration,

such as epidermal growth factor (EGF), nerve growth factor (NGF), and transforming

growth factors (TGF-a and TGF-0); and mucinous lubricating agents. For the most part,

both tear and saliva secretions serve similar functions and contain many of the same

protein constituents, e.g., EGF, NGF, TGF-a, lactoferrin, lysozyme, and immunoglobulins

(9,10). At the same time, however, saliva and/or tear specific secretary proteins, as

evidenced by salivary amylase and digestive enzymes, provide for specialized physiological

functions of the individual secretary fluids (9).

The clinical presentation of sicca syndrome is associated with the loss of both the

fluid and proteinacious phases of saliva (1). This is demonstrated by the fact that

supplemental tear and saliva substitutes lacking the protein constituents of naturally-

occurring exocrine fluids are not complete remedies for patient discomfort associated with

dryness (11). Similarly, complaints of chronic dryness are often displayed in a portion of

autoimmune diabetic patients with normal flow rates (12,13). This indicates that the









protective protein and mucous constituents in addition to fluid saliva are essential for the

maintenance of oral and ocular health.

Disruption of exocrine secretion has severe clinical implications. In addition to

patient discomfort, corneal damage due to lack of lubricating fluids and chronic ocular

infections can lead to blindness in severely affected patients (14. Similarly, loss of

protective saliva and hydration provided by mucinous A*oatings leads to rampant

periodontal disease, caries, candidal infection, and backingg and loss of teeth despite

rigorous dental treatment regimens (15). Drypess and severe cracking of the tongue in

many patients leads to difficulties in speaking, chewing and swallowing most foods. As in

many autoimmune diseases, Sj6gren',patients often develop a waxing and waning in the

severity of symptoms, leading t periods of moderate health followed by chronic episodes

(16). To combat dryness, ~tificial saliva, tears, and ointments can provide symptomatic

relief for many patient; however, severe cases are also treated with anti-inflammatory

drugs (16).

Numerous systemic complications frequently appear in Sjogren's patients. The

most severe of these is a marked increase in the risk (>40 fold) of developing non-

Hodgkin's lymphomas in the salivary glands or cervical lymph nodes (17). In 1991, Fox

et al. demonstrated that salivary gland lymphomas predominately involve a t(14:18)

translocation which allows for increased synthesis of the proto-oncogene bcl-2 (17).

Over-expression ofbcl-4 is known to rescue activated lymphocytes from apoptotic death

(18).

Other symptoms include chronic fatigue, itchy skin attributed to both dryness and

vasculitis, and digestive disorders. Many patients appear to have central nervous system








disorders, which, in addition to chronic fatigue, further adds to the systemic nature of this

autoimmune disease (19).



Modem Diagnosis of Sjgiren's Syndrome



New advances in our immunologic and molecular understandings have led to an

expansion of the criteria determining the diagnosis of SjOgren's Syndrome. Today,

Sjogren's syndrome is subdivided into primary and secondary classifications. Primary

Sjogren's syndrome is defined as autoimmune sicca syndrome in the absence of other

autoimmune conditions (1). Secondary Sjogren's syndrome occurs in the presence of

other autoimmune connective tissue diseases, such as rheumatoid arthritis, systemic lupus

erythematosis, or scleroderma. The presence of dry eyes is measured through use of the

Schirmer's test of tear production, and Rose Bengal corneal-staining dye, once used by

Henrik Sjogren, is still used to visualize corneal damage. Salivary gland scintigraphy is

used to evaluate saliva flow rates. In 1960, Bloch et al. detected the presence of anti-

nuclear antibodies in the sera of approximately 70% of Sjogren's patients (20). This

finding was further expanded to identify two specific ribonucleoprotein antigens named

SS-A/Ro (60 and 52 kDa isoforms) and SS-B/La (47 kDa) (21). Antibodies directed

against SS-A and SS-B are present in approximately 90%/ and 70%, respectively, of sera

of Sjogren's syndrome patients (22). Rheumatoid factor is also present in 70%/ of

patients, including those that do not display joint involvement (23). Therefore, presence of

these autoantibodies has been used as a critical criteria for diagnostic purposes.








In the United States, the San Francisco criteria is used to diagnose candidate

patients displaying sicca syndrome (1). Under this criteria a positive diagnosis includes the

detection of lymphocytic infiltration in labial lip biopsies. Unfortunately, different

standards for diagnosis in the international community have led to great controversy in

regards to the patient populations used in clinical and basic research studies (24,25). In

the European (EEC) criteria, for example, examination of labial lip biopsies for presence

of lymphocytic infiltration is not essential for diagnosis. Thus, only an estimated 15% of

EEC patients would be diagnosed with Sjogren's Syndrome under the San Francisco

criteria (25). Since the patient populations are more restrictive in the San Francisco

criteria, comparison of international studies remains controversial.



Genetic Factors in Susceptibility



While the initiating environmental triggers of Sjogren's syndrome are unclear,

intrinsic genes contributing to disease susceptibility are thought to be critical potentiators

in the development of autoimmune disease. Approximately 12% of SS Sj6gren's

syndrome patients have a relative with this disease, demonstrating a familial aggregation

(26). The strongest disease associations appear related to specific alleles of the human

leukocyte antigen (HLA) complex or the corresponding major histocompatibility complex

(MHC) in murine models (27). These highly polymorphic gene products serve to bind and

present peptide epitopes to lymphocytes. A specific allele has unique peptide binding

specificities which are dependent upon the polymorphic amino acid composition in the

binding/anchoring clefts of the molecule. The subsequent presentation of peptides serves








as a foundation for the development of both tolerance to self tissue antigens and the ability

to bind and present novel exogenous peptides to the immune system (28). Therefore, it is

theorized that individuals with specific HLA alleles may be predisposed to selected tissue

specific autoimmunity based on the capacity of tissue autoantigens to bind in the HLA

binding clefts. In support of this theory is the finding that different autoimmune diseases

often correlate to the presence of specific HLA alleles.

In Caucasians with primary Sjogren's syndrome, an increased frequency of HLA-

B8 is found in -60% of Sjogren's patients and only 20% in controls (29). Additionally,

83% of primary Sjogren's syndrome patients displayed HLA-DW3 as compared with 24%

in control populations (30). Racial distinction in patient populations is also detected, as

Japanese patients display higher frequencies of HLA-DRW52 (31). While these findings

were generally true for primary Sjogren's patients, secondary Sjogren's syndrome

associated with rheumatoid arthritis did not display an association with either HLA-DW3

or HLA-B8, indicating genetic distinction between patient groups (29,30). Similarly,

Sjogren's syndrome associated with systemic lupus erythematosis appears related to DR3,

DQw2, and C4AQ0 (complement gene mapped to the HLA region) (31). Specific HLA

haplotypes are further associated with differences in the levels of anti-Ro antibody

production or high autoantibody levels in general. HLA-DW3, for instance, is associated

with decreased salivary flow rates and larger focal infiltration among patients (32).

Therefore, the presence of particular HLA alleles may not only contribute to disease risk,

but also to specific characteristics of an individual patient's disease.

The importance of non-HLA related genes appears to be important as well. A

review of familial studies by Goldstein et al. suggested the presence of a dominant, non-








HLA linked gene (33). In addition, discordance of Sjogren's Syndrome among identical

twins suggests that an environmental trigger(s) plays a critical role as well. The complex

polygenic nature of this disease may explain the widely varying complaints and disease

severity among patients afflicted with Sjogren's syndrome.



Infiltrating Lymphocytes



One advantage to the clinical study of Sj6gren's Syndrome as compared to many

other autoimmune diseases is the routine accessibility of tissue biopsies. Focal infiltrates

of the salivary glands are predominately CD4' T lymphocytes, with a smaller percentage

of CD8 T cells than detected in peripheral tissues (11). Helper T-cells are predominately

of a memory phenotype. Although polyclonal activation of B-lymphocytes is a hallmark

feature of this disease, only 10-15% of salivary leukocytic infiltrates are comprised of B-

lymphocytes. Interestingly, lacrimal gland infiltrates contain nearly double (-30%) the

numbers of B-cells as seen in the salivary glands (34). Whether the increased percentage

of B lymphocytes within the lacrimal glands is due to increased B-cell proliferation or

active recruitment remains unknown. However, detection of CD23 (blast-2 antigen) in the

lacrimal infiltrates of Sjogren's Syndrome patients support the possibility of

lymphoproliferation.

Diversity of the T-cell receptor (TCR) repertoire is achieved through the random

arrangements of the genes encoding the a and P chains. While numerous TCR a chains

are available to generate unique VDJ rearrangements, less than thirty P chains are

available to compliment the heterodimeric formation of the TCR. Therefore, it was








questioned whether the generation of autoimmune activity against selected tissue antigens

involved the preferential usage of specific variable P (Vp) chains which had a biased

affinity for tissue antigen epitopes (35). This idea is supported by the expansion of

dominant VB and Vo T-cells specific for defined experimental antigens, such as myelin

basic protein (36). In theory, if a biased TCR VP gene predominated a given autoimmune

disease, specific TCR depletion therapies could be used to slow disease progression

without completely immuno-compromising the patient.

In primary Sjogren's patients, a preferential usage of several TCR Vp genes has

been demonstrated in salivary gland biopsies (35,37,38). However, a similar study of

lacrimal tissues showed a highly diverse TCR repertoire, providing a potential indication

of the involvement of multiple tissue antigens. Unfortunately, the study of T-cell

phenotypes in human patients is hampered by the fact that tissue samples are from the late

stages of autoimmune progression. Therefore, the identification of the lymphocyte subsets

critical for the initiation of disease necessitates the use of animal models.



Cvtokines in Siogren's Syndrome



The immunoregulatory effects of cytokines have stimulated great interest among

researchers. Of note, several studies using immunohistochemistry, in situ RT-PCR, or

RT-PCR methods have shown that production of inflammatory cytokines is not restricted

to infiltrating immunological cells. Indeed, the exocrine tissues appear to produce IL-1

and IL-6 which may contribute to inflammation (39,40). Constitutive production of

transforming growth factor beta (TGFI), a strong immunosuppressive factor, is also








found in these tissues (41). Furthermore, production of IFNy by infiltrating immune cells

has been shown to induce the expression of HLA-DR on the surface of salivary gland

epithelium (42). Cytokine production is also important in upregulating expression of

ICAM-1 and E-selectin on the surface of salivary endothelial cells in Sjogren's patients

(43). Together, these findings have led to the current hypothesis that exocrine cells were

not merely passive targets of an aggressive immune system, but may be active participants

in the autoimmune process. Whether aberrant tissue expression of HLA protein is

involved in stimulating or propagating the autoimmune response has yet to be resolved.

In addition to IL-1 and IL-6, high levels of IL-2, IL-10, IL-12, TNFa and IFNy

are detected in Sjogren's biopsies (11). Levels of IL-4 are typically beyond detection.

While much emphasis has been placed upon delineating whether Thl (cytotoxic response)

or Th2 humorall) T-helper responses predominate in Sjogren's Syndrome, a clear picture

of the complex cytokine interactions is still beyond reach. The presence of IL-10,

described to direct the immune response to Th2 phenotype, in addition to IL-12 and IFNy,

which mediates the opposing Thl phenotype, suggests that the distinction of a Thl or Th2

response may not be applicable for this disease (44). Indeed, the cytokine profiles are

highly suggestive of a generalized pro-inflammatory process. Nonetheless, potential

clinical usage of cytokine-blocking antibodies or cytokine therapy to direct the immune

response to a beneficial state is of extreme interest among researchers.

In addition to immunoregulation, cytokines may play a more direct role in tissue

destruction. In vitro studies have now shown that IFNy is toxic to a human salivary gland

ductal cell line (HSG) (45). Similarly, TNFa, whose receptor bears homology to the

death domain associated with FAS-induced apoptosis, may play a critical role in mediating








both inflammation and apoptotic events (46). Wu et al. demonstrated that TNFa, while

not able to induce significant death of the HSG line, was able to act synergistically with

IFNy to induce rapid cell death (45). Whether these cytokines directly serve to induce

exocrine cell death in the autoimmune state has yet to be determined.



Implications of Viral Expression



Despite extensive research, the initiating triggers for this disease remain a mystery.

Discordance between identical twins and the late disease onset suggests that an

exogenous agent, such as glandular trauma or viral infection, may potentiate

autoaggression in susceptible individuals. Much interest has focused on the potential role

of virus in the initiation of Sjogren's Syndrome. Hepatitis C and HIV infection often

result in dry eye and mouth syndromes, but do not have the same histological and

autoimmune phenotypes of Sjogren's patients (47,48). Transgenic expression of the tax

gene from the HTLV-1, a human leukemia virus, in mice results in lymphocytic infiltration

of the exocrine glands (49). Detectable lax gene sequences were evident in 25% of labial

salivary gland biopsies of Sjogren's patients (50). Epstein-Barr Virus (EBV) antigens and

genomic DNA has been detected in the lacrimal tissue of Sjogren's patients, however high

levels in normal biopsies complicates these findings (51). Of particular interest is the

finding that the SS-B/La nuclear autoantigen is associated with viral translation and may

also be redistributed to the cytoplasm and cell surface following infection (52). Whether

the generation of autoantibodies against the Ro and La ribonucleoproteins is potentially

due to their association with viral RNAs or molecular mimicry of viral epitopes is








currently a hot topic among researchers. Thus, the potential involvement of viral infection

in the initiation or exacerbation of this disease is intriguing; however, researchers have yet

to demonstrate a primary etiologic role of virus in Sj6gren's Syndrome.



Autoimmune Disruption of Exocrine Secretion



Stimulation of exocrine secretion is under the control of both sympathetic and

parasympathetic innervation. Parasympathetic nerves, primarily allowing for the release of

watery phase of saliva, are responsible for basal levels of saliva production as well as

mechanically stimulated saliva flow, such as when eating (53). Parasympathetic

stimulation leads to the release of electrolytes which form the osmotic gradient needed for

salivation. Mucin rich saliva secretion is mediated through sympathetic stimulation.

Pilocarpine, a muscarinic receptor agonist, is a drug currently used to treat patients

through parasympathetic stimulation (54). In our studies, pilocarpine was used to

stimulate watery saliva, while the B-adrenergic receptor agonist, isoproterenol, was used

to stimulate proteinacious saliva flow.

Disruption of the naturally occurring secretary process is a frequent consequence

of pharmaceutical drugs or the effects of irradiation in anti-cancer therapies (1). While the

pathogenic mechanism of exocrine dysfunction in Sjogren's Syndrome is unclear,

autoimmune disruption of the secretary process may occur through degeneration of

glandular innervation, blockage of receptor stimulation, cellular destruction, or

degeneration of the secreting cells. It is generally believed that immune destruction of the

exocrine cells is not the primary mechanism for the loss of exocrine secretion (11). This is









in stark contrast to Type 1 insulin dependent diabetes, for instance, where disease onset

occurs when over 90% of the insulin secreting 3 cell mass is eliminated (55). Histological

evaluation of exocrine biopsies reveals that only approximately 10% of the tissue area is

replaced by lymphocytic infiltration (56). In areas not in direct contact with lymphocytic

foci, large areas of seemingly normal tissues are present. Furthermore, the severity of

sialadenitis does not appear correlated with measured loss of glandular secretions. Equally

perplexing is the fact that loss of glandular secretion appears inversely related to the

presence ofanti-ductal cell antibodies in patient sera (57). These findings have led to the

current theory that the exocrine glands of Sjogren's patients are "loafing"(l 1). Chronic

destruction of the normal glandular architecture could lead to tissue dedifferentiation and

loss of secretary function.

The classical waxing and waning of symptoms in Sjogren's patients suggests that

lymphocytes play a direct role in effecting the loss of salivary function. For instance, if

glandular innervation was effectively destroyed, abrogation of the immune response would

not be expected to restore function. Therefore, it is possible that cytokines and antibodies

released by immune components are playing an active role in disrupting neuro-glandular

stimulation. Autoantibodies against the Ro and La antigens, while prevalent in Sjogren's

patients, have yet to be directly implicated in the loss of exocrine function; although high

levels of maternal autoantibody correlates to an increased risk of severe heart defects in

children (1).








Animal Models of Sjogren's Syndrome

The inherent scientific and ethical limitations in the study of human subjects has

forced basic researchers to turn to the study of animal models which display specific

disease traits. Several murine models for Sjogren's Syndrome have been suggested based

on the presence oflymphocytic infiltration in the salivary glands, including the NZB/NZW

F1, MRLApr, NFS/sld, and TGFp knockout congenic mice (58-61). The TGF3 knockout

mouse develops extensive systemic autoimmunity including anti-exocrine infiltration and

typically dies within one month of birth (61). In 1994, Haneji et al. detailed anti-exocrine

autoaggression in NFS/sld mice thymectomized at birth, however exocrine dysfunction

was not evaluated in this study (60). The NFS/sld mouse was shown to contain a single

recessive gene defect in sublingual gland development, indicating that abnormalities in

glandular development may contribute to autoimmune tissue targeting.

The NZB/NZW Fi and the MRL/lpr mice display lymphocytic infiltration of both

the lacrimal and salivary glands in addition to lupus-like disorders, and have therefore been

studied as models for secondary Sjogren's Syndrome (58,59). Interestingly, the presence

of lymphocytes in these tissues and apparent histological changes in the exocrine glands of

these mice does not correlate to severe loss of glandular function. Only NZB/NZW mice

>4 months of age displayed slightly abnormal Schirmer tests, and none of these strains

showed a loss of salivary flow (54). Walcott et al. recently described progressive

degeneration of glandular innervation in the lacrimal glands of NZB/NZW mice over 6

months of age (62). Similar to that of human disease, focal infiltration of the exocrine

tissues in NZB/W and MRIJIpr mice was more prominent in female than male mice,









occupied no more than 30% of tissue sections, and intensity of the infiltration did not

correlate to glandular dysfunction (54).

Lymphocytes infiltrating the exocrine tissues of MRL/Ipr mice, which have a

genetic disruption of Fas-FasLigand-mediated apoptosis, are predominately CD4' T-cells

(63). Analysis of TCR Vp expression revealed a diverse T-cell repertoire, however, a

skewing of the population to V18.1.2 and V36 was detected (64). Purified CD4', but not

CD8' lymphocytes isolated from MRL/lpr salivary glands are able to transfer sialadenitis

to immunocompromised CB17-scid mice (65). Interestingly, specific depletion of donor

VP38 or VP6* lymphocytes prior to transfer significantly inhibited sialadenitis in recipient

mice.

Both the NZB/NZW Fl and MRL/lpr mice display similar cytokine profiles as

those detected in human tissue biopsies (66). Therefore, since these mice do not develop

severe functional deficiency, it is unlikely that the cytotoxic effects of hallmark cytokines

such as IFNy are primary effectors of exocrine dysfunction in murine models. Together

these findings demonstrate that the mere homing of leukocytes to the exocrine tissues is

not sufficient to cause the loss of exocrine function. In 1992, an advance in the search for

an animal model for Sjogren's was made by Hu el al. in their description of secretary

dysfunction in autoimmune NOD (nonobese diabetic) mice (67).



NOD Mouse



First introduced in 1980, the inbred non-obese diabetic (NOD) mouse exhibits a

strikingly similar pathology to that of human Type 1 insulin dependent diabetes mellitus









(IDDM) (55). Lymphocytic destruction of the p-cells of the islets of Langerhans results in

a loss of blood glucose regulation due to the loss of insulin secretion. Immune infiltration

of the pancreas can begin in NOD mice as early as two weeks of age and begins with the

appearance of Class 11 monocytes and CD8 T lymphocytes (68). Overt diabetes in NOD

mice generally begins between 8-12 wks of age and occurs when >90% of the B-cell mass

is destroyed. By 30 weeks of age roughly 80% of female and 20% of male mice become

diabetic (55). Interestingly, NOD mice kept in specific pathogen free (SPF) colonies

develop diabetes more frequently and at an earlier age that those kept in traditional

colonies (69). This indicates that, similar to the human condition, environmental factors

play a role in the development of autoimmunity in these mice.

The observation of the lymphocytic infiltration in the NOD mouse is not confined

to the pancreas, however, but is also observed in the salivary and lacrimal glands of this

strain (70). Interestingly, the parotid gland does not develop extensive lymphocytic

infiltration. The first appearance of periductal and perivascular lymphocytic infiltration in

the submandibular and lacrimal glands begins at 8-10 wks and 10-12 wks of age,

respectively (71). By 18 week of age, the exocrine glands display focal lymphocytic

lesions with an observable disorganization of normal acinar structure. Despite the early

appearance of lymphocytic infiltrates within the submandibular and lacrimal glands, loss

of saliva flow and tear production does not typically occur until 14-16 wks of age (67).

This result was expanded to show that NOD mice lose approximately 90%/ of their saliva

flow and 30% of tear flow between 8 and 20 weeks of age (71). Unlike diabetes in the

NOD, both male and female mice develop infiltration of the exocrine tissues at similar

rates (70). These findings reveal that the NOD mouse represents the first-described animal









model for the spontaneous autoimmune-induced loss of both saliva flow and tear

production, and, as such, is emerging as an excellent model for the study of Sjogren's

Syndrome in humans.



Immunogenetics of the NOD Mouse



The NOD mouse arose during the selective breeding of the inbred cataract

Shionogi (CTS) strain from outbred ICR mice (55). In 1974, a single female mouse

exhibiting polyuria, severe glycosuria, and weight loss was discovered, and, after extensive

inbreeding, the NOD strain was introduced in 1980. Since this time, at least 13 genetic

loci have been discovered which contribute to the development of diabetes in NOD mice

(72). An additional two genetic loci, Idd-7 and Idd-8 found in the B10 background, are

also able to increase diabetes progression when bred onto the NOD background (69).

Genetic influence contributes to both the incidence of diabetes, as in Idd-l, as well the

timing of disease onset, as in Idd-2 and Idd-4.

The MHC-linked locus, Idd-1, is essential for the development of diabetes, as

replacement of the of the NOD MHC loci with the B10-derived allele completely inhibits

both diabetes and insulitis, but not sialadenitis (73). The NOD MHC, H-287 haplotype

contains several unique features, including a structurally distinct I-A molecule and a

deletion of I-E expression (74). Conserved serine-aspartic acid or proline-aspartic acid at

the A3 amino acid positions 56 and 57 are replaced with histidine and serine in the NOD

allele (75). In humans, replacement of the aspartic acid with uncharged amino acids at

position 57 of the human DQP has been implicated in increased diabetes risk (76).









Transgenic expression of I-Ak in NOD mice inhibits insulitis and diabetes, however

sialadenitis was not affected (77). The lack of I-E expression is not unique to the NOD

strain, but is also a feature of inbred C57BL/6, SJL, ACA, and DBA/I mice (78). Since

the B10 mice used in generating the NOD.B0I-H-2b congenic are also I-E negative, the

impact of 1-E expression on the NOD strains was not investigated in this model.

Generation of transgenic NOD mice which express the I-E molecule has determined that I-

E expression is protective against diabetes and insulitis (79). Sialadenitis was not

investigated in these mice. Interestingly, Faustman found occasional lymphocytic

infiltration in the submandibular glands in the majority of the I-E negative strains

mentioned above (78). The I-E molecule is responsible for the deletion of TCR vI35 T-

lymphocytes, suggesting that vp5' lymphocytes may play a role in the development of

sialadenitis. However, NOD mice backcrossed with SWR mice, which contain a deletion

in v35, vp8 and vp11 gene segments, still develop typical frequencies of insulitis and

diabetes (80). Together these findings demonstrate that both the unique NOD I-Ag7

molecule and the lack of I-E expression are important for diabetes, however exocrine

gland infiltration is only associated with the lack of I-E expression.

The contributions of non-MHC genes to the development of diabetes and

sialadenitis is an area of intense interest. Garchon et al. linked a centromeric locus on

chromosome 1 with diabetes susceptibility (81). Interestingly, a second susceptibility

locus on the same chromosome was linked to sialadenitis, peri-insulitis, and hyper-IgG

production. The development of sialadenitis was found to be a dominant trait in these

studies (81). To evaluate immune and non-immune components of autoimmunity in the

NOD mouse, Leiter et al. bred the scid mutation onto the NOD genetic background,









creating the NOD-scid strain which was extensively used in the current studies (82).

Homozygosity at the scid locus leads to the loss of functional T and B lymphocytes, and,

therefore, these animals do not develop insulitis, sialadenitis, or diabetes. This study also

demonstrated that, similar to parental NOD mice, NOD-scid mice have complement

deficiency, functionally immature macrophage populations, and decreased natural killer

cell activity (82). The sum results of these immunogenetic findings are promising in that

they strongly suggest that the ultimate goal of genetically separating diabetes from

exocrine gland autoimmunity to develop a primary marine model for Sjogren's Syndrome

will be attainable.



Role of Helper T-Lymphocvtes



Prior to this study, the lymphocyte phenotypes infiltrating the exocrine glands were

largely unexplored. Therefore, our knowledge of autoimmunity in the NOD mouse comes

almost exclusively from diabetes research. While autoimmune infiltration includes several

immune cell types, researchers have predominately focused on the role of CD4' T

lymphocytes which represent the largest component of the infiltrate. Neonatal

thymectomy of NOD mice generally inhibits insulitis and diabetes, and athymic nude NOD

mice do not develop either phenotype (83,84). Similarly, injection of anti-Thyl.2, anti-

CD4, or anti-CD3 antibodies suppresses diabetes (55). A direct role of lymphocytic

involvement is demonstrated by the ability of splenic or tissue infiltrating lymphocytes

from diabetic donors to rapidly transfer both diabetes and sialadenitis to non-diabetic

NOD or NOD-scid recipients (85). As opposed to rapid transfer of disease, splenic









lymphocyte preparations from prediabetic mice suppressed disease onset. Therefore, the

ability to successfully transfer disease is not only dependent upon the presence of CD4* T

lymphocytes, but also their activation state.

When transferred to immunocompromised recipients, purified naive CD4+ T cells,

represented by the CD4'CD45RBhi phenotype, induce a wasting disease associated with

intestinal inflammation (86). Interestingly, co-transfer ofCD4+CD45RBo memory T cells

suppresses the wasting disease (87). Two distinct populations of CD4' memory T cells

with opposing diabetogenic potential have been separated from prediabetic and diabetic

NOD mice based upon their cytokine expressions (87). Memory cells producing high

levels of IFNy (Thl) and low levels of IL-4 transfer diabetes rapidly to recipient mice.

However, memory cells producing low amounts of IFNy and high levels of IL-4 (Th2)

confer active suppression (87). This indicates that the ability to progress from a protective

to a pathogenic state may lie in the relative levels of cytokines present in the autoimmune

lesion. This is supported by similar findings using diabetogenic CD4' T-cell clones.

Haskins et al. demonstrated that shifting high IFNy producing T cell clones to IL-4

producers in long term culture suppressed their ability to transfer disease (88).

Furthermore, successful transfer of diabetes with CD4' T cell clones into NOD-scid

recipients demonstrates the potential of CD4' cells to transfer diabetes in the absence of

either CD8' T cells or B lymphocytes.

Analysis of TCR V3 usage in the pancreas of NOD mice shows heterogeneous

TCR populations. However, much like the MRL/lpr mouse, a preferential usage of V08

and Vp6 genes is present in diabetogenic T cells (89). Edouard et al. showed that

selective deletion of Vp6' or Vp38 T cells prior to adoptive transfer significantly lowered









the ability to transfer diabetes to irradiated NOD recipients (90). Although suggestive of a

pathogenic role for Vp6' and VB8* T cells, NOD X SWR mice develop insulitis and

diabetes despite deletions in Vp5, V38, and Vl 31 gene segments (80). In addition, TCR

usage from islet specific T cell clones of diabetic NOD mice include Vp4, Vp6, V18.2,

Vp112, Vp16, and V319 gene usage, further indicating that glandular specific TCR gene

usage is not restricted (55). Recent findings by Anderson (personal communication) have

indicated that TCR V1 restriction may be present in a CD3*CD4"CD8 "double negative"

T cell population which is present in early islet infiltrates; however, this has yet to be

confirmed.



CD8' T Lvmphocvtes and B Lymphocytes



While the necessity of CD4' lymphocytes is readily apparent, the role of CD8' T

cells and B cells in the initiation of autoimmunity is less clear. Transfer of purified CD8* T

cells is typically insufficient to cause diabetes, however a recent report demonstrated that

diabetes can be transferred by a CD8+ T cell clone (85,91). Using a gene knockout

strategy, Wicker et al. determined that disruption of the 0-2 microglobulin gene, which

results in a specific loss of CD8 T-cells, inhibits both insulitis and diabetes in NOD mice

(92). However, in prediabetic mice, co-transfer of both CD4+ and CD8' populations is

necessary to initiate diabetes (85). Therefore, while an aggressive CD4* or CD8' T cell

clone may be able to cause diabetes, both cell types appear essential for the initiation of

disease.








Similarly, despite the fact that autoantibodies are detected on the surface of islet

cells prior to leukocytic infiltration, the role of B-cells has been largely downplayed in the

literature (93). Renewed interest in the B cell component is being generated with the

discovery that NOD mice containing a p-heavy chain knockout which halts the generation

of mature B lymphocytes do not develop insulitis or diabetes (94). While exocrine

dysfunction has yet to be evaluated in these mice, several studies have indicated that the B

cell component may be important for exocrine dysfunction in NOD mice. In addition to

the presence of antibodies directed against islet cell components, NOD sera contains both

anti-nuclear antibodies and antibodies directed against salivary gland cells (95,96). In an

analysis of the signal transduction pathways responsible for secretion from salivary and

lacrimal glands, Humphreys-Beher et al. identified a subclass of autoantibodies directed

against the muscarinic and p-adrenergic cell surface receptors that initiate this process

(97,98). The density of these cell surface receptors is reduced on both the parotid and

submandibular glands, along with secretary response to 0-adrenergic,

muscarinic/cholinergic and neuropeptide agonists during disease. Thus, it is possible that

soluble factors such as autoantibodies may ultimately be responsible for the loss of

secretary function in NOD mice. This may also explain how parotid gland function is

inhibited despite the lack of focal lymphocytic infiltration. Together, these findings

suggest that the initiation of autoimmunity in the NOD mouse involves multiple immune

components and numerous cell types.









Potential Dichotomy of Diabetes and Sialoadenitis



Approximately 30% of autoimmune diabetic patients suffer concomitantly from

xerostomia and xerophthalmia due to insufficient glandular secretions (13). These clinical

symptoms are thought to result from poorly maintained blood glucose regulation, lack of

insulin secretion, or neuropathy (99). Expansion of the autoimmune processes into the

exocrine glands of diabetic patients is rarely encountered and generally not considered to

be the causative factor for exocrine dysfunction in diabetics (12). In support of this, tight

glucose control generally alleviates the feeling of dryness in most patients. Therefore, is it

possible that the loss of exocrine secretion in NOD mice is due to the loss of blood

glucose regulation, and not anti-exocrine autoaggression?

In addition to the immunogenetic studies discussed above, several studies have

illustrated a dichotomy between autoimmune diabetes and autoimmune sialadenitis in the

NOD mouse. Loss of secretary function in NOD mice, while more severe after diabetes

onset, is present in both male and female prediabetic mice, which lose 50-70% of

stimulated salivary flow between 8 and 20 weeks of age (67). Similarly, injection of

diabetic mice with daily insulin injections does not restore secretary function. Recent

findings using NOD.B10-H-2b mice have shown that the loss of secretary function occurs

in the absence of either insulitis or diabetes (unpublished observations). Treatment of

NOD mice with antibodies against alpha 4-integrin and L-selectin was able to significantly

inhibit both insulitis and diabetes, however, sialadenitis was unaffected (100). In humans,

increased expression of ICAM-1 and E-selectin is detected on salivary epithelial cells (43).

From the standpoint of tissue tolerance, intrathymic injection of islet cell homogenates or









transgenic expression of proinsulin II in NOD mice prevents diabetes but fails to protect

against sialadenitis (101,102). These studies suggest that the antigens involved in the anti-

exocrine response are distinct from those in the islet, and that loss of exocrine gland

tolerance is not secondary to p cell autoimmunity.



Changes in Saliva Proteins in the NOD mouse



In addition to loss of secretary function in NOD mice, the protein constituents of

NOD saliva change over time. This is reflected by reductions in amylase activity (>50%)

and loss of ductal cell secretion of EGF (67). In newly diabetic male NOD mice, over

97% of EGF production is lost. In this dissertation, specific changes in both parotid

secretary protein (PSP) and proline rich proteins (PRP) are observed in both NOD and

NOD-scid animals which parallel changes in EGF and amylase. The proline rich proteins

are latent in normal mice; however, they comprise >70% of the protein in human saliva,

where they serve to bind both calcium and hydroxyapatite (103). Expression of the PRPs

can be induced in both the marine parotid and submandibular glands by chronic B-

adrenergic stimulation or the introduction of high levels of tannic acid in the diet (104). In

these studies we demonstrate the abnormal presence of PRPs in both submandibular and

parotid glands of aging NOD mice.

In normal mice, PSP is described as a 20 kDa, leucine-rich glycoprotein (-23%

leucine; 235 amino acids) of unknown function that is secreted predominately by amylase

producing acinar cells of the parotid gland (105). Studies have shown a developmental

coordination of murine PSP and salivary amylase expression in the parotid gland of adult









mice where the two proteins appear in constant ratios (106). Coordinate expression,

however, is not determined by the rate of gene expression, since developmental expression

of the PSP gene occurs before amylase expression (107). Although PSP is specific to the

parotid and sublingual glands of adult mice, PSP is expressed in the developing

submandibular gland up to 5 days of age (108). This is important in that abnormal re-

expression of PSP in the submandibular gland is detected in aging NOD mice and may

indicate dedifferentiation of the glandular acini. While the function of PSP is unknown,

our findings suggest that it may play a role in anti-microbial binding. To date, PSP has not

been detected in human saliva (our anti-mouse PSP antibody does not crossreact),

however a single copy of the PSP gene is present in human cells and PSP mRNA has been

detected in human parotid tumors (109).

Results in this dissertation show that PSP is enzymatically cleaved in older NOD

saliva. The cleavage occurs between leucine and asparagine at the 26th and 27"' amino

acids of the protein. Database searches uncovered no known enzymes which may be

responsible for this cleavage. However, since PSP cleavage corresponds to the time of

dramatic acinar cell loss in the NOD mouse, we investigated the potential of both matrix

metalloproteinases and apoptotic cysteine proteases to cleave PSP through bystander

activity. These proteins, involved in glandular restructuring and apoptotic cell death,

respectively, may play a key role in the loss of submandibular acini in NOD and NOD-scid

mice.








Matrix Metalloproteinases and Cysteine Proteases



Cellular homeostasis depends on regulated cell proliferation coupled to cell death

(110). The matrix metalloproteinases (MMPs) are a class of zinc-dependent enzymes

which, in conjunction with their specific inhibitors (TIMPS), are responsible for

restructuring and maintenance of the extracellular matrix. Over 12 MMPs have been

described and include the collagenases, gelatinases (collagen type IV), stromalysins

(laminases), and elastins which are able to cleave virtually all components of the

extracellular matrix (111). Expression of specific MMPs during the developmental

process is responsible for sculpturing the extracellular environment and thereby dictating

cellular turnover and differentiation. While much research has focused on their association

with tissue homeostasis and tumor metastasis, a prominent role of MMPs in autoimmune

processes has recently been described (111). Monocyte production of MMPs is involved

in dictating the extent of tissue damage caused by inflammation and may additionally be

critical for matrix degradation necessary for lymphocyte chemotaxis. Inhibitors of MMPs

have been shown to significantly reduce the appearance and severity of EAE in animal

models (112). This effect was attributed to the inhibition of monocyte-derived gelatinases

responsible for the degradation of the blood-brain barrier basement membrane. Similarly,

MMP inhibitors significantly reduce joint inflammation and tissue destruction in collagen-

induced murine model of rheumatoid arthritis (113). Studies have further demonstrated

that MMP activity is connected to the apoptotic pathway through the ability to cleave cell

surface FAS on apoptotic target cells (111). In humans, SjOgren's patients have elevated

levels of collagenase (MMP-1) in their saliva, and supematants from excised biopsies









contain elevated MMP activities (114). Whether elevated or aberrant MMP production

precedes or is a result of tissue inflammation in autoimmune target tissues is still unknown,

however the extensive changes in glandular architecture seen in tissue biopsies may, in

part, be attributed to MMP activity.

Extracellular signaling molecules are capable of regulating glandular cell

populations through a series of intracellular events termed programmed cell death or

apoptosis (110). Apoptosis is distinct from cell lysis in that the target cell actively and

efficiently mediates its own death. This process involves intracellular activation, reduction

of cell volume, chromatin condensation, and endonuclease cleavage of DNA within the

cell membrane (115). By this process, the apoptotic cell contents are kept safely inside the

cell membrane until phagocytized by macrophages or neighboring cells. Immune system

molecules capable of mediating apoptosis include the cytokine tumor necrosis factor

(TNF) and the related protein CD95 (FAS) (115). Mice that lack functional FAS surface

protein develop a lymphoproliferative disorder and lupus-like pathology associated with

the inability to delete lymphocytes during the immunological education process (116). In

Sjogren's patients, it is hypothesized that dysfunction in apoptosis results in abnormal

longevity of both T and B lymphocytes and may underlie polyclonal B-cell activation.

Elevated levels of bcl-2, which is able to rescue target cells from apoptotic death, are

detected in salivary gland biopsies (117). Interestingly, 2-fold increases in surface FAS

expression are detected in peripheral blood T cells from both Sjogren's Syndrome and

SLE patients than controls which correlated with accelerated apoptosis of these cells in

vitro (118). Therefore, it is unlikely that defective FAS molecule is directly responsible

for Sj6gren's Syndrome or SLE in most diseased patients.









Activation of the FAS or TNF receptors leads to a proteolytic cascade involving

members of the cysteine protease family (110). These proteases cleave important cellular

proteins including pro-enzymes of other members of this class of proteases and

endonucleases responsible for DNA cleavage. A prototype protein of the cysteine

proteases is interleukin-l -converting enzyme (ICE) which has been characterized as the

activator of the cytokine interleukin-lp through cleavage of its precursor at

Aspl 16/Alal 17 (119). All members of the ICE-related cysteine protease family, including

Nedd-2 and apopain/cpp32, cleave their substrates after an aspartate residue followed by a

small amino acid residue which is important for substrate consensus recognition (120).

Expression of ICE in neuroblastoma or fibroblast cell lines leads to apoptotic death (110).

Therefore, high levels of cysteine protease activity may be indicative of both the

activation of apoptotic mechanisms as well as the processing of proinflammatory cytokine

precursors. Since both MMPs and cysteine proteases appear involved in regulated cell

proliferation and cell death, it is possible that upregulation of these enzymes in the NOD

mouse are directly involved in acinar cell death and the aberrant cleavage of additional

salivary proteins.





Further Development of the NOD Mouse Model of Soigren's Syndrome



The physiological loss of secretary function makes the NOD mouse an ideal animal

model for the study of human Sjogren's Syndrome. Despite the limited amount of

exocrine gland research, the most exciting aspect of the NOD mouse is the ability to apply









the findings of over 15 years of diabetes research to the understanding of autoimmune

sicca syndrome. Additionally, the seeming dichotomy between sialadenitis and diabetes

suggests that the study of congenic NOD mice, such as the NOD.BIOH-2b, will lead to the

development of a primary model to study anti-exocrine autoimmunity. To begin these

studies, however, it is necessary to determine the immunological and physiological

processes underlying exocrine dysfunction in the NOD mice. Therefore, the studies of this

dissertation explore the following specific aims:

1. To determine the temporal changes in exocrine gland histology, infiltrating lymphocyte

populations, and cytokine production spanning the initiation of the autoimmune

process to the final stages of glandular destruction.

2. To investigate exocrine gland abnormalities in immunodeficient NOD-scid mice in

order to separate immune and non-immune components of the disease process.

3. To investigate the tissue specificity and function of parotid secretary protein, which is

found to be aberrantly expressed and processed in aging NOD mice.

4. To evaluate the proteolytic activity in NOD saliva responsible for the aberrant

cleavage of PSP and investigate candidate enzymes for PSP cleavage.














CHAPTER 2
CHARACTERIZATION OF THE CHANGING LYMPHOCYTE POPULATIONS AND
CYTOKINE EXPRESSION IN THE EXOCRINE TISSUES OF AUTOIMMUNE NOD
MICE

Introduction


During the past several years significant interest has developed in detailing the

autoimmune destruction of the salivary and lacrimal tissues in the NOD mouse.

Pioneering studies have demonstrated that the lymphocytic infiltration of the salivary and

lacrimal glands correlates with a functional decline in saliva flow and tear production

independent of the loss of blood glucose regulation observed in NOD mice (67,71).

Lymphocyte transfer studies have now shown that the induction of thymic tolerance to the

pancreatic islets does not confer immunologic tolerance to the salivary tissues, suggesting

a potential dichotomy of disease states between these target tissues (101). This is

supported, as well, by immunogenetic studies linking sialoadenitis, hyper-IgG production,

and peri-insulitis to a centromeric loci on Chromosome 1, while insulitis is linked to a

telomeric loci on the same chromosome (81). In addition to anti-pancreatic and anti-

insulin autoantibodies, NOD sera has been shown to contain autoantibodies targeting the

acinar and ductal cells of the submandibular and parotid glands, including those directed

against the muscarinic and P-adrenergic receptors responsible for the generation of saliva

flow (97,98). The decline in saliva output is accompanied by changes in salivary protein

content as well. Both alterations appear to be the result of down-regulation of signal









transduction components of the salivary glands (97). Abnormalities in gene transcription

and protein processing in the salivary glands of immunodeficient NOD-scid mice suggest

that glandular dysfunction in NOD mice may precede lymphocytic infiltration and provide

a potential trigger for the autoimmune targeting of the salivary glands (121).

The NOD mouse represents the first-described animal model for the spontaneous

autoimmune-induced loss of both saliva flow and tear production, and, as such, is

emerging as an excellent model for the study of Sjogren's Syndrome in humans. Sjogren's

Syndrome is an autoimmune disease characterized by dry eye and dry mouth syndromes

due to the destruction of exocrine tissue. Clinical patients develop chronic lymphocytic

infiltration of the salivary and lacrimal glands as well as a cell-mediated and autoantibody

response against the exocrine tissue (11). Diagnosis of Sj6gren's Syndrome often relies

on the detection of lymphocytic infiltration in labial lip biopsies excised from patients,

which have revealed a predominance of CD4' T lymphocytes with an oligoclonality of the

T-cell receptor repertoire (38). In a majority of patients, autoantibodies to ribonuclear

protein antigens are also present (20). In addition, increases in IFNy, TNFa, IL-2, IL-6,

and IL-10 cytokine production have been described in biopsy tissues (11). However, since

patient samples are from a late stage, the initial events surrounding the development of

autoimmune infiltration and destruction of tissue remain unknown.

In this study I have characterized the infiltrating lymphocyte repertoire and

cytokine mRNA profile of the lacrimal, parotid, and submandibular glands of NOD mice

during the course ofimmunopathogenesis. Spleen and islet-infiltrating cells were analyzed

as control lymphocyte populations throughout the time course of disease progression.

This has allowed for a qualitative comparison of lymphocyte populations infiltrating the









pancreas, submandibular and lacrimal tissues of NOD mice, as well as provide a detailed

account of the anti-exocrine autoimmune response which can be compared to that seen in

Sjogren's Syndrome.


Materials and Methods



Animals

Female NOD mice (7 wks of age) were purchased from Jackson Laboratories (Bar

Harbor, ME) and maintained throughout the course of the study in the animal facility at

the Health Science Center at the University of Florida (Gainesville, FL). Diabetes was

diagnosed by elevated blood glucose levels using Chemstrip bG reagent strips (Boehringer

Mannheim, Indianapolis, IN). Mice with blood glucose >200 mg/dL were given insulin

injections (lU/mouse/day ip) (67). Evidence of diabetes was first noted at 14 weeks of

age. At 16 weeks, 3 of 5 mice were diabetic; at 18 weeks, 1 of 5, and at 20 weeks, 2 of 5

mice were diabetic.



Antibodies

Monoclonal antibodies used in this study were purchased from PharMingen (San

Diego, CA) and are as follows; CD3e (clone 145-2cll), CD4 (RM4-5), CD8a (53-6.7),

CD45RB/B220 (RA3-6B2), CD45RB (23G2), TCR Vp3 (KJ25), TCR VI6 (RR4-7),

TCR V08.1, 8.2 (MR5-2), TCR V39 (MR10-2), TCR V31l (RR3-15), and TCR Vpl7a

(KJ-23).









Tissue Preparation

Spleen, pancreas, lacrimal, parotid and submandibular glands were removed at

each harvest. A small piece was cut from each tissue, placed in 10% buffered formalin

and submitted to the Diagnostic Referral Laboratory at the University of Florida

(Gainesville, FL) for histologic sectioning and staining. The remainder was processed as a

pool from the 5 mice per group. A small aliquot of each pool was removed for RNA

isolation and all remaining tissue prepared for flow cytometric analyses.

Single cell suspensions of splenic leukocytes were obtained by gently pressing

spleens through wire mesh screens and washing in PBS (68). Red blood cells were lysed

with 0.84% ammonium chloride. After washing, the remaining leukocytes were aliquoted

at 1 x 106/tube and washed with FACS buffer (PBS with 0.1% NaN3 (FISHER Scientific,

Orlando, FL) and 0.5% BSA (SIGMA Chemical Co, St. Louis, MO) prior to antibody

staining.



Tissue Digestion

Except for spleen, all pooled tissues were dissociated by gentle mincing followed

by digestion at 37C in a shaking water bath for 15 minutes in a mixture of 4 mg/ml

collagenase Type V (SIGMA) + 100 U/ml DNase Type II (SIGMA). Digested tissue was

further dissociated with vigorous pipetting, removed (after allowing large, undigested

pieces to settle) and placed in ice-cold HBSS with 2% FBS (GIBCO/BRL, Grand Island,

NY). Digestion was continued using a mixture of 2 mg/ml collagenase + 100 U/ml DNase

in 5 min incubations at 37C in a shaking water bath and repeated until completion.





34


















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Islets were picked manually from digested pancreatic tissue under a dissecting

microscope and collected in ice-cold IMDM (GIBCO/BRL) with 1% NMS (68). Islets

were collected by centrifugation, supernatant discarded and the cells dispersed by

continuous pipetting in 10 ml of a solution containing trypsin/EDTA (GIBCO/BRL) +

2000 U DNase (SIGMA) at 37"C for 10 min. Digestion was stopped by adding ice-cold

IMDM with 1% NMS.

Digested lacrimal, parotid and submandibular tissue and dispersed islets were

washed and then separated by centrifugation through a 55% Percoll (SIGMA) gradient.

The infiltrating cells were collected in the pellet and contaminating red blood cells lysed

with 0.84% ammonium chloride. After washing, cells were divided into aliquots

containing approximately 1 x 106 cells and washed in FACS buffer prior to antibody

staining.



Flow Cvtometry

Aliquots of all cell populations were resuspended in 100 il FACS buffer and

stained with antibody at 1 .tg/106 cells. Cells were stained first with anti-CD3 for 40 min

at 4C, washed with FACS buffer and then stained with the appropriate second antibody in

a 40-min incubation at 4C. After a final wash, cells were suspended in FACS buffer for

analysis.

Flow cytometric analyses were performed using a FACScan flow cytometer

(Becton Dickinson, Mountain View, CA) equipped with a 15 milliwatt, 488nm air-cooled

argon-ion laser and using LYSYSTM II software (68). Ten thousand events were collected









per sample from a population gated on a window encompassing the splenic lymphocyte

population.



RNA Isolation and RT-PCR Detection of Cvtokine mRNA

Pooled tissues were minced in PBS, placed in lysis buffer and mRNA isolated

using a Micro-FastTrack" Kit (Invitrogen). Isolated mRNA was stored at -70C in

ethanol until all samples were collected. mRNA was pelleted by centrifugation and cDNA

prepared by reverse transcription using Superscript IIT Reverse Transcriptase

(GIBCO/BRL). cDNA was quantified using a DNA DipstickT Kit (Invitrogen, San

Diego CA). Equal quantities of cDNA from each sample (50 ng per reaction) were

amplified by PCR for 40 cycles at 60C annealing (1 min) and 72'C elongation (2 min)

using cytokine primer pairs shown in Table 1. PCR products were separated on 1.2%

agarose gels and transferred to positively charged nylon membranes (Boehringer

Mannheim) by Southern blotting (122).



Detection of Cvtokine mRNA

Specific PCR products were identified using the Genius" system of

nonradioactive DNA labeling and detection (Boehringer Mannheim) according to the

manufacturer's protocols. Briefly, internal oligonucleotide probes specific for each

cytokine (Table 1) were labeled by random primed incorporation of digoxigenin-labeled

deoxyuridine-triphosphate. After overnight hybridization at 65"C, the PCR products were

detected colorimetrically using an anti-digoxigenin alkaline phosphatase conjugate in an

enzyme-linked immunoassay. Concanavalin A (SIGMA)-stimulated mouse splenocytes









were used as a positive control for the primers and probes and G3PDH was used as a

positive control for the isolation of nRNA (123). All nucleotide primers and probes were

synthesized in the Interdisciplinary Center for Biotechnology Research DNA Synthesis

Core Laboratory at the University of Florida (Gainesville, FL).



Densitometric Analysis

Semi-quantitative analyses of cytokine PCR products was done using densitometry

and One-Dscan software. Sample calculations were standardized using G3PDH values to

ensure that equal amounts of nRNA were present in each sample thus allowing for semi-

quantitative comparison between sample bands (G3PDH values for 8 wk lacrimal and 10

wk submandibular samples are reported as raw data since they did not appear to be

consistent with experimental samples). Lanes which did not exceed background were

given a value of zero and subsequent sample values were determined using auto-

background calculations.



Results



Tissue Histology

Lacrimal, submandibular, parotid and pancreatic tissues were surgically removed

from groups of 5 NOD mice at 2 wk intervals from 8 through 20 weeks of age. A small

piece of each tissue was stained with hematoxylin/eosin(H&E) and examined for leukocyte

infiltration. As expected, leukocytic infiltration was observed in the pancreatic islets of 8

wk old mice and increased in severity over time. By 18 wks, few islets remained due to

















































Figure 1 Histological profile of tissues showing lymphocytic infiltrates of the exocrine
tissues and insulitis in the NOD mouse. Tissue sections were stained with
hematoxylin/eosin. (A) 10 wk pancreas; (B) 12 wk submandibular gland; (C) 12 wk
lacrimal gland; (D) 16 wk parotid gland.





















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Figure 3 Histogram of the temporal expression of CD4' and CD8' T-cells in lymphocytic
infiltrates of submandibular (SMG), lacrimal (LAC), parotid (PAR) glands and spleen
(SPL) from NOD mice. Data was obtained from flow cytometric analyses of infiltrating
cells using FITC-conjugated CD3 and PE-conjugated CD4 or CD8. All values are
expressed as a percent of CD3' cells.


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autoimmune destruction. At 8 wks of age the submandibular glands of 2 of the 5 mice

showed small focal areas of infiltration while the lacrimal and parotid glands remained

normal. By 12 wks, the lacrimal glands showed infiltration, but their glands had fewer and

smaller lymphocytic foci than the submandibular glands which were heavily infiltrated in

all mice by 14 wks. In contrast, the parotid glands showed no focal lymphocytic

infiltration up to 20 wks of age, although a few mononuclear cells were occasionally seen

in some mice from 14 to 20 wks of age. Representative histological profiles of glandular

infiltrates are shown in Fig 1.



Flow cytometric analysis of glandular infiltrating lymphocytes

Monoclonal antibodies to cell surface molecules have been widely used as

phenotypic markers corresponding to functionally distinct subsets of lymphocyte

populations. We have used flow cytometric analysis to determine the cellular phenotypes

of lymphocytes infiltrating the pancreas, lacrimal, parotid and submandibular glands of the

NOD mouse from the first appearance of lymphocytes at 8 wks through 20 wks, which is

4 wk beyond the detection of exocrine gland dysfunction. Gates set on the NOD spleen

lymphocyte populations were used to select the infiltrating populations of the other tissue

samples.

The percentage of the cell populations falling in this gated window remained fairly

constant throughout the time course of the study averaging approximately 73% for spleen,

47% for islets, 59% for submandibular gland, 24% for lacrimal gland and 17% for parotid

gland. Within these gated populations the percentage of CD3' cells also remained

relatively constant over the time course of the study at 48% for spleens, 41% for islets,









37% for lacrimal glands and 39% for parotid glands. However, in the submandibular

gland, there was an increase in CD3* cells from 41% at 8 wks to 70% at 20 wks.

Double labeling of the infiltrating populations with CD3/CD4 or CD3/CD8 is

shown in Fig 2 for the 12 wk time point. Similar profiles were obtained for each time

period to determine the percent of CD4 or CD8 cells (Fig 3). The CD4* population in the

submandibular gland approximated that in the spleen: 68% and 62% respectively. In the

lacrimal gland, the CD4* population was about half that of the spleen at 8 wks (-30%) and

increased over time to the level of the spleen. The parotid gland showed a decrease from

84% CD4' cells at 8 wks to -50% at 20 wks. However, the low total number of

lymphocytes detected in the parotid glands as well as their similarity to splenic profiles

may be indicative of contamination by lymphatic vessels which are interspersed throughout

the gland. There was greater variability in the infiltrating CD8' populations of the

exocrine glands than seen in CD4' cells (Fig 3). Generally, fewer than 10% of the CD3*

population were CD8' in these glands at 8 wks -- considerably lower than the 27% found

in the spleen. As the disease progressed, the percentage of CD8* cells increased in all 3

glands, but it is unclear if the apparent decrease at 20 wks is significant.

As indicated by the spleen population, the ratio of peripheral CD4' to CD8* cells

remained relatively constant at ~2.5:1. In the submandibular gland, the CD4:CD8 ratio

was 6:1 at 8 wks, and decreased to an average of 3.5:1 from 12 wks on. Both the lacrimal

and parotid glands also had higher ratios in early infiltrates, but decreased to spleen levels

at 12 wks.

The CD4* and CD8' populations did not account for all of the CD3' cells. The

CD3', CD4CD8 population accounted for 5% of the spleen cells, 8% of the




























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Figure 4 Histogram of selective TCR Vp3 distribution in infiltrating lymphocytes isolated
from spleen, pancreatic islet, submandibular, and lacrimal glands of NOD mice. Data from
flow cytometric analyses of 8 through 20 wk samples were averaged for each tissue.
Spleen-solid bar; Pancreatic islet-horizontal striped bar, Submandibular gland-clear bar;
and Lacrimal gland-diagonal striped bar. Data are presented as percentage of CD3' cells
+ SE of the mean.









submandibular cells, 15% of the lacrimal cells and 9% of the parotid cells. This indicates

an increase in the double negative population in the exocrine tissues over that of the

periphery.

The infiltrating populations of the submandibular and lacrimal glands were further

characterized using antibodies against the B-cell marker B220 (63) and the

activation/memory marker CD45RB (123). As shown in Table 2, the CD3-B220' B-cell

population in the submandibular gland increased from -1% at 8 wks to -15% at 10 wks

and maintained that level throughout the rest of the study. In the lacrimal gland, CD3

B220* cells also started at 1% at 8 wks and increased to -33% at 12 weeks. Whether the

fluctuation seen between 10 and 14 weeks is meaningful is unknown at this time. In

addition, the presence of a CD3'B220* cell population was detected in both of these

tissues. This phenotype did not appear in significant numbers until 16 wks of age in either

gland and never exceeded 10% in the submandibular gland or 5% in the lacrimal gland.

The CD3'B220' phenotype has been reported as a double negative population in MRL/lpr

mice (63) and as a lymphokine activated killer phenotype (LAK cell) in other studies

(124).

CD45RB staining presented a much more complex picture (Table 3). In

submandibular glands at 8 wks, -3% of the CD3' cells were CD45RB"' (naive T-cell)

and from 10 wk on, this CD45RB'" population remained relatively stable at -16%. The

percentage of CD3'CD45RB"+ (memory T-cells) population was 15% at 8 wks, showed a

1.8-fold increase to 27% at 12 wks and remained at that level through 20 wks. The CD3

CD45RB"' population in the submandibular gland remained constant at -25% from 10 -

20 wks. In the lacrimal gland, the picture was less clear due to a smaller sample size









(Table 3). In general, the CD45RBhil population (both CD3' and CD3) increased to a

maximum of -55% at 12 wks, plateaued and then decreased at 18 and 20 wks to -24%.

The CD3'CD45RB'* population remained fairly constant at ~20% from 10 wks on.

Analysis of TCR VB usage was done using the monoclonal antibodies listed in the

Materials section. It has been previously reported that the majority of the infiltrating

populations of the submandibular and lacrimal glands are TCRoap (64). In the VPs tested,

there was no appreciable time-related variation in response, therefore, the data from all

time points were averaged for VB distribution (Fig 4). The submandibular and lacrimal

glands showed a similar distribution pattern to that seen in the spleen. Both Vp6 and V38

were significantly increased over background as represented by VB3, while, to a lesser

extent, V39 and Vp17 were also increased. These increases were much more dramatic in

the submandibular gland than in the lacrimal gland which paralleled the response in the

periphery as represented by the spleen.



Cvtokine mRNA expression in salivary and lacrimal tissues

Temporal expression of mRNA transcripts for selected cytokines expressed within

the infiltrates of lacrimal and submandibular tissues from each experimental age group was

determined through the use of semi-quantitative RT-PCR. PCR bands were quantified

using densitometry and compared against PCR bands of G3PDH to provide a

measurement of temporal changes in the production of mRNA transcripts and provide a

detailed analysis of cytokine expression throughout the progression of the autoimmune

activity. As presented in Fig 5, mRNA transcripts for a number of interleukins were

detected generally at both an earlier age and with greater intensity in the lacrimal glands











SUBMANDIBULAR


IL-IP
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IL


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IL-10










Figure 5 Interleukin mRNA expression of lacrimal and submandibular glands as
determined by RT-PCR and Southern blotting. Blots were scanned and analyzed by
densitometric comparison using G3PDH as a standard. Values were plotted by arbitrary
density units and analyzed using a best-fit polynomial or exponential trendline.


LACRIMAL


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SUBMANDIBULAR






















AGE IWM


Figure 6 Proinflammatory mRNA expression of lacrimal and submandibular glands as
determined by RT-PCR and Southern blotting. Blots were scanned and analyzed by
densitometric comparison using G3PDH as a standard. Values were plotted by arbitrary
density units and analyzed using a best-fit polynomial or exponential trendline.









than submandibular glands. In the lacrimal tissue, IL-Il, IL-5, IL-6, IL-7, and IL-10

mRNA transcripts were detected as early as the 8 wk as well as at most later time points.

In the submandibular gland infiltrates, detectable interleukin mRNA transcripts were

usually not observed before 14 wk of age. Of note, mRNA transcripts for IL-4 were

absent from lacrimal glands, while IL-4 and IL-5 were absent from the submandibular

glands. Both interleukin transcripts were detected in control mRNA obtained from ConA

stimulated NOD splenocytes, while IL-4 is commonly seen in islet infiltrates (122). mRNA

transcripts for three effector cytokines, IFNy, TNFa and iNOS were detected in both

lacrimal and submandibular gland infiltrates of mice aged 8 and 12 wks, respectively (Fig

6). All three cytokines exhibited increased levels of mRNA transcription through 16-18

wks. Cytokine mRNA transcripts were rarely detected in control parotid tissues.

Considered in lolo, these results indicate that major temporal changes in the cytokine

profiles occur in the lacrimal and submandibular glands of NOD mice between 12 and 16

wks of age. In the case of the submandibular glands, this is several weeks following the

first appearance oflymphocytic infiltration.



Discussion



Overt diabetes in our NOD mouse colony generally begins at 12 wks of age, while

the first appearance of focal lymphocytic lesions in the submandibular and lacrimal glands

begins at 8-10 wks and 10-12 wks of age, respectively. Despite the early appearance of

lymphocytic infiltrates within the submandibular and lacrimal glands, loss of saliva flow

and tear production does not occur until 14-16 wks of age (71). For this reason, the









argument has been advanced that the autoimmune attack against the salivary and lacrimal

tissues is merely secondary to the loss of immunological tolerance for a pancreatic 0 cell

antigen also expressed on the salivary and lacrimal glands. A second argument suggests

that the decreased saliva and tear flow is a consequence of the loss of blood glucose

regulation.

Several recent studies have raised serious questions with both arguments. First,

Leiter et al. (101) have reported that development of immunological tolerance against the

P cell following intrathymic injection of islet cell homogenates into neonatal NOD mice

prevented the development of diabetes, but not the autoimmune attack against the salivary

glands. Second, Garchon et al (81) have linked sialoadenitis, hyper-IgG production and

peri-insulitis to a telometric locus, but insulitis and diabetes to a centromeric locus on

chromosome 1. Third, I have observed (unpublished data) that loss of secretary function

in the salivary glands proceeds the onset of changes in blood glucose levels and diabetes in

NOR/chr 9 mice, a strain which has delayed onset diabetes (85). These studies indicate

diabetes and salivary/lacrimal gland complications in NOD mice are most likely distinct

entities.

In the present study, I have attempted to define the temporal development of the

autoimmune attack against the salivary and lacrimal glands of NOD mice. A unique

feature of this pathogenesis, and comparable to Sjogren's Syndrome in humans, is the loss

of secretary function resulting in clinical presentations of xerostomia and xerophthalmia

(71,11). As such, I believe these data will allow for a comparison of the autoimmune

processes, e.g., lymphocyte phenotypes, activation states and cytokine profiles, within









several organs of the same host as well as a comparison between this animal model and

Sjogren's Syndrome.

Analyses of the lymphocytic infiltrates of the salivary and lacrimal glands

uncovered a relatively high percentage of CD4' T-cells, as might be expected from

previous studies on the islet infiltrating lymphocyte populations (68). In contrast, only

10% of the T-cells detected were CD8', and even fewer were CD3'/CD4"CD8" double-

negative T-cells. While generally accepted that CD4* T-lymphocytes are necessary for the

immunopathogenesis of diabetes in the NOD mouse, the role of the CD8' population,

particularly in the initiation of the disease, has remained more elusive (92). T-cell transfer

studies in NOD mice have confirmed the necessity of CD4* T-cell populations to transfer

autoimmunity, whereas neither purified CD8' T nor B-cells were capable (85).

It is becoming increasingly apparent that the activation state of transferred

lymphocyte populations is critical. Using the CD45RB as a marker of lymphocyte

activation, 1 observed that the majority of T-cells present in the infiltrates of both

submandibular and lacrimal glands were either of a memory (CD45RB') phenotype or a

phenotype (CD45RBh") suggesting transition to an activated stage. The presence of a

distinct CD45RB" population throughout the time course of this study suggests that a

population of naive T-cells are being actively recruited to the salivary and lacrimal glands.

Interestingly, a recent study has shown that the ability to transfer diabetes in NOD mice

is associated with a CD45RB'" memory T-cell population and a concomitant increase in

the 1FNy to IL-4 ratio (87). T-cells that produce L-4 have consistently failed to transfer

disease. Markedly increased levels of IFNy mRNA transcripts were observed in both the









submandibular and lacrimal glands, possibly indicating a switch in the cytokine production

by activated CD45RBI T-cells.

In addition to exploring the lymphocytic activation states, I have also investigated

selected Vo TCR phenotypes of the infiltrating cells. While most VD TCR populations

were detected in the salivary and lacrimal gland infiltrates (as expected in the NOD

mouse), an increased percentage of Vp6 and Vp8 expression was present, consistent with

previous observations of preferential TCR usage observed immunohistochemically (89).

The importance of V38 and Vp6 lymphocytes in the development of autoimmunity in both

NOD and MRLlpr mice has been demonstrated through the use of both T-cell transfer

and specific anti-Vp therapy (64,65,125).

Differences in the numbers of B lymphocytes infiltrating the salivary and lacrimal

glands were also observed: approximately 15% in the submandibular gland, but 33% in

the lacrimal tissue. Whether the increased percentage of B lymphocytes within the lacrimal

glands is due to increased B-cell proliferation or active recruitment remains unknown.

However, detection of CD23 (blast-2 antigen) in the lacrimal infiltrates of Sjogren's

Syndrome patients support the possibility of lymphoproliferation (34).

In this study, I have also documented several age-dependent increases in cytokine

mRNA expression in the salivary and lacrimal glands. Of particular interest is the

increased expression of IFNy, TNFa, and iNOS in both salivary and lacrimal tissues

starting at 12-14 wks of age and increasing to maximally detected levels by 20 wks of age.

By 16 wks, high levels of L-10 mRNA production was detected as well. The dramatic

rise of each of these transcripts coincides with the time of the first evidence of the loss of

saliva flow as reported in previous studies (67). Whether these cytokines are involved in









effecting loss of exocrine function directly via a cytotoxic pathway or their presence is

necessary to initiate lymphocyte-mediated cell killing is not yet known.

Many recent studies have focused on the potential roles of cytokines as cytotoxic

effectors of exocrine tissue destruction. Evidence indicates that IL-1, IFNy, TNFt and

nitric oxide may be key mediators in the pathogenic process of islet cell destruction in both

humans and NOD mice (126-129). Of particular interest, production of high levels of

IFNy by islet infiltrating cells has been repeatedly demonstrated in NOD mice and appears

tightly linked to P cell destruction (127,130). Recent studies by Mathis et al.

demonstrated that an islet-specific Thl T-cell clone producing high levels of IFNy is able

to rapidly transfer diabetes to young NOD recipients (88). However, when shifted in vitro

to an IL-4 producing phenotype, the same T-cell clone was unable to transfer diabetes.

Furthermore, IFNy has been noted to induce cell death of both islet and salivary gland

cells in vitro, and may represent a non-specific mediator of exocrine cell destruction in

vivo (45). However, a caveat to this argument is the detection of high levels of IFNy and

TNFa in the salivary glands of MRL/lpr mice which do not lose secretary function

(66,54).

Unexpectedly, cytokine mRNA expression in the lacrimal glands typically appeared

at an earlier age and in larger quantities than in the submandibular gland. This occurred

despite the fact that both fewer and smaller focal lymphocytic lesions were present in the

lacrimal glands in the earlier age groups, thus suggesting that lymphocytes infiltrating the

lacrimal tissue are activated at an earlier stage than those in the submandibular gland.

Alternatively, a lag time between the detection of focal lymphocytic lesions in the

submandibular gland (8-10 wks) and the detection of increased cytokine mRNA








expression (12-16 wks) may indicate that the submandibular gland-infiltrates remain

functionally quiescent and require a signal of activation.

In conclusion, this data demonstrate numerous similarities in both lymphocyte

phenotypes (CD4/CD8 ratios, V3 TCR restriction, and B220 populations) and cytokine

expression (increased IL-Il, IL-2, IL-10, TNFa and IFNy) between the NOD mouse and

other animal models for autoimmune sialoadenitis as well as Sjogren's Syndrome.

Although the initiating agent for Sjogren's Syndrome remains unknown, it is often

believed that extrinsic factors, i.e., viral agents, may be responsible for the breakdown of

tolerance in immunologically susceptible individuals. In NOD mice, however, the

necessary intrinsic elements for the breakdown of salivary gland tolerance exist in their

genetic background. Using the NOD-scid mice, I have recently described multiple salivary

gland abnormalities of NOD-scid mice that do not appear to be immunologically related

(121). These alterations are detectable starting at 8-10 wks of age and include

morphological abnormalities, aberrant gene expression, and increased proteolytic activity.

While any number of immunological changes can result in aggregation of lymphocytes in

exocrine tissues, such as the Ipr/ gld mutation of MRL mice, graft vs. host models (131),

TGF-P knockouts (61), and bcl-2 overexpression (18), only with the appearance of

developmental defects or extrinsic destruction of the exocrine tissue leads to the

development of secretary dysfunction through loss of immune regulation.














CHAPTER 3
GENETICALLY PROGRAMMED DEVELOPMENT OF SALIVARY GLAND
ABNORMALITIES IN THE NOD (NON-OBESE DIABETIC)-SCID MOUSE IN THE
ABSENCE OF DETECTABLE LYMPHOCYTIC INFILTRATION: A POTENTIAL
TRIGGER FOR SIALOADENITIS OF NOD MICE


Introduction



The recent development of the NOD-scid congenic strain provides a unique model

to investigate the role of the immune response in the pathogenesis of Sjogren's Syndrome

(82). The NOD-scid mouse is homozygous at the scid (severe combined

immunodeficiency) locus and thus lacks functional T and B lymphocytes. The scid

mutation prevents the spontaneous development of sialoadenitis, insulitis, and diabetes in

these mice; however, the transfer ofT lymphocytes from diabetic NOD mice to recipient

NOD-scid mice can restore an autoimmune phenotype (85). In addition, because of its

NOD background, the NOD-scid has impaired NK (natural killer) cell and reduced

complement activity (82). Since NOD-scid mice share the same NOD genetic

background, this model is ideal for studying the non-immune genetic factors that

contribute to the development of autoimmunity. Previously, the dramatic changes in

exocrine gland histology, protein synthesis, and secretary dysfunction are thought to be a

direct result of the autoimmune lymphocytic component (67). By investigating exocrine

gland function in the absence of functional lymphocytes, this specific aim demonstrates

that temporal changes in salivary gland function occur in the absence of overt









autoimmunity. This indicates that the resulting immune response may actually be

triggered by aberrant physiological changes in exocrine gland homeostasis and function.



Materials and Methods



Animals

BALB/cJ, C3H, CBA, and NOD/Uf mice were bred and maintained in the

Department of Pathology's mouse facility (University of Florida, Gainesville, FL).

NOD-scid mice were bred and housed in the Department of Pathology's transgenic mouse

breeding colony. Both male and female mice ranging in age from 3 to 30 weeks were

used. Maintenance of the scid mutation was assessed in experimental animals by flow

cytometry of spleen cells and RT-PCR analysis of CD4, CD8, and TCR VP repertoire

from isolated submandibular gland total RNA. NOD mice were routinely tested for blood

glucose levels using Chemstrip bG reagent strips (Boehringer Mannheim, Indianapolis,

IN). Consecutive elevated fasting blood glucose levels >240 mg/dl were considered onset

of diabetes, after which time, the mice were maintained on daily insulin injections.



Saliva Collection and Flow Rate

Saliva was collected from control and experimental groups of male and female

mice following stimulation of secretion using isoproterenol (0.20 mg/100 g body weight)

and pilocarpine (0.05 mg/100 g body weight) (Sigma Chemicals, St. Louis, MO) dissolved

in saline. The secretogogue cocktail was injected (0.1 ml volume) intraperitoneally and

saliva was collected, starting 1 min post-injection, for 10 min from the oral cavity by









micropipette and placed into chilled 1.5 ml microcentrifuge tubes (67). Volume was

determined by measurement with 200 pl micropipettes. Saliva samples were collected

from groups of 7 male or 5 female mice then frozen at -700C until analyzed for temporal

protein changes by enzyme assay, radio-receptor assay, SDS polyacrylamide gels and

Western blotting



Total Salivary Protein and a-Amvlase Analysis

Saliva samples were analyzed for total protein using bovine serum albumin as the

standard (132). Amylase was determined by its ability to hydrolyze starch according to

published protocols (133). In brief, 500-1000 fold dilutions of saliva in

phosphate-buffered saline (PBS) were added to a solution containing 0.4 g soluble starch

in 60 mM tris(hydroxymethyl)aminomethane (Tris)-HCI, 0.15 M NaCI and 3 mM CaCI2.

The reaction was stopped after 5 or 10 min by the addition of 0.045% 12, 0.045% KI, and

0.03 N HC1. Absorbance was measured at wavelength 620 nm. One unit of amylase was

defined as the amount that hydrolyzed 1 mg starch/min/mg protein at 370C.



EGF Analysis

Salivary epidermal growth factor (EGF) was estimated by the procedure of Booth

et al. (134). Saliva was diluted 10 fold in PBS containing 0.2 mg/ml bovine serum

albumin (BSA). Reactions consisting of 100 pl diluted saliva, 100 pl human placental

microvilli membranes and 100 pl 1251-labeled human EGF were incubated for 24 hr at

4C (67). Each reaction was then diluted with 3.5 ml ice-cold 0.1% BSA in PBS solution,

centrifuged for 20 min at 7,000 g in an RC-3B Sorvall centrifuge and the quantity of








radiolabel associated with the membrane pellet determined using a Beckman gamma

counter. The concentration of EGF was compared to a standard curve generated with

dilutions of known quantities of human recombinant EGF. Membrane binding competition

is independent of species origin for the source of EGF (135).



Isolation of Salivary Gland Tissues.

Parotid and submandibular glands were excised from mice killed by cervical

dislocation. Each gland was freed of connective tissue, fat, and any lymph nodes, then

homogenized in 10 mM Tris buffer (Ph 7.4) containing 100 pM phenylmethylsulfonyl

fluoride, 1 liM leupeptin, and 100 lM benzamidine. The slurry was then centrifuged at

100,000 g for 30 min to recover total membrane (67). The supernatant was saved and

frozen until analysis for a-amylase activity.



Polvacrylamide gel electrophoresis and Western blot analysis

Total saliva proteins (5 pg of total protein or 5 pl of total saliva volume) were

subjected to electrophoretic separation on a 1.5 mm thick 10% SDS-polyacrylamide gel

(12% SDS for Western blots) using a modified Tris-Glycine system of Pugsley and

Schnaitman (136). Western blots of gland lysates contained 20 lg protein per lane. Gels

were fixed and stained using Coomassie Brilliant Blue R-250 or transferred to

Immobilon-P membranes (Millipore, Boston, MA) for 2 hr at 70v for Western blotting

(137,138). The blocking buffer consisted of 3% nonfat dry milk and 3% BSA in

Tris-buffered saline. Polyclonal rabbit anti-mouse parotid secretary protein (mPSP) IgG

antibody (139), kindly provided by Dr. William Ball (Dept. of Anatomy, Howard









University) or polyclonal rabbit anti-rat proline rich protein (140), kindly provided by Dr.

David Castle (Dept. of Anatomy and Cell Biology, University of Virginia), was incubated

with each membrane for 2 hr at 250C. Following three 10 min washes, the membranes

were incubated in alkaline phosphatase conjugated goat anti-rabbit immunoglobulin

(Sigma Chemical Co.) and exposed to substrate as previously described (95).



Protein Sequencing

Salivary proteins were subjected to electrophoresis on 10% SDS-polyacrylamide

gels as described above and transferred to Immobilon-P membranes. Selected protein

bands were carefully cut from the membranes and subjected to N-terminal sequencing

using Applied Biosystems Model 470A Gas Phase Protein Sequencer with Model 120A

on-line PTH analyzer (University of Florida ICBR Protein Sequencing Core Laboratory).

Protein sequences for the first 12 amino acids of each protein were entered into a protein

database for comparison with known protein sequences.



Histology

Freshly excised submandibular glands were placed immediately into 10% PBS-

buffered formaldehyde (pH 7.2). Each tissue was embedded in paraffin, sectioned, then

stained with hematoxylin/eosin dyes (UF Diagnostics Referral Laboratory, Gainesville,

FL). The stained sections were viewed using light microscopy.









Statistical Analyses

All measures of variance are given as standard deviations of the mean. Tests of

significance for differences between independent means were performed with the unpaired

Student I-test. Results in which p < 0.05 were considered significant.



Results



Analysis of NOD-scid Saliva

To determine the impact on salivary function of the scid mutation in the NOD

mouse, I analyzed whole saliva samples from individual NOD-scid mice for total volume,

total protein, amylase activity and EGF concentration. Saliva from 10-12 wk old

NOD-scid mice, an age at which salivary glands in NOD mice are devoid of detectable

lymphocytic infiltration, was compared to that of >20 wk old NOD-scid mice, an age

when lymphocytic infiltration is present in the salivary glands of NOD mice (Table 4).

While there was no significant age or sex differences observed for salivary flow rates

(P>0.05), significant sex-related differences were observed for protein content (P<0.05).

Saliva from female NOD-scid mice contained approximately 40% less total protein than

that of male mice in both experimental groups.

Saliva from NOD-scid mice were analyzed for two proteins, EGF (a product of

submandibular gland ductal cells) and amylase (a product of parotid gland acinar cells),

normally secreted in high quantities. As presented in Table 4, the quantity of EGF in the

saliva from >20 wk old male NOD-scid mice showed a 21% decline compared to the

amount present in 10-12 wk old male mice (P<0.002). A comparison of amylase activity

















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Figure 7 Cytoplasmic amylase activity of parotid and submandibular glands.
Homogenates of parotid and submandibular glands were centrifuged at 100,000 x g.
Lysates prepared from the glands of 10-12 wk old NOD-scid (open bars), >20 wk old
NOD-scid (hatched bars), and 20 wk old BALB/cJ mice (solid bars) were tested for
amylase activity in a starch hydrolysis assay. Values are expressed as means of 4
experimental animals performed in triplicate : the standard deviations.



















96

68

AMY

45



32

24


STD 6 wks 10 wks 15 wks 20 wks


Figure 8 Temporal changes in the protein profiles of saliva from NOD-scid mice. Saliva
was collected from 6 wk old NOD-scid (Lanes 1-3), 10 wk old NOD-scid (Lanes 4 and
5), 15 wk old NOD-scid (Lanes 6 and 7), and 20 wk old NOD-scid mice (Lanes 8 and 9)
following isoproterenol/pilocarpine stimulation. Salivary protein (5 p-g protein in 35 pl
SDS-PAGE sample buffer containing p-mercaptoethanol) was loaded on each lane and
separated by electrophoresis through a 10% polyacrylamide gel. The gel was stained with
Coomassie Brilliant Blue R-250. Prestained molecular weight markers (STD) were: 96
kDa, Phosphorylase B; 68 kDa, bovine serum albumin; 45 kDa, ovalbumin; 32 kDa,
carbonic anhydrase; 24 kDa, soybean trypsin inhibitor.



















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in the saliva of old versus young male and female NOD-scid mice (Table 4) also revealed

significant decreases over time. Again, no sex-related differences in amylase activity were

observed, with both male and female NOD-scid mice showing approximately 50%

declines in activity between the two age groups (P<0.0002).



Analysis of Ctoplasmic Amylase

The temporal decline in amylase activity present in saliva of NOD-scid mice was

also reflected in the amylase activity present in parotid and submandibular gland lysates

(Fig 7). Cytoplasmic fractions from parotid gland lysates showed a 50% reduction in

amylase activity in the >20 wk old NOD-scid mice when compared to those of 10-12 wk

NOD-scid mice and a 90% reduction when compared to levels obtained from parotid

gland lysates of BALB/c mice. As expected, submandibular gland lysates exhibited low

levels of amylase activity in all experimental groups.



Temporal Changes in NOD-scid Salivary Proteins by SDS-PAGE

To evaluate temporal changes of salivary proteins in NOD-scid mice, we

compared electrophoretic separations in SDS-polyacrylamide gels of three individual

saliva samples collected from 6 wk old female NOD-scid mice, plus two samples collected

from each of 10 wk, 15 wk, and 20 wk old female animals (Fig 8). Although the total

protein concentration in each saliva sample is constant, the gel profiles revealed substantial

differences in protein composition between animals of the various age groups. These

protein profiles were similar whether constant protein (5 gg) or constant volumes (5 il)

were applied to the SDS-PAGE. The temporal decline (then precipitous disappearance









from the saliva samples of 20 wk old NOD-scid mice) of the protein observed at

approximately 55 kDa, shown to be amylase (105), is consistent with reported changes

observed for salivary amylase in NOD mice when analyzed on SDS-PAGE gels.

Two additional protein bands, one at 32 kDa and one at 20 kDa (indicated by

arrows in Fig 8), present in relatively high amounts in saliva of NOD-scid mice of 10 wks

of age or less, virtually disappear from saliva of NOD-scid mice 15 wks of age and older.

Interestingly, the decline of these two proteins appeared to correlate with the emergence

of a third protein band at approximately 27 kDa (indicated by an asterisk in Fig 8). By 20

wks of age, this 27 kDa protein band appears to be one of the most abundant proteins in

the salivary samples.



Sequence Homology of the 20 kDa. 27 kDa and 32 kDa Proteins with Parotid Secretory

Protein

N-terminal amino acid sequence analyses of the 20 kDa, 27 kDa and 32 kDa

protein bands indicated that all three were homologous to parotid secretary protein (PSP)

(Fig 9). Interestingly, the N-terminal amino acid residues of both the 32 kDa and 20 kDa

proteins, the two isoforms observed prior to the time NOD mice present with

sialoadenitis, were not only identical to each other, but were also identical to the published

murine PSP sequence beginning at the signal cleavage site of the secretary form (108;141-

142). In contrast, the N-terminal sequence of the 27 kDa protein, appearing at a time

NOD mice begin exhibiting sialoadenitis, matched an internal portion of the PSP starting

at the 27th amino acid after the signal cleavage site. The secretary form of PSP has been








reported to be a 20 kDa, leucine-rich glycoprotein that is abundant in the secretary fluids

of the parotid gland (105).



Antigenic Cross-reactivity of Salivary PSP from NOD-scid. NOD and BALB/c Mice

To confirm the identity of the 20 kDa, 27 kDa and 32 kDa protein bands as PSP,

Western blots of SDS-PAGE separated saliva from 10 wk old NOD-scid, 20 wk old

NOD-scid, diabetic NOD and normal BALB/c mice were treated with anti-PSP polyclonal

antibody. As shown in Fig 10, anti-PSP antibody detected each of the three isoforms.

Saliva from BALB/c and 10 wk old NOD-scid mice contained predominately the 20 and

32 kDa isoforms, while saliva from diabetic NOD contained the 27 and 20 kDa isoforms

and lacked the 32 kDa isoform. Saliva from 20 wk old NOD-scid mice contained each of

the isoforms, but had greatly reduced levels of both the 20 and 32 kDa isoforms compared

to the BALB/c and 10 wk NOD-scid mice.



PSP Detected in the Cytoplasmic Fractions of Salivary Glands of NOD-scid Mice

Cytoplasmic lysates of the submandibular and parotid glands were analyzed on

Western blots for the presence of PSP using anti-PSP antibody (Fig 11). Lysates of

submandibular glands from 10 wk old NOD-scid mice revealed the presence of all three

PSP isoforms, and each isoform increased in quantity by 20 wks of age (Lane 2 versus

Lane 3). No PSP was present in the lysates of BALB/c submandibular glands (Lane 1).

Lysates of parotid glands from 10 wk old NOD-scid mice contained the 32 kDa

protein band plus a comparatively low level of the 20 kDa protein (lane 5). In contrast, by

26 wks of age, parotid gland lysates from NOD-scid mice exhibited all three isoforms














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Figure 10 Identification of parotid secretary protein in saliva using polyclonal anti-PSP
antibody on Western blots. Protein (5 gg/lane) in whole saliva from 20 wk old diabetic
NOD (Lane 1), 20 wk old BALB/c (Lane 2), 10 wk old NOD-scid (Lane 3, and 20 wk old
NOD-scid mice (Lane 4) was separated using SDS-PAGE, electroblotted to Immobilon-P
membranes, and reacted with rabbit anti-mouse PSP antibody. The Western blots were
then developed using alkaline phosphatase-conjugated anti-rabbit IgG plus substrate. The
32 kDa and 20 kDa bands are indicated with arrows and the 27 kDa protein with a star.
Prestained molecular weight markers are 45 kDa, ovalbumin; 32 kDa, carbonic anhydrase;
and 24 kDa, soybean trypsin inhibitor (Lane S).
















Submandibular Parotid



s o o o o

z Z E Z
120
96

68



45
32 O Q




24
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Figure 11 Representative Western blot analysis of cytoplasmic fractions of submandibular
and parotid cell lysates using anti-PSP polyclonal antibody. The cytoplasmic proteins (20
gg/lane) of either submandibular gland lysates prepared from 26 wk old BALB/c (Lane 1),
10 wk old NOD-scid (Lane 2), and 26 wk old NOD-scid mice (Lane 3) or parotid gland
lysates prepared from 26 wk old BALB/c (Lane 4), 10 wk old NOD-scid (Lane 5), and 26
wk old NOD-scid mice (Lane 6) were separated on a 12% polyacrylamide gel and
transferred to Immobilon P membranes. Membranes were treated with alkaline
phosphatase-conjugated rabbit anti-mouse PSP and substrate. The 32 kDa and 20 kDa
protein bands are indicated with arrows, while the 27 kDa protein band is shown with a
star. Prestained molecular weight markers are the same as for Fig 9.









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Figure 12 Western blot analysis of saliva and cytoplasmic fractions of parotid and
submandibular cell lysates using anti-Proline-Rich Protein polyclonal antibody. Panel A,
10% SDS-PAGE of constant saliva protein of 5 pg from mouse strains C3H, BALB/c,
CBA, NOD, and NOD-scid. P-DM, prediabetic NOD; DM, diabetic NOD. Panel B,
represents the antibody staining of PRP in parotid (PAR) and submandibular (SM) gland
lysates from BALB/c, P-DM and DM NOD, and NOD-scid mice. Each lane contained 20
pg of protein from total cell lysate.

















































Figure 13 Morphological changes in the salivary glands of NOD-scid mice.
Hematoxylin/eosin-stained tissue sections of submandibular glands from 10 wk old NOD-
scid (A: 100X, B: 200X), 30 wk old NOD-scid (C: 100X, D: 200X) and 20 wk old NOD
mice (E: 100X, F: 200X).








(lane 6). Again, lysates ofBALB/c parotid glands contained only the 32 kDa protein (lane

4), comparable with the profile of young NOD-scid mice. Interestingly, the anti-PSP

antibody bound to a protein band of approximately 65 kDa present in the parotid gland

lysates of both BALB/c and 10 wk old NOD-scid mice; however, this protein band has

not been studied. These results show the temporal nature of the appearance of the 27 kDa

isoform of PSP in the salivary glands and the abnormal expression of PSP by

submandibular glands of NOD-scid mice.



Detection of Proline-Rich Protein (PRP) in the Saliva and Salivary Glands of NOD and
NOD-scid mice

The PRPs are a set of proteins whose synthesis are induced in the salivary glands

of mice following chronic p-adrenergic agonist treatment or by the introduction of

deleterious dietary changes (104). Saliva from NOD-scid, diabetic or prediabetic NOD

mice, as well as BALB/c, CBA, and C3H strains were evaluated by Western blot for the

presence of PRP. As indicated in Fig 12A, antibody to rat PRP was able to cross-react

with a protein of approximately 33 kDa in saliva of diabetic NOD, 10 wk and 25 wk

NOD-scid mice. In contrast, very little of this protein was detectable in the saliva from

control BALB/c and prediabetic NOD mice. No reactivity was detected in the saliva of

C3H or CBA mice. N-terminal amino acid sequence analysis confirmed the identity of this

protein as PRP (data not shown). An examination of parotid and submandibular gland

lysates revealed that PRP expression was occurring in both glands of the NOD-scid,

prediabetic, and diabetic NOD mice, but not in control BALB/c animals (Fig 12B).









Histological Changes in the Submandibular Glands of NOD-scid mice

Histological sections of submandibular glands from 15 individual NOD-scid mice

ranging in age from 3 to 30 wks were evaluated for structural changes. Figure 13 shows

hematoxylin/eosin stained sections of submandibular glands from 10 wk (Fig 13A & 13B)

and 30 wk (Fig 13C & 13D) old female NOD-scid mice. A comparison of these two time

points reveal a marked, progressive loss of acinar tissue and a decline in the acinar to

ductal cell ratio. The histology of the parotid gland was also examined. However, there

were no observable changes in the cellular or glandular morphology between 10 wk and

30 wk NOD-scid mice (data not shown) as was seen in the submandibular gland. As

expected, the NOD-scid tissue displayed no signs of immune cell infiltration. In contrast,

submandibular gland tissue from diabetic NOD mice, which appear to have only slight loss

ofacinar tissue, is highly infiltrated with mononuclear cells (Fig 13E & 13F).



Discussion



The clinical symptom of xerostomia, or oral dryness, associated with human

Sjogren's Syndrome is commonly attributed to two problems: the loss of the fluid phase

of saliva and changes in salivary protein composition (1). Both problems are considered a

result of a progressive autoimmune response against the salivary glands. This hypothesis

is supported by studies using the NOD mouse model in which changes in both salivary

flow rates and protein composition are associated with the appearance of immune cells in

the salivary glands (67). Unfortunately, these studies in man and the NOD mouse fail to









determine if the loss of salivary function is a direct result of an autoimmune attack or if the

autoimmunity is in response to specific changes in the exocrine glands.

In the present study, I have attempted to dissociate immune and non-immune

factors which may contribute to the loss of salivary gland function by using the NOD-scid

mouse. As expected, histological analyses of salivary glands from NOD-scid mice did not

reveal the presence oflymphocytic infiltrates. Furthermore, total salivary flow and protein

concentration measured in NOD-scid mice appeared similar to the published values of

BALB/c mice and prediabetic NOD mice (67). The composition of saliva proteins, as

shown by EGF and amylase concentration in NOD-scid saliva, however, showed

significant changes with age, whereas the levels detected in BALB/c saliva remained

relatively constant over the same time period (67,71). This was further reflected by

greatly reduced amylase activity (90% reduction) in parotid gland lysates of NOD-scid

mice as compared to BALB/c. In addition, amylase activity detected in NOD-scid parotid

lysates declined nearly 50% between 10 and 25 wks of age. Temporal analyses of

NOD-scid saliva by SDS-PAGE again revealed the age related decline of amylase as well

as several additional proteins. Age-related changes in marine saliva volume and

composition in non-NOD strains does not typically occur until after 12 months of age

(54).

The most striking findings, however, were the discovery of a novel expression of

PRP and an internally cleaved PSP isoform (27 kDa) which was prominent in 15 wk

NOD-scid saliva but was not detected in saliva from BALB/c mice or younger NOD-scid.

The abundant appearance of these proteins may explain why total saliva protein

concentration remained constant despite the decline in other major proteins. Proline-rich








protein is a latent constituent of murine saliva which is induced in response to glandular

trauma (104). In normal mice, PSP is a 20 kDa, leucine-rich glycoprotein of unknown

function that is secreted predominately by amylase producing acinar cells of the parotid

gland (105). Studies have shown a developmental coordination of murine PSP and

salivary amylase expression in the parotid gland of adult mice where the two proteins

appear in constant ratios (107,109). With the onset of diabetes in NOD mice (>14 wks)

and the aging of NOD-scid mice (>15 wks), both the 32 kDa and 20 kDa isoforms of PSP

are replaced with a 27 kDa protein band detected with the anti-PSP antibody. Sequence

analysis of the N-terminal amino acid residues of the 27 kDa isoform revealed that the

protein started at an internal region (+27 aa) of the PSP protein. Interestingly, all three

isoforms of PSP appeared in the lysates of the submandibular glands of older NOD-scid

mice but not of normal BALB/c mice. These findings suggest that, first, the glandular

specificity of PSP gene expression is lost over time in NOD and NOD-scid mice, and

second, the transition to a new isoform of PSP might involve differential splicing and/or

alteration ofpost-translational modifications.

Taken together, these findings suggest that both submandibular and parotid gland

function, as shown by alterations in gland specific PSP and PRP gene regulation and

salivary EGF and amylase concentrations are altered in NOD-scid animals between 10 and

25 wks of age. Therefore, changes in saliva protein content in the NOD-scid mice are

probably not due to the insufficiencies of an individual gland but are multiglandular and

potentially affecting other exocrine gland function. The low level of detection of amylase

activity in parotid gland lysates, ectopic expression of PRP, and the appearance of a novel








isoform of PSP in both the parotid and submandibular glands further suggest that these

changes take place at the intracellular level.

Histological examination of the submandibular gland, but not the parotid gland, of

aging NOD-scid mice revealed a remarkable decline in the acinar to ductal cell ratio. This

may be due to acinar cell loss, the hyperproliferation of ductal cells, or a combination of

both. Acinar cell loss in the submandibular gland may be triggered by the observed

defects in protein synthesis or processing which could potentially lead to cell death. Since

whole salivary flow rates in the older NOD-scid animals do not decline despite this

apparent loss of submandibular acini, it is possible that the parotid, sublingual, and minor

salivary glands in the oral mucosa have increased salivary output in order to compensate

for this loss of acinar cells (143,144). An interesting alternate hypothesis to acinar cell

loss is the hyperproliferation of the submandibular ductal cells, which are known to have

self-renewing capacity. Hyperplasia and metaplasia of the salivary ductal epithelium is a

hallmark feature seen in labial salivary biopsies of human Sjogren's patients (15). It should

be noted too that abnormal proliferation of differentiated tissue is often accompanied by

the loss of differentiated cell protein synthesis and function (145). This hypothesis is

especially attractive in light of studies showing that the ductal cells of the NOD exocrine

pancreas can undergo hyperproliferation (146,147). Therefore, it is altogether possible

that similar developmental abnormalities are occurring in the submandibular gland and

pancreas of NOD mice that may precede the development of sialoadenitis and insulitis

respectively, and be inherently involved in the pathogenesis of these autoimmune lesions.

The de novo production of the 27 kDa PSP isoform in aging NOD-scid mice

suggests that aberrant proteolytic processing may play a role in the generation of









otherwise hidden cryptic antigens, priming the immune system for an autoimmune

response. The time at which this new isoform of PSP appears in the saliva and

submandibular glands of NOD-scid mice coincides with the time of appearance of

lymphocytic infiltrates in the salivary glands of NOD mice. In addition, morphological

changes in the salivary glands of NOD-scid mice involving loss of acinar tissue are

detectable by histology in the absence of lymphocytic infiltrates. These observations

suggest that changes in the salivary glands of NOD mice occur independently of

lymphocytic infiltration and that development of the associated autoimmune activity may

actually occur in response to these changes. This is consistent with the proposed model

for the induction of human Sjogren's Syndrome (1), except that the initial "injury" to the

gland is intrinsic i.e. genetically programmed in the NOD mouse rather than extrinsic i.e. a

consequence of viral infection or other insult.

A newly emerging model of pathogenesis of autoimmune sialoadenitis in the NOD

mouse suggests that the initial trigger may reside in a defect in salivary gland homeostasis,

leading to the production of new or altered proteins. As antigenic forms of cellular and

secretary proteins are released, the immune system responds by initiating the homing of

immune cells to the exocrine tissue. Production of cytotoxic cytokines and direct cell

killing by activated T cells may play a role in furthering glandular damage. However, the

presence of a subclass of autoantibodies recognizing the j-adrenergic and muscarinic-

cholinergic receptors alludes to a potential mechanism for the decline in saliva production

and protein output which involves antibody-mediated impairment of neuro-glandular

stimulation. Thus, the presence of autoantibodies may be responsible for secondary

effects on gland function following a primary immunological disturbance generated by a









genetic defect in the salivary glands. This model is useful in explaining why first, the

presence of activated lymphocytes is necessary to develop the loss of the fluid phase of

saliva in the NOD mouse while NOD-scid salivary flow rates remain normal, and second,

how an autoimmune response against relatively small portions of the total area of the

submandibular gland may dramatically affect the total salivary output of the parotid,

sublingual and submandibular glands combined.

In conclusion, by using the NOD-scid mouse model, I have begun to dissociate

elements of salivary dysfunction in the NOD mouse attributable to the progression of the

autoimmune disease from normal/abnormal events dictated by the genetic background of

the animal. These findings are striking in that they suggest an underlying defect in salivary

gland homeostasis in NOD mice which results in a histopathological and functional

phenotype similar to human Sj6gren's syndrome.














CHAPTER 4
EXPRESSION OF PAROTID SECRETARY PROTEIN (PSP) IN MURINE
LACRIMAL GLANDS AND ITS POSSIBLE FUNCTION AS A BACTERIAL
BINDING PROTEIN IN EXOCRINE SECRETIONS


Introduction



The protein and mucin-rich secretions derived from the salivary, lacrimal, and

other minor exocrine tissues, i.e.labial and hardarian glands, are essential for maintaining

the health and integrity of the oral and ocular surfaces (9). For the most part, both tear and

saliva secretions serve similar functions and contain many of the same protein constituents,

e.g., EGF, NGF, TGF-a, lactoferrin, lysozyme, and immunoglobulins (9,10). At the same

time, however, saliva and/or tear specific secretary proteins, as evidenced by salivary

amylase and digestive enzymes, provide for specialized physiological functions of the

individual secretary fluids (9).

Chapter 3 of this dissertation documented unique changes in the synthesis of

several proteins noted specifically in saliva, including de novo synthesis of PSP as well as

decreased concentrations of amylase and EGF in NOD mice (121). Similarly, protein

expression abnormalities were detected in NOD-scid mice. These findings suggest that

changes in saliva protein constituents in the NOD strain are independent of the

autoimmune destruction of the glands. The aberrant synthesis of PSP in the

submandibular glands of NOD mice led us to examine the question of whether PSP may









be abnormally synthesized in other exocrine tissue of NOD mice, and whether

transcriptional splicing is likely to account for the novel PSP isofonn described in Aim 3.

In this specific aim, I show that while PSP is synthesized in the lacrimal glands of NOD

mice, it is detected in the lacrimal glands in several other laboratory mouse strains as well.

As a constituent in saliva and tears, I provide evidence of a potential anti-microbial

function for PSP which may explain its normal synthesis by both the salivary and lacrimal

glands. In addition, NOD mice were found to have lost the normal regulation of PSP gene

transcription in a number of organs/tissues. No alternatively spliced mRNA was detected,

however, the NOD PSP gene contains numerous base pair changes indicative of strain

specific differences between mice.



Materials and Methods



Animals

BALB/c, CBA/J, and NOD/Uf mice were bred and maintained under SPF

conditions in the mouse colony of the Department of Pathology and Laboratory Medicine

at the University of Florida, Gainesville, FL. C3H/HeJ and NOD-scid mice were

purchased from The Jackson Laboratories (Bar Harbor, ME). Both male and female mice

ranging in age from 8 to 25 weeks were used. NOD mice were routinely tested for blood

glucose levels using Chemstrip bG reagent strips (Boehringer Mannheim, Indianapolis,

IN). Consecutive elevated fasting blood glucose levels >240 mb/dl were considered onset

of diabetes, after which time the mice were maintained on daily insulin injections (67).









NOD mice of 15 weeks of age were separated into diabetic and prediabetic sample groups

for protein studies.



Bacteria

Lysteria monocytogenes and Streplococus mulans were gifts from Dr. F.

Southwick, Department of Infectious Diseases, University of Florida, and Dr. A. Bleiweis,

Department of Oral Biology, University of Florida. Actinobacillus

actinomycelenmcomitans was generously provided by Dr. P. Fives-Taylor, Department of

Microbiology, University of Vermont.



Isolation of Tissues for Protein Studies

Tissues were excised from mice killed by cervical dislocation. Glands were pooled

from a minimum of two age- and sex- matched mice of each strain, freed of connective

tissue, fat, and any lymph nodes, then homogenized in 10 mM Tris buffer (pH 7.4). The

slurry was centrifuged at 500 g for 15 min to pellet cellular debris and protein

concentrations of the resulting supernatant were determined by the method of Bradford

(132) using bovine serum albumin as the standard. Supernatants were frozen at -700 C for

Western blot analysis.



Isolation of total RNA

Excised tissues were immediately homogenized in 2 ml of 4M Guanidinium

Isothiocynate (GITC) as described by Chirgwin et al. (148). Briefly, 100 pl of 10%

Sarkosyl and 14.2 il of 2-Mercaptoethanol were added to homogenates and total RNA








was isolated by centrifugation at 35,000 g over a 2 M CsCI gradient. RNA pellets were

washed in 200 pl of 80/20% ethanol/Diethyl Pyrocarbonate (DEPC) treated water and

then resuspended in 200 1l of DEPC-treated water. RNA was precipitated through the

addition of 500 pl of 100% ethanol and 8 p. of 5M NaCI. Quantitation of total RNA was

determined by spectrophotometric analysis at 260 nm wavelength.



RT-PCR and Southern Blot Detection ofPSP PCR Products

Total tissue RNA (2 pg) was pelleted by centrifugation and reverse transcribed

using Superscript II Reverse Transcriptase (Gibco BRL) (122). The resulting cDNA was

amplified by PCR for 40 cycles using a 94C denaturation (1 min), 600C primer annealing

(1 min) and 72C elongation (2 min) using 5'GCAGAGAAACAAGGATCTCG and 3'

CACTGGAGAGTAGCCAGCAGG PSP-specific primers. These primers spanned a

region preceding the start codon and extending beyond the translation termination site

(149). PCR products were separated on 1.2% agarose gels and transferred to nylon

membrane by Southern blotting. To confirm the identity of specific PCR products,

hybridization of digoxigenin-labeled oligonucleotide internal probe (5'

AATGCGACCGTTCTTGCC) specific for PSP cDNA was carried out using the Genius

system ofBoehringer-Mannheim (Indianapolis, IN) (150). Briefly, oligonucleotide probes

were labeled with digoxigenin using terminal transferase. Southern blot membranes were

baked at 80C for I hour, blocked with pre-hybridization buffer (Genius Kit), and

hybridized with labeled probes overnight at 650C. Colorimetric detection of PSP product

was assayed using an anti-digoxigenin alkaline phosphatase conjugated antibody according

to manufacturer's instructions. Primers and probes specific for G3PDH were used as









positive controls for all PCR reactions. All nucleotide primers and probes were

synthesized in the University of Florida's ICBR DNA Synthesis Laboratory (Gainesville,

FL).



Polvacrylamide eel electrophoresis and Western blot analysis

Gland lysates (30 pg of total protein per lane) were subjected to electrophoretic

separation on a 12% SDS-polyacrylamide gel using a modified Tris-Glycine system of

Pugsley and Schnaitman (136). Gels were stained by using Coomassie Brilliant Blue R-

250 (137) or transferred to Immobilon-P membranes (Millipore, Boston, MA) for 2 hr at

70v for Western blotting (138). The blocking buffer consisted of 3% nonfat dry milk and

3% BSA in Tris-buffered saline. Polyclonal rabbit anti-mouse parotid secretary protein

(mPSP) IgG antibody, kindly provided by Dr. William Ball (Dept. of Anatomy, Howard

University) (139) was incubated with each membrane for 2 hr at 25"C. Following three

10 min washes, the membranes were incubated in alkaline phosphatase conjugated goat

anti-rabbit immunoglobulin (Sigma Chemical Co.) and exposed to substrate as previously

described (95).



Purification and Radiolabeling of PSP

Parotid secretary protein was purified using a one-step procedure by

electrophoretic separation of saliva proteins on a 3 mm 10% polyacrylamide prep gel.

Four hundred Il of whole saliva from C3H/HeJ or BALB/c mice was separated using a

single large well in the stacking gel. Molecular weight standards (Bio-Rad) and 0.5 cm of

the sample well were cut and transferred to PVDF membrane for Western blotting.








Following detection of PSP migration, the similar region on the remaining unfixed gel was

removed, macerated by an electric homogenizer in 3 ml PBS containing 0.02% NaN3 and

0.2% SDS, and placed on an orbital shaker overnight at 4C. The extracted protein was

dialyzed against ddH20 and lyophilized to concentrate the purified protein. Typically, 1.5-

2 .ig of pure PSP was recovered for 400 pl of saliva protein. Purity of PSP was

determined by electrophoresis and staining of polyacrylamide gels with Coomasie Brilliant

Blue R-250, autoradiography and Western blot. Pure PSP, M, 25,000 was radiolabeled

using chloramine-T and Na1251 obtained through Amersham (Arlington Heights, IL).

Radiolabeled PSP was purified from free [1251], potentially other contaminating proteins

and radiolabeled BSA in the incubation buffer by molecular sieve chromatography on

Sephadex G-75 obtained from Pharmacia (Uppsala, Sweden).



PSP Binding to Bacteria

Four strains of bacteria, L. monocytogenes, E. coli, S. mutans, and A.

actinomycetemcomitans were grown in overnight cultures at 370C. The bacteria were

pelleted by centrifugation at 40C for 5 min at 15,000 x g and washed in PBS buffer

containing 0.5% BSA, 0.02% NaN3, 1 mM CaCI2, 1 mM MgC2, and 1 mM ZnCl2. The

bacteria were resuspended in 1.0 ml of the above buffer at 108 cells/ml. [1251]- Labeled

PSP (104 cpm) was added to the cells and incubated at 370C or 230C for 2 hr on an orbital

shaker. The cells were pelleted by centrifugation, washed twice, and the radiolabel bound

to the bacteria quantitated by a Beckman gamma counter. Specificity of PSP binding was

determined by pre-incubation of the cells with 10 pg/ml of unlabeled PSP. Cation-








dependent binding was determined by incubation where one or all of the salts were altered

in the incubation buffer.

Ligand blot assays were conducted as described previously (151). In brief,

bacteria were lysed by sonication (4C in PBS), and the membrane fraction collected

following centrifugation. The membranes were sonicated a second time (in buffer

containing 0.5% SDS), followed by centrifugation and the soluble material heated to 1000

C for 5 min. Sample (the optical density adjusted to A2s0= 2.6 units/ml: 10pil (-0.1 units)

was mixed with SDS containing sample buffer, separated on 10% polyacrylamide gel, and

transferred to PVDF. The membrane was blocked using a modification of the method of

Hossenlopp et al. (151). Briefly, the blot was incubated for 30 min in 10 mM Tris-HCI,

pH 7.4 containing 150 mM NaCI, 0.02% NaN3, and 3% NP-40, 30 min in Tris buffer

containing 1% BSA in place of detergent, and 30 min in Tris buffer containing 0.1%

Tween-20. The membrane was incubated overnight in TBS containing 1% BSA, 0.1%

Tween-20, and 10' cpm ['25I]-PSP. The blot was washed 3 times in TBS and exposed to

X-ray film for 12 hr at -800 C using Kodak XAR-5 film. Specificity of PSP binding to

bacterial proteins was determined by pre-incubation of the filter with unlabeled protein.

Ligand binding blots were run on 3 separate occasions for reproducibility using 2 separate

preparations of ['25I]-PSP from C3H/HeJ and BALB/c saliva.



Amylase Assay

The activity of human salivary amylase (SIGMA Chemical) was determined in the

presence of varying concentrations of pure PSP, proline-rich protein (PRP), and BSA.

Amylase activity was determined by the method of Bernfeld using starch as a standard









substrate (133). Human amylase (lgg/ml) was resuspended in PBS. The incubation

solution was comprised of 0.4 g soluble starch in 60 mM Tris-HCI containing 0.15M NaCI

and 3 mM CaC12 and ZnCI2. The stop solution consisted of 0.45% 12, 0.045% KI and

0.03 N HCI. After termination of the reaction at 5, 10, 15, and 20 min, the enzyme

activity was defined as the amount that hydrolyzed 1 mg starch/min/mg of protein. All

values are expressed as mean + S.E. for 3 separate determinations.



Results



Detection ofPSP RNA Transcripts in Murine Lacrimal Gland

Total RNA derived from lacrimal tissue isolated from NOD, BALB/c and

C3H/HeJ mice was assayed for the presence of PSP RNA transcripts by RT-PCR and

Southern-blotting. As shown in Fig 14A, a strong PCR band was observed at 785 bp in

lacrimal RNA of NOD but not C3H/HeJ or BALB/c mice. When hybridized with a PSP-

specific internal oligonucleotide-probe, both NOD and BALB/c developed a band at the

expected base pair size (Fig 14B) while the C3H/HeJ remained negative. Interestingly, the

lack of PSP expression in C3H/HeJ mice is in concordance with previous reports by

Hjorth et al (105) describing the parotid specificity of PSP gene expression using

C3H/HeJ mice. Detection of the housekeeping gene, G3PDH, was used as a positive

control to indicate that similar amounts of RNA was utilized in each PCR reaction (Fig

14C). G3PDH bands of 983 base pairs were present in all samples.















A
(j;


800 bp
700 bp



B

800 bp
700 bp




C

1,000 bp
900 bp


- PSP






PSP


a n r- G3PDH


Figure 14 RT-PCR and Southern blot detection of PSP mRNA isolated from murine
lacrimal glands. Total tissue RNA (2gg) was reverse-transcribed and resulting cDNA
amplified by PCR using PSP-specific primers spanning the entire of length of the PSP
transcript (785 bp). Panel A; Ethidium bromide-stained agarose gel containing PSP-
amplified PCR product (10 l/lane) from 8 week C3H/HeJ (Lane 1), NOD (Lane 2), and
BALB/c (Lane 3) lacrimal tissues. Panel B; Agarose gel (from Panel A) was transferred
to a nylon membrane by Southern blotting and hybridized with a digoxigenin-labeled
oligonucleotide probe specific for an internal PSP sequence. Blots were developed using
an alkaline phosphatase labeled anti-digoxigenin antibody. Panel C; Positive control of
RT-PCR and Southern Blot procedure using G3PDH-specific primers (983 bp product)
and probe for C3H/HeJ (Lane 1), NOD (Lane 2), and BALB/c (Lane 3) RNA samples.


























I 'b ol
0 0
. 0
Q:~~ Z s


00
zz
Z Z


84.0 -
53.2 0

34.9 h1
28.7 Z. *
kDa "


Figure 15 Western blot detection of parotid secretary protein in murine lacrimal glands.
Total protein in pooled tissue lysates (n=3 mice/sample) from C3H/HeJ parotid gland
(Lane 2; 1 pg/lane), was compared with pooled lacrimal gland lysates (30 jg/lane) from
C3H/HeJ (Lane 3), CBA/J (Lane 4), BALB/c (Lanes 5 and 6), NOD (Lanes 7-10) and
NOD-scid (Lane 11) mouse strains. Age (in weeks) and sex of sample groups are labeled
above, with 15 week NOD mice being diabetic. Briefly, proteins were separated on 12%
SDS-PAGE gels under reducing conditions, transferred to Immobilon P membranes, and
incubated with rabbit anti-PSP antibody. Blots were developed with alkaline phosphatase-
conjugated goat anti-rabbit antibody and substrate as per methods section. Prestained
molecular weight markers are as follows; Bovine serum albumin, 84.0 kDa; Ovalbumin,
53.2 kDa; Carbonic Anhydrase, 34.9 kDa; and Soybean trypsin inhibitor, 28.7 kDa.




























1
C',
116-
84-

53-


35-
28-
kDa


S12
116-
84-

53-

35-
28-
kDa


Figure 16 Purification of Parotid Secretory Protein. Four hundred I. of total saliva was
separated in a prep gel well (Panel A). Parotid secretary protein was identified by
Western blot using antibody to PSP (Panel B) and cut from the gel. The purified protein
was dialyzed, radiolabeled, and reanalyzed for purity by autoradiography and Western blot
reactivity (Panel C lanes 2 and 3, respectively). Molecular weight standards (Bio-Rad) are
Phosphorylase B, 116 kDa; Bovine Serum Albumin, 84 kDa; Ovalbumin, 53 kDa;
Carbonic Anhydrase, 35 kDa; and Soybean Trypsin Inhibitor, 28 kDa.


1
116 l
84 -db

53 -i

35-4hq
28
kDa




Full Text

PAGE 1

ANTl-EXOCRfNE AUTOIMMUNITY IN THE NOD MOUSE MODEL OF S JO GREN S S YN DROME By CHRISTOPHER P ROBINSON A DISSERTATION PRESENTED TO THE GRADUATE SCI-IOOL OF THE UNIVERSITY OF FLORIDA IN P ARTlAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHJLOSOPHY UNIVERSITY OF FLORIDA 1997

PAGE 2

ACKNOWLEDGEMENTS No usual tl1anks are suffici e nt to ack11owledge 111y debt a11d sincere appreciation to both of my co-1nentors, Dr Micl1ael G Humpl1reys-Beher ar1d Dr Am111on B. Peck Tl1eir i11sigl1t has i11flue11ced both my personal a11d profe ss ional progression tl1rough tl1is graduate program 111 addition, I would like to thank Dr Linda Brinkley, Dr Micl1ael Clare-Salzler, and Dr Joel Schitfenbauer for tl1eir advice and contributions toward this proJect I am deeply grateful to Janet Corn e lius, Micah Kerr Jeff At1derson, Elizabeth Bowen, Jason Brayer and Kim Nguye11 for pro v iding tech11icaJ as s istance on thi s project Special thanks to all of my friends in the Peck, Humphreys-Beher, Clare-Salzler, and Hillman l a boratories who l1ave made these past four years a genuine pleasure. I would like express n1y s incere gratitude to my parents, Paul and Wesley R o bi11son, without who s e love encoura g ement and support I would not be the person that I am today Lastly, I wi s h t o thank my wife, Meryl, for l1er love, understa n ding, and confidence in me . 11 I I

PAGE 3

TABLE OF CONTENTS ACKNOWL E DGEMENTS ............. ...... ... ...... .... ............. .... .......... LIST OF TABLES .... ... .. ... . ... ... ........ .. .... ...................... ... ...... ... LIST OF FIGUR E S .................................... ......................... ........ ABSTMCT ................................................................. .. ................ .... .. CHAPTERS 1 INTRODUCTION . .. .... ... ................. . ...... .. ... ..... .... 2 CHARACTERIZATION OF THE CHANGING L YMPHO CYTE POPULATIONS AND CYTO.KlNE EXPRESSION 3 IN THE EXO C RINE TI SS UES OF AUTO N OD MI. CE .............. ................................................ Introduction .... . ..... ........... .... ....... .................... Materials a nd Methods .. ..... ................................ Results ..... ...... ... . .. ................... .. .... . ....... ... . D . 1 s cus s 1on ....... .. .. ....... .. .. .................. .. . ....... GENETICALLY PROGRAMMED DEVELOPMENT OF SALIVARY GLAND ABNORMALITIES IN THE NOD (NON OBESE DIABETIC)-S C /D MOUSE IN THE ABSEN CE OF DETECT ABLE L YMPHOCYTIC INFIL TRATION : A POTENTIAL TRIGGER FOR SIALOADENITIS OF NOD MICE .. .. ....................... ............. Introduction ....................................................... Materials and Methods .............. .. .. .... .. . ............. Results . . ... ...... .. ........ .. ...... .. ................ ..... D . ISC USSlOfl ............... .. ..... .. ............ ... .. ..... .... . . . lll Page .. u V Vl lX 1 30 30 32 37 50 56 56 57 61 74

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4 5 6 EXPRESS ION OF PAROTlD SECRETORY PROTEIN fN MURINE LACRlMAL GLANDS AND ITS POSSIBLE FUNCTION AS A BACTERIAL BIND I NG PROTEfN ........... l11troduction ... . .. . .. .... ... .......... ... ....... .. .. . .... .. . Materials and Methods .... ... .............. .... ............. .. . Results ............. .. .... ........ . ... .. .... . ... ... .... ..... .... D . 1 scu ss Ion .. ... ............ ... .............. .. . ..... ... . ..... EV ALUATlON OF NOVEL PROTEOLYTIC ACTIVITY DETECTED IN THE SALIVA OF AGING NOD MICE .......... l11troduction ... .... ........ . .. .............. .. ... ............ . Materials and Meth o ds . .. ... ..... .. .... .... ... . .. . . ..... . Re s ults .. . ..... .. ...... ..... ... .... ... ....... .. .... ...... .. ...... D . 1 s cuss1on ... ...... ......... .. ... . .......... ..... .. ............ CONCLUSION AND FUTURE DIRECTION . ................. . . 80 80 81 87 I O I 105 105 1 07 116 124 1 29 REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136 BIOGRAPHICAL SKETCH .. . .... .... ...... . ... .............. . . ... ................ 150 IV I

PAGE 5

LIST OF TABLES Table l Murine Prin1er ai1d Probe Sequences .. . .. .......................................... 2 Percentage of CD3 and 8220 in the Lyn1phocytic Infiltrates of Submandibular (SMG) and Lacrimal (LAC) Glands of NOD Page 34 Mice . ............ .... ....... ....... .... ... ........... ... .......... . ....... ..... 43 3 CD45RB Expression in Lyn1pl1ocytic l11filtrating Cells of Submandibular (SMG) and Lacrimal (LAC) Glands of NOD Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 4 Analysis of NODsc id Saliva ......... .... .. ....................... ........ ....... 62 5 Cysteine Protease Activity in Saliva ... . . . . . . . . . . . . . . . . . . . . . . . 115 V

PAGE 6

LIST OF FIGURES Figure Page 1 Histological Profile of Tissues Showing Lympl1ocytic Infiltrate s of tl1e Exocrine Tissues and Insulitis in the NOD Mouse .. . . . . . . . . . . . . . 3 8 2 Flow Cytometric Analysis of CD4 + and CDS + T Cell Populations in Lymphocytic I11filtrates in Ti s sues From 12 wk NOD Mice .... .. ........ 39 3 Hi s togram of the Temporal Expre s sion of CD4 + and CDS + T cells in Lympl1ocytic Infiltrates of Submandibular (SMG) Lacrimal (LAC), Parotid Glands (PAR) and Spleen (SPL) from NOD Mice ... . . . . . . . . 40 4 Histogram of Selective TCR VP Distribution in Infiltrating Lympl1ocytes Isolated From Spleen Pa11creatic Islet, Submandibular and Lacrimal Glands of NOD Mice .... .... .......... ... ..... ... .......... . ... ......... 45 5 Interleukin mRNA E x pre ss ion ofLa c rimal ru1d Submandibular Glands a s Determjned By RT-PCR and Soutl1em Blotting ... .. . . . . . . . . . . . . . 48 6 Proinflan1matory mRNA Expre s sion ofLacrimal and Sub111andibular Glands as Determined by RT-PCR and Southern Blotting .. . . .............. 49 7 Cytoplasmic Amylase Activity of Parotid and Submandibular Glands ... . . . . . 63 8 Temporal Changes in the Protein Profiles of Saliva Frotn N OD-s id Mice ......................... ...... ........ ..... .... ... . .... .. ................. 64 9 N-Terminal Amino Acid Residue Sequences of the 32 kDa 27 k.Da, and 20 kDa Protein Bands (Shown on Fig 8) and Tl1eir Alignment With the Published Sequence of Murine Parotid Secretory Protein (PSP) ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 65 10 Identification of Parotid Secretory Protein in Saliva Using Polyclonal Anti-PSP Antibody on Western Blot ... . . . . . . . . . . . . . . . . . . . . . . 69 11 Western Blot Analysis of Cytoplasmic Fractions of Submandibular and Parotid Cell Lysates Using Anti-PSP Polyclonal Antibody ... . . . . . . . . . 70 ; V1 I

PAGE 7

12 We s tern Blot Analysis of Saliva and Cytoplasn1ic Fractions of Parotid and Submandibular Cell Lysates Using Anti-Proline-Rich Protein Polyclonal Antibody ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 13 Morpholo g ical Changes in tl1e Salivary Glands of NOD-s c id Mice ................. 72 14 RT-PCR and Southern Blot Detection of PSP nlRNA Isolated From Murine Lacrimal Glands .............. ..................... ........... ...... ....... 88 15 Western Blot Detection of Parotid Secretory Protein in Murine Lacrimal Glands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 16 Purificatio11 of Parotid Secretory Protein . . . . . . . . . . . . . . . . . . . . . . . 90 17 Amylase Enzyn1e Activity in the Presence of Saliva Proteins . . . . . . . . . . . . 91 18 Bacterial Binding of PSP ................................................................. 93 19 Autoradiograph of PSP Binding to Bacterial Membrane Proteins . . . . . . . . . 94 20 RT-PCR and Southern Blot Detection of PSP Transcripts in NOD and C3H/HeJ Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 21 Western Blot Detection of Parotid Secretory Protein in 8 wk NOD and C3 H/HeJ Tissue Lysates . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 7 22 Sequen c e of Parotid Secretory Pro t ein cDNA Derived From the Parotid Gland mRNA From the NOD Mouse Strain . . . . . . . . . . . . . . . . . . . 98 23 Autoradiogram of Differential PSP Migration Following Incubation With Saliva or Salivary Gland Ly sates . . . . . . . . . . . . . . . . . . . . . . 111 24 Western Blot of Differential PSP Migratio11 Following Incubation of NOD and BALB / c Saliva ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 25 Western Blot Depicting EDTA Inhibition of the Proteolytic Cleavage ofPSP .......... .... ................................... ..... ........... ... ......... 113 26 Western Blot Depicting Proteolytic Cleavage of PSP in the Presence ofEGTA ..... ... ....................................................... ............. 114 27 Histogram of Cysteine Protease Activity in Salivary Gland Lysates ...... . . . . . 119 28 Western Blot Analysis of Saliva for the Presence of Apoptotic Proteases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 Vll

PAGE 8

29 Western Blot Analysis of Saliva for tl1e Presence of tl1e Cysteine Protease Inhibitor~ Cystatin ...... .................. .. ...... ....... . ...... .... .. . 121 30 Zyn1ogram Gel Showing Distinct MMP Activities in NOD ar1d BALB/c Saliva .. ..... ... ........... .............. .............. .... ... ... .. .. .............. 122 ... Vlll

PAGE 9

Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulftllment of the Requiren1ents for the Degree of Doctor of Plulosophy CHARACTERIZATION OF ANTI-EXOCRINE AUTOIMMUNITY [N THE NOD MOUSE MODEL OF SJOGREN'S SYNDROME Chairman : Dr Ammon B Peck By Christopl1er P Robinson May 1997 Cochairman : Dr Michael G Humpl1reys-Bel1er Major Department : Pathology and Laboratory M e dicine The primary goal of this dis s ertatio11 is to elucidate key features of tl1e pathogenesis of anti-e x ocrine autoimn1unity i11 the NOD (non-obese diabetic) mouse In addition to autoiminune islet cell destruction, the NOD mouse develops chronic lymphocytic infiltration of the salivary and lacrin1al tis s ues leading to dramatic declines in exocrine gland secretion. The finding that secretory dysfunction in the NOD mouse correlates to the presence of leukocyte infiltration of tl1e exocrine tissues prompted the study of tlus strain as a novel n1odel of secor1d a1 y Sjogren's Syndrome To further develop this model, the studies p resented in thi s di ss ertation inve s tigate the anti-exocrine inftltrating cell populations, pl 1 y s iological and biocl1emical alterations in NOD exocrine tissues, and the contribution of the NOD ge11etic background to the development of antiIX

PAGE 10

exocrine at1toin11nunity. By flow cytometric ar1alysis, the infiltrati11g lymphocyte populations are sl1own to be quantitatively and qualitatively similar to tl1at observed i11 exocrine biopsies from Sjogren's patients and other anirnal n1odels of this disease Si1nilarly, RT-PCR detection of cytokine gene expression demo11strates similar cytoki11e profiles in tl1e NOD exocrine tissues as that see11 in human patients and other disease models The investigation of the NOD-scid strain shows tl1at morphological and biochen1ical alterations of the salivary glands of NOD mice occur in the absence of ly1nphocytic infiltration, su gges ting that intrinsic alterations of the salivary glands may underlie autoimmune invasion. Furthern1ore aberrant gene expression and proteolytic processing of the s alivary protei~ PSP, were detected in botl1 NOD and NOD-scid animals This protein, previously reported as salivary gland specific, is further found to be expressed in tl1e lacrimal glands of mice The novel cleavage of PSP is the result of a proteolytic activity uniquely present in the saliva of aging NOD and NOD-scid animals Both cystei11e protease and matrix metalloprotei11ase activities were detected in older NOD and NOD-scid samples, potentially indicating increased apoptotic activity and glandular restructuring, respectively. Novel proteolytic activity ma y explain the loss of submandibular acini in NODsc id mice, as well as potential candidate e11zymes for PSP cleavage. As such, the findings presented in this dissertation lay the foundation for future genetic and molecular studies investigating the underlying pathogenesis of autoimn 1 une Sicca synd r ome X

PAGE 11

CHAPTER 1 INTRODUCTION Sjogren's Syndrome is a progressive, debilitating disorder in which the body s in1mune system destroys the n1ucinous secreting exocrine tissues resulting in the hallmark features of dry mouth and dry eyes ( sicca syndro111e) ( 1 ) While classified as an orphaned autoim111une disease, the estin1ated 11umber of cases of Sjogren's Syndrome in the United States is believed to range between 200,000 to 4 million (2) This wide range i11 estimated case nun1bers is attributed to the difficulty in properly di a gnosing the see1ningly ubiquitous n ature of many of tl1e patient compla i nts. 111 Europe, where the criteria for diagnosis is less stringent thai1 i11 the United States, the prevalence is estimated at 0 1 to 1 Oo/o of the population. As typical of n1any autoimmune diseases, over 90/4 of Sjogren s Syr1drome patients are won1en, most of which are po s t-menopausal (2) Common complaints include 'trouble chewing or parched moutl\' ' 'bumi11g throat," ''grit or glass in the eyes," 'blurred vision," ''itching skin, or ''debilitating fatigue''(3) Indeed, Sjogren s has been termed the 'great mimicker'' since many symptoms, often considered minor or vague, resemble those seen in other disease states such as hepatitis C infection, autonomic neuropathy, or drug treatment (I). Since symptoms are often reported to medical specialists such as dentists or optometrists Sjogren' s Syndron1e is often misdiagnosed and symptoms treated as individual entities instead of systemic disorders These complications in diagnosis have led to the consensus described by the National Organization for Rare l

PAGE 12

2 Disorders (NORD) president Abbey Meyers tl1at ' Sjogre11's Sy11dro111e isn't a rare disease ; it' s just ma ss ively under-di ag 11osed' '. The etiology of Sjogren' s is unknown, a11d there are no known cures It was n o t until 1933 how ev er, tl1at su fficient interest led to tl1e landmark studies presented to the medical con1munity by Swedish physician, Henrik Sjogre11 ( 4) Henrik Sjogren Individual case studies of dry mouth or dry eye patients were presented as early as the late 1800's (5) By 1927 Gougerot made the connection between sa livary and lacrimal si cca syn1ptoms, and the linkage of these symptoms to artliritic disease was first presented (6, 7) Tying these findi11gs together Henrik Sjogren studied a patient group predominately con s i st ing of women over 40 years of age and displaying keratoconjuncti v itis sicca ( 4) Sjogren noted the appearance of lymphocytic infiltrates in both the sa livary and Jacrimal ti ss ues, as well as salivary gland swelling. Over 50/4 of the patients had a history of arthritis For the next 20 y e ars, little a dvancement was made beyond these descriptive observations In 1951, after publishing numerous supplemental studies, Sjogren concluded that the major criteria for diagnosis were keratoconjunctivitis sicca, xerostomia, and polyarthritis (8). While tl1e etiology of disease remained unknown, Sjogren's microscopic and descriptive findings laid the foundation for the diagnostic criteria which is still in use today

PAGE 13

3 Clinical Presentation and Co111plications of Sjogre11 's Syr1drome The protein and mucin-rich secretions derived fron1 the salivary, lacrimal, a11d otl1er rninor exocri11e tissues, i e labial and hardarian glands are essential for mai11taining tl1e health and integrity of tl1e oral and ocular surfaces These secretions provide not only the fluid and electrolytes necessary for tissue l1omeostasis, but also contain several additional clas s es of protein constituents (9) These con s tituents include important anti microbial defense 1necl1anisms against pathogens such as immunoglobulins, iron chelators, and proteases~ growth factors important for mucosa} tissue n1aintenance and regeneration, such as epidermal growtl1 factor (EGF), nerve growtl1 factor (NGF), and transfo11ning growth factors (TGF-a and TGF-P)~ and mucinous lubricating agents For the most part, both tear and saliva secretions serve similar functions and contain n1any of the same protein constituents, e g ., EGF, NGF, TGF-a, lactoferrin lysozyme, and immunoglobuli11s (9, I 0). At the same tin1e, however, saliva and/or tear specific secretory proteins, as evidenced by salivary an1ylase and digestive enzymes, provide for specialized physiological functions of the individual secretory fluids (9). The clinical presentation of sicca syndrome is associated with the loss of both the fluid and proteinacious phases of saliva (1). This is demonstrated by the fact that supplemental tear and saliva substitutes lacking the protein constituents of naturally occurring exocrine fluids are not complete remedies for patient discomfort associated with dry11ess ( I I) Similarly, complaints of chronic dryness are often displayed in a portion of autoimmune diabetic patients with nom1al flow rates (12, 13). This indicates that the

PAGE 14

4 protective protein and mucous constituents in addition to fluid saliva are essential for tl1e n1aintenance o f oral and ocular health Disruption of exocrine secretion l1as severe clinical implications In addition to ,. patient discomfort, corneal damage due to lack of lubricating fluids a119 chronic ocular ~-r" inf ections can lead to blindness in severely affected patients ( 1 4:-J Similarly, lo ss of protective saliva and hydration provided by mucinou ~ ( eatings leads to rampant ,,., 4' periodontal disease, caries, candidal infection, and efaclcing and loss of teeth desp it e fi' ,.~ rigorous de11tal treat1nent regimens (15). Dryp l ss and severe cracking of the tongue in ,,., ,;, many patients leads to difficulties in spea ki g, chewing and swallowing most foods As in { p n1any autoimmune diseases, Sjogren' patients often develop a waxing and waning in the ,,/' severity of sy1nptoms, leading tQ l ~ eriods of moderate health followed by chronic episodes I ( 16) To con1bat dryness, ttificial sa liva, tears and ointmer1ts can provide symptomatic drugs ( 16). Numerous systemic complications frequently appear in Sj ogre n 's patients Tl1e most severe of these is a marked increase in tl1e risk ( >4 0 fold) of developing non Hodgkin's lympl1omas in the salivary glands or cervical lytnph nodes (17) In 1991, Fox et al. demonstrated that salivary gland lymphomas predominately involve a t(l4 : 18) tran s location which allows for increased synthesis of the proto -oncoge ne bc/-2 (17). Over-expression of1 bcl-2 is kt1own to rescue activated lympl1ocytes from apoptotic death \_.) (18). Other symptoms include clrronic fatigue, itchy skin attri buted to both dryness and vasculitis, and digestive disorders Many patients appear to have central nervous system

PAGE 15

5 disorders, which, in addition to chronic fatigue further adds to the systemic nature of tl1is auloimn1une disease ( 19) Modern Diagnosis of Sjogren's Syndrome New advances in our imn1unologic and molecular understandings have led to an expansion of the criteria determining the diagnosis of Sjogren's Syndrome. Today, Sjogren's syndrome is subdivided into prin1ary and seco11dary classifications. Primary Sjogren's syndrome is defined as autoi1nmu11e sicca syndrome in the absence of other autoimmune conditions (1 ). Secondary Sjogren's syndrome occurs in tl1e presence of other autoimmune connective tissue diseases sucl1 as rheumatoid artlrritis systemic lupus erythen1atosis, or sclerodern1a The pre se nce of dry eyes is 111easured tlirough use of tl1e Schir111er' s test of tear productio n, and Rose Bengal corneal-staining dye, once used by Henrik Sjogren is still used to visualize corneal da111age Salivary gland scintigraphy is used to evaluate saliva flow rates In 1960, Bloch et al detected the presence of anti nuclear antibodies in the sera of approximately 70% of Sjogren's patients (20) This finding was further expanded to identify two specific ribonucleoprotein antigens named SS-A/Ro (60 and 52 kDa isoforms) and SS-B/La (47 kDa) (21) Antibodies directed against SS-A and SS-B are present in approximately 903/o and 70%, respectively, of sera of Sjogren s sy ndrome patients (22) Rheumatoid factor is also present in 70% of patients, including tl1ose that do not display joi~t involvement (23) Therefore, presence of these autoantibodies has been u s ed as a critical criteria for diagnostic purposes

PAGE 16

6 In the United States, the San Francisco criteria is used to diagnose candidate patie11ts displaying sicca syndrome ( 1 ). Under this criteria a positive diagnosis includes the detectio11 of lymphocytic i11filtration in labial lip biopsies Unfortunately, different standards for diagnosis in the international comn1unity have led to great controversy in regards to tl1e patient populations used in clinical and basic research studies (24,25) In the European (EEC) criteri~ for example, examination of labial lip biopsies for presence of lyn1phocytic infiltration is not essential for diagnosis Tl1us, only an estimated l 5o/o of EEC patients would be diagnosed with Sjogren s Syndron1e under the San Francisco criteria (25) Since the patient populations are more restrictive in the San Franci sc o cr it eria, comparison of international studies remains controversial Genetic Fact or s in Su s ceptibility While the initiating environmental triggers of Sjogren s syndrome are unclear intrinsic genes contribu t ing to disease susceptibility are thought to be critical potentiators in the developtnent of autoin1mune di s ease Approximately 12% of SS Sjogren s syndrome patients have a relative with th.is disease demonstrating a familial aggregation (26) The strongest disease associations appear related to specific alleles of the human leukocyte antigen (HLA} complex or the corresponding major histocompatibility complex (MHC) in murine models (27) These highly polymorphic gene products serve to bind and present peptide epitope~ to lympl1ocytes. A specific allele has unique p e ptide binding specificities which are dependent upon the polymorphic amino acid composition in the binding/anchoring clefts of the molecule. The subsequent presentation of peptides serves

PAGE 17

7 as a four1dation for the developme11t of both tolerar1ce to self tissue antigens and the ability to bi11d and present novel exogenous peptides to tl1e im111une syst e m (28) Therefore, it is tl1eorized that individuals with specific HLA alleles may be predisposed to selected tissue specific autoimmunity based on the capacity of tissue autoantigens to bind in the HLA binding clefts In support of this theory is the finding tl1at different autoimmune diseases often correlate to the presence of specific HLA alleles In Caucasians with primary Sjogren's syndrome, an increased frequency of HLA B8 is found in -60% of Sjogren's patients and only 20% in controls (29) Additio11ally, 83o/o of primary Sjogren's syndrome patients displayed HLA-DW3 as compared with 24% in co11trol populations (30). Racial distinction in patient populations is also detected, as Japanese patie11ts display higher frequencies of HLA-DRW52 (31 ). While these findings were generally true for primary Sjogren 's patients, secondary Sjogren's syndro 1 ne associated with rheun1atoid artluitis did not display an association with either HLA-DW3 or HLA-B8, indicating genetic distinction between patient groups (29,30) Similarly, Sjogren s syndrome associated with systemic lupus e rythen1atosis appears related to DR3, DQw2, and C4AQO (complement ge11e mapped to the HLA region) (31) Specific HLA haplotypes are further associated with differences in the levels of anti-Ro antibody production or high autoantibody levels in general HLA-DW3, for instance, is associated with decreased salivary flow rates and larger focal infiltration among patients (32) Tl1erefore, the presence of particular HLA alleles may not only co11tribute to disease ris~ but also to specific characteristics of an individual patient's disease The importance of non-filA related genes appears to be important as well. A review of familial studies by Goldstein et al. suggested the presence of a dominant, non

PAGE 18

8 HLA li11ked ge11e (33) In addition, discordance of Sjogren's Syndron1e an1011g identical twins suggests tl1at an enviro11mental trigger(s) plays a critical role as well Tl1e complex polyge11ic nature of this disease may explain the wid e ly varying con1plaints a11d disease severity among patients afflicted with Sjogre11's syndrome. Infiltrating Lymphocytes One advantage to the clinical study of Sjogren's Syndron1e as compared to n1any other autoin1mur1e diseases is the routir1e accessibility of tissue biopsies Focal infiltrates of the salivary glands are predominately CD4 + T lymphocytes, with a sn1aller percentage of CDS + T cells than detected in peripheral tissues ( 11 ). Helper T-cells are predominately of a memory pl1enotype. Althougl1 polyclonal activation of B-lymphocytes is a hallmark feature of tl1is disease, only 10-1 So/o of salivary leukocytic infiltrates are comprised of lymphocytes lnte r esti11gly, lacrimal gland infiltra te s contain nearly double ( ~ 300/4) the numbers of B-cells as seen in the salivary glands (34) Whether the increased percentage of B lymphocytes within the lacrimal glands is due to increased B-cell proliferation or active recruitment remains unknown. However, detection of CD23 (blast-2 antigen) in the lacrin1al inftltrates of Sjogren's Syndrome patients support the possibility of ly1nphoproliferation. Diversity of the T-cell receptor (TCR) repertoire is achieved through the random arrangements of the genes encoding the a and P chains. While numerous TCR a chains are available to generate unique VDJ rearrangements, Jess than thirty P chains are available to compliment the heterodimeric formation of the TCR. Therefore, it was

PAGE 19

9 qt1estioned whether the generation of autoimmune activity against selected tissue antige11s i11volved tl1e preferential usage of specific variable P (VP) chains which l1ad a biased affinity for tissue antigen epitopes (35) This idea is supported by the expansion of domi11a11t VP a11d Va T-cells specific for defi11ed experimental antigens, such as myelin ba s ic protein (36) In tl1eory, if a bia se d TCR VP gene predominated a given autoimn1u11e disease, specific TCR depletion therapies could be used to slow disease progression without co1npl e tely immuno-compronusing the patie11t ln primary Sj og ren's patients a prefere11tial usage of several TCR VP genes l1as been demonstrated in salivary gland biopsies (35,37,38) How ev er, a similar study of lacrimal tis s ues showed a highly diverse TCR repertoire, providing a potential indication of the involvement of multiple tis s ue anti gen s Utlfortunately the study of T-cell phenotypes in human patients is hampered by the fact tl1at tissue samples are from the late stages of a utoimmune progression The r e f ore, the identification of the lympl1ocyte s ubsets criti ca l for th e initiation of disease necessitates the use of a nimal n1odels. Cytokines in Sjogren s Syndrome The immunoregulatory effects of cytok.i11es have stimulated great interest among researchers Of note, several studies u s ing immunohistochemistry, i11 sit1, RT-PC~ or RT-PCR methods have shown that production of inflammatory cytokines is not restricted to infiltrating immunological cells Indeed, the exocrine tissues appear to produce IL-1 and IL-6 which may contribute to inflammation (39,40) Constitutive production of transforming growth factor beta (TGFf3), a strong immunosuppressive factor, is also

PAGE 20

l (> found in tl1e s e tissues ( 41 ) Furthermore productio11 of lFNy by infiltrating immune cells l1as been sl1 o wn to i11duce tl1e expression of HLA-DR on the surface of salivary gla11d epithelium ( 42) Cytokine production is also important in upregulating expre ss ion of lCAM-1 and E-selectin on the surface of salivary endothelial cells in Sj o gren's patients ( 43) Together these findi11gs have led to the current hypothesis that exocrine cells were not merely passive targets of an aggressive immune system, but may be active participants in the autoimn1une process W11ether aberrant ti s sue e x pression of HLA protein is involved in stimulating or pr o pa g ating the autoimmune response l1as yet to be resolved 1n addition to IL-1 and IL-6, high levels of IL-2 ILI 0, IL-12, TNFa and IFNy are detected in Sjogren' s biopsies ( 1 1 ) Levels of IL-4 are typically beyond detection While much emphasis has bee11 placed upon delineating whether Thl (cytotoxic re s p o nse) or Th2 (humoral) T-helper respo11ses predominate in Sjogren's Syndrome, a clear picture of the complex cyt o kine inter a ctions is still be yo nd reach Tl1e presence of IL-10, described to direct the immune re s ponse to T h2 phenotype, i11 addition to IL-12 and IFNy which mediates the oppo s ing Th 1 phenotype suggests that the distinction of a Thl or T h2 response may not be applicable for this disease ( 44) Indeed, the cytokine profiles are highly suggestive of a generalized pro-inflammatory process Nonetheless, potential clinical usage of cytokine-blocking antibodies or cytokine therapy to direct the immune re s pon s e to a beneficial state is of extreme interest among researchers In addition to immun o re g ulation, cytokines may play a n1ore direct role i11 tis s ue destruction In vitro studies have now sh o wn that IFNy is toxic to a human salivary g land ductal cell line (HSG) ( 45) Similarly, TNFa, whose receptor bears homology to the death domain associated with FAS-induced apoptosis, may play a critical role in mediating

PAGE 21

I I both i11tlammation a11d apoptotic events ( 46) Wu el al. demon s trated that TNFa, wl1ile not able to induce significant death of the HSG line, was able to act synergistically with lFNy to indt1ce rapid cell death ( 45) Whether these cytokines directly serve to induce exocrine cell death in the autoinunune state has yet to be determined Implications of Viral Expression Despite extensive research, the initiating triggers for this disease remain a mystery Discordance betw e en identical twins a11d tl1e late di s ease onset s uggests that an exogenous agent, such as gla11dular traun1a or viral infection, may potentiate autoaggression in s usceptible individuals Much interest has focused on the potential role of virus in the initiation of Sjogren's Syndrome. Hepatitis C and I-ITV infection often result in dry eye and n1outh syndromes, but do 11ot have the srune histological and autoin1mune phenotypes of Sjogren's patients ( 47 48) Transgenic expression of the lax gene fron1 the HTL V -1 a human leukenua virus, in mice results in lymphocytic inftltration of the exocrine glands ( 49) Detectable tax gene sequences were evident in 25/o of labial salivary gland biopsies of Sjogren's patients (50) Epstein-Barr Virus (EBV) antigens and ge11omic DNA has been detected in the lacrimal tissue of Sjogren's patients, however high levels i11 normal biopsies complicates these findings (51 ) Of particular interest is the finding that tl1e SS-B/La nuclear autoantigen is associated with viral tran s lation and may also be redistributed to the cytopla s n1 and cell s urface following infection (52) Whether the generation of autoantibodies against the Ro and La ribonucleoproteins is potentially due to their association with viral RNAs or molecular mimicry of viral epitopes is

PAGE 22

12 curre11tly a hot topic a111ong re s earchers Thus, the potential involveme11t of viral infection ir1 tl1e initiation or exacerbation of this disease is i11triguing ~ however researcl1ers have yet to d e monstrate a prin1ary etiologic role of virus in Sjogren's Syndron1e Autoirnmune Disruption of Exocrin e Secretion Stimulation of exocri11e secretion is under tl1e control of both sympathetic and parasympatl1etic innervation Parasy111pathetic 11erves, primarily allowing for tl1e release of watery phase of saliva, are responsible for ba s al levels of saliva production as well as mechanically stimulated saliva flow, such as whe11 eating (53) Parasympathetic stimulation leads to the release of electrolytes which form the osmotic gradient needed for salivation Mucin rich saliva secretion is mediated tl1rough sympathetic stimulation Pilocarpine a muscarinic receptor agonist is a drug currently used to treat patients through parasympathetic stimulation (54) In our studies, pilocarpine was used to stimulate watery saliva, while the B-adrenergic receptor agonist isoproterenol, was used to stimulate proteinacious saliva flow Disruption of the naturally occurring secretory process is a frequent consequence of pl1armaceutical drugs or the effects of irradiation in anti-cancer therapies ( 1 ) While the pathogenic mechanism of exocrine dysfunction in Sjogren' s Syndrome is unclear, autoimmune di s ruption of the secretory process may occur tl1rough degeneration of glandular innervation, blockage of receptor stimulation, cellular destruction, or degeneration of the secre t ing cells It is generally believed that immune destruction of the exocrine cells is not the primary mechanism for the loss of exocrine secretion ( 11 ) This is

PAGE 23

13 in stark co11trast to Type 1 insulin dependent diabetes, for insta11ce, where di s ease 011set occurs whe11 over 903/o of tl1e insulin secreting P cell n1ass is elimi11ated (55) Histological evaluation of exocrine biopsies reveals that only approximately l 0% of the tissue area is replaced by lymphocytic infiltration ( 56 ) lt1 areas not in direct co11tact with lyn1phocytic foci, large areas of seen1ing]y normal tis s ue s are present Furthermore, the severity of sialadenitis does not appear correlated with measured loss of glandular secretions. Equally perplexing is tl1e fact that loss of glandular secretion appears inversely related to the presence of anti-ductal cell antibodies in patient sera (57) These findings l1ave led to the current theory tl1at the exocrine glru1ds of Sjogren's patients are '' loafing''(l l) Chronic destruction of the normal glandular architecture could lead to tissue dedifferentiation and loss of se c retory function The classical waxing and wruung of symptoms in Sjogren's patients suggests that lyn1phocytes play a direct role in effecting the loss of salivary fu1 1 c tion For instance, if glandular innervation wa s effectively destroyed, abrogation of the imn1une response would not be expected to restore function Therefore, it is pos s ible that cytokines and antibodies released by immune components are playing an active role in disrupting neuro-glandular stimulation Autoantibodies against tl1e Ro and La antigens while prevalent in Sjogren's patients, have yet to be directly implicated in the loss of exocrine function; although high levels of maternal autoantibody correlates to an increased risk of severe heart defects in children (1)

PAGE 24

14 Anin1al Models of Sjogre11's Syndrome The inherent scie11tific and etl1ical lirnitat i o ns i n the study of l1u1nan subjects has forced basic researchers to tum to tl1e study of animal models wl1icl1 display specific disease traits Several murine n1odels for Sjogren's Syndrome l1ave been suggested based on tl1e presence of lymphocytic inftltration in tl1e salivary gla11ds, including the NZB/NZW F 1 MRIJlpr, NFS/sld and TGFP knockout congenic mice (58-61) The TGFP knockout mouse develops extensive systemic autoimn1unity including anti-exocrine infiltration and typically dies within one month of birth (61 ) In 1994, Haneji et al. detailed anti-exocrine autoaggression in NFS / sld mice thymectomized at birth_, however exocrine dysfunction was not evaluated in this study (60) The NFS/sld n1ouse was shown to contain a single recessive gene defect in sublingual gland development, indicating that abnormalities in glandular development may contribute to autoimmune tissue targeting The NZB/NZW F 1 and tl1e MRL/lpr nuce display lymphocytic infiltration of both tl1e lacrimal and salivary glands in addition to lupus-like disorders, and l1ave tl1erefore been studied as models for secondary Sjogren's Syndrome (58,59) Interestingly, tl1e presence of lyn1phocytes in these tissues and apparent histological changes in the exocrine glands of these mice does not correlate to severe loss of glandular function Only NZB/NZW nuce >4 months of age displayed slightly abno11nal Schirmer tests, and none of these strains showed a loss of salivary flow (54). Walcott et al. recently described progressive degeneration of glandular innervation in the lacrimal glands of NZB/NZW mice over 6 mo11ths of age (62) Sitnilar to that of human disease, focal infiltration of the exocrine tissues in NZB/W and MRL/lpr mice was more prominent in female than male mice

PAGE 25

15 occupied no more tl1an 30% of tissue sectior1s, and intensity of the infiltration did not correlate to gla11dular dysfunctior1 (54) Ly111phocytes infiltrating the exocrine tissues of MRL / lpr nuce, which have a genetic disruption of Fas FasLigand-mediated apoptosis, are predominately co4 T-cells (63) Analysis of TCR VP expre s sion revealed a diverse T-cell repertoire however a skewing of the population to VP8 l 2 and VP6 was detected (64) Purified CD4 but not CD8 + lymphocytes isolated from MRL / lpr sa livary glands are able to transfer sialade11itis to inununo c ompronused CB 11-scid mice (65) Intere s tingly, specific depletion of donor vps t or VP6 + lymphocytes prior to transfer significantly inhibited sialadenitis in recipient mice Both the NZB/NZW FI and MRL/lpr mice display similar cytokine profiles as those detected in human tissue biopsies (66) Therefore si11ce these mice do not develop severe functional deficiency, it is unli k ely that the cytotoxic effects of hallmark cytokines such as IFNy are primary effectors of exo c rine dysfunction in murine models. Together these findings demonstrate that the mere honung of leukocytes to the exocrine tissues is not sufficient to cause the loss of exocrine function In 1992 an advance in the searcl1 for an arnmal model for Sjogren's was made by Hu et al. in tl1eir description of secretory dysfunction in autoimtnune NOD (nonobese diabetic) nuce (67) NOD Mouse First introduced in 1980, the inbred non-obese diabetic (NOD) mouse exhibits a strikingly similar pathology to that of human Type I insulin dependent diabetes mellitus

PAGE 26

16 (IDDM) (55) Lympl1 ocy tic destruction of tl1e P-cells of the islets of Langerl1ar1s results in a loss of blood glucose regulation due to tl1e loss of i11sulin secretion ln1n1une infiltration of tl1e pancr e as can begin in NOD mice as early as two weeks of age and begins witl1 tl1e appearance of Class II + monocytes and CD8 + T lyn1phocytes (68) Overt diabetes in NOD mice generally begins b et ween 8-12 wks of age and occurs when >9 0% of tl1e P-cell 111ass is destroyed By 30 weeks of age roughly 80% of female and 20% of male mice beco1ne diabetic (55). Interestingly, NOD mice kept in specific pathogen free (SPF) colonies develop diabetes more frequently and at an earlier age that those kept in traditional colonies (69) This indicates tl1at, similar to the human condition, environmental factors play a role in tl1e development of autoin1mt1nity in these mice The observation of the lymphocytic infiltration in the NOD n1ouse is not confined to the pancreas, however but is also observed in the sa livary a11d lacrimal glands of this s train (70) Interestingly, tl1e parotid gland does not develop extensive lymphocytic infiltration The first appearance of periductal and perivascular lymphocytic infiltration in the submandibular and lacrin1al g Jands begins at 8-10 wks and 10-12 wks of age, respectively (71 ) By 18 week of age the exocrine glands display focal lymphocytic lesions with an observable disorganization of nom1al acinar structure Despite the early appearance of lymphocytic infiltrates within the submandibular and lacrimal glands, loss of saliva flow and tear production does not typically occur until 14-16 wks of age ( 6 7) This result was expanded to show that NOD mice lose approximately 90% of their saliva flow and 30% of tear flow between 8 and 20 weeks of age (71) Unlike diabetes in the NOD, both male and fen1ale mice develop infiltration of the exocrine tissues at similar rates (70). These findings reveal that the NOD mouse represents the first-described animal

PAGE 27

17 1nodel for the spontaneous autoir11mune-indt1ced loss of both saliva flow and tear producti o n a11d as s uch is emerging as an excell e nt n1 ode l for the s tudy of Sjogr e n s Syndr o tne in hun1ans Immunog e n e ti cs of tl1e NOD Mouse The NOD mouse arose dur i ng tl1e selective breeding of the inbred cataract Shionogi (CTS) s train from o utbr e d ICR mice (55) In 1 9 74, a single female mouse exhibiting polyuria s evere glycosuria, an d weight los s was discovered, and, after exten s ive inbreeding, the NOD strain was introduced in 1 98 0 Sin c e this time at least 13 genetic l oc i have been di scove red which contribute to the devel op ment of diabetes in NOD mice (72) An a dditional two genetic lo c i Jdd7 and ldd-8 found in the BIO ba ckg r ou nd are also able to increase diab etes progre ssion when bred onto the NOD background (69) Genetic influence contributes to both the incidence of diabetes as in Jdd-1, as well tl1e timin g of di sease o n se t, as in Jdd-2 and ldd-4. The MI-IC-linked lo c us ldd-1 is essential for tl1e devel o pment of di abe te s, as replacement of the of the NOD MHC loci with the B 10-derived allele completely inhibits both diabetes and insulitis, but not sialadenitis (73) The NOD MHC, H-2 87 haplotype contains several unique features, incl u din g a structurally distinct I-A molecule and a del et ion of 1-E expression (74) Conserved seri11e-aspartic acid or proline-a s partic acid at the AP amino acid positions 56 and 5 7 are replaced with h ist idine and serine in the NO D allele (75). In humans, replacem e nt of the aspartic acid with uncharged amino acids at po s ition 57 of the human DQJ3 has been implicated in increased diabetes risk (76).

PAGE 28

18 Tra11sgenic expres s ion of 1-Ak in NOD mice i11l1ibits in s ulitis a11d diabetes, however sialadenitis was not affected (77) The lack of 1-E expres s ion is not unique to the NOD strain, but is also a fe a ture of inbred C57BL/6 SJL, ACA, and DBNI mice (78) Since the B 10 mice used in generating the NOD B 1 O-H-2b congenjc are also 1-E negative, the i1npact of 1-E expression on the NOD strains was not investigated in trus model Generation of tra11sgenic NOD mice wlucl1 express the 1-E molecule has determined tl1at E expression is protective against diabetes and insulitis (79) Sialadenitis was not investigated in these mice Interestingly, Faustman found occasional lymphocyti c infiltration in the submandibular glands in tl1e majority of the 1-E negative strains n1entioned above (78). The 1-E molecule is responsible for the deletion of TCR vPS + lymphocytes suggesting that vps + lyn1pl1ocytes 1nay play a role in tl1e development of s ialadenitis However NOD mice backcrossed with SWR mice, which contain a deletion in vPS vP8 and vP 11 gene segments, still develop typical frequencies of insulitis and diabetes (80) Together these findings demonstrate that both the unique NOD I-Ag7 1nolecule and tl1e lack of 1-E e x pression are i1nportru1t for diabetes, however exocrine gla11d infiltration is o n ly associated with the lack of 1-E expression The contributions of non-MHC genes to the developtnent of diabetes and sialadenitis is an area of intense interest. Garchon et al linked a centromeric locus on chromosome 1 witl1 diabetes su s ceptibility (81 ) Intere s tingly, a second susceptibility locus on the same cl1romosome was linked to sialadenitis, peri insulitis, and hyper-IgG production The d e velopment of sialaden.itis was found to be a domin a nt trait in tl1ese studies (81) To evaluate immune and non-immune components of autoimmunity in the NOD mouse, Leiter et al. bred the scid mutation onto the NOD genetic background,

PAGE 29

19 creating the NOD-scid strai11 which was extensively used in the current studies (82). H o 111ozygosity at the ~ c id locus leads to the loss of fu11ctional T and B lymphocytes, and tl1erefore, tl1ese ani111als do not develop insulitis, sialadenitis, or diabetes This study also demonstrated that, similar to p a rental NOD mice, NOD-scid mice have complement deficier1cy fi111ctionally iinm a ture n1acropl1age populations and decreased natural k i ller cell activity (82) The sum results of these immunogenetic findings are promising in that they strongly suggest that the ultimate goal of genetically separating diabetes from exocrine gland autoimmunity to develop a prin1ary rnurine m o del for Sjo g ren' s Syndrome will be attainable Role of Helper T-Lyn1phocytes Prior to this study, the l y mphocyte phenotype s infiltrating the exocrine glands were largely unexplored Therefore our knowledge of autoimmunity in the NOD mouse comes almost exclusively from diabetes research While autoimmune i1lfiltration it1cludes several immune cell types researchers have predominately focu s ed on the role of CD4 .. T lymphocytes which represent the largest cornponent of the infiltrate Neonatal thymectomy of NOD mice generally inhibits in s u li tis and diabetes, and athymic nude NOD 1nice do not develop either phenotype (83 84) Similarly injection of anti-Thy I 2, anti CD4, or anti-CD3 antibodies suppresses d ia betes (55) A direct role of lympho c ytic involvement is demonstrated by the ability of splenic or tis s ue infiltrating lymphocytes from di a betic donors to rapidly transfer both diabetes and s ialadenitis to non-diabetic NOD or NOD-s c id recipients (85) As opposed to rapid transfer of disease, splenic

PAGE 30

20 lyn1pl1ocyte preparations from prediabetic mice suppressed disease onset Tl1erefore the ability to successfully tr a 11sfer di sease is not only dep e 11de11t upo11 tl1e presen ce of CD4 + T lyn1phocytes but also their activatior1 state W11en transferred to irnmunocompromi se d recipie11ts, purified na ive CD4+ T cells, represented by the CD4 + CD45RB 11 i phenotype, i11duce a wa sti n g disease associated with intestinal inflrunmation (86) Interestingl y co-transfer of CD4 4 CD4SRB 10 memory T ce lls suppresses the wasting disease (87) Two distinct p op ulations of CD4 + memory T ce lls with opposing di abe togenic potential )1ave been separated from prediabetic and diabetic NOD mice b ase d up o n their cytokine expressio11s (87) Memory cells pr od ucing hi g h levels of IFNy (Th I) and low levels of IL 4 transfer diabetes rapidly to recipient mice However, memory cells produci11g low amounts of IFNy and lugh levels of IL-4 (T1t2) confer active suppression (87) Tllis indicates that tl1e ability to progress from a protective to a patl1ogenic s tate may lie in the r e l a tive l eve ls of cytokines pre s e11t in the autoimmune le sio n This is supported by sinlilar findings using diab etoge ruc CD4 + T-cell clones Haskins et al demon s trated that shifting high IFNy produ c ing T cell clones to IL-4 produ ce rs in long term culture suppressed their ability to tran s fer di s ea s e (88) Further1nore, successful transfer of diabetes with CD4 + T cell clones i11to NOD-scid recipients demonstrates the potential of CD4 + cells to tr ans fer diabetes in the absence of either CDS + T cells or B lymphocytes Analysis of TCR VP usage in the pancreas of NO D mice shows heterogeneous TCR popu la tions However, much like the MRL/lpr mouse, a preferential u sage of vps and VP6 genes is present in diab e togenic T cells (89) Edouard et al. showed that selective deletion of Vp6+ or vps + T cells prior to a doptive transfer significantly lowered

PAGE 31

21 tl1e ability to transfer diab e tes to irradiated NOD recipients (90) Although suggestive of a pathoge11ic role for VP6 .. and vps + T cells, NOD X SWR n1ice develop insulitis a11d diabetes despite deletio11s in VP5, VJ38, and VP 11 gene segn1e11ts (80) In addition, TCR usage from islet specific T cell clones of diabetic NOD mice include VP4 VP6, VP8 2, VP 12, VP 16, and VP 19 gene usage, further indicating that glandular specific TCR ge11e usage is not restrict e d (55) Recent findings by A.J1derso11 (perso11al communicatio11) have indicated that TCR VP restriction may be present in a CD3+CD4-CD8. ''double negative" T cell population which is present in early islet infiltrates; however, this has yet to be confirmed CDS + T Lymphocytes and B Lympl 1oc ytes Wlule the nece ss ity of CD4+ l y n1pl1ocytes is readily apparent, the role of CD8 + T cells and B cells in tl1e ini t iation of autoimn1unity is less clear. Transfer of purified CDS + T cells is typically insufficient to cause diabetes, however a recent report demonstrated tl1at diabetes can be transferred by a CD8+ T cell clo11e (85,91). Using a gene knockout strategy, Wicker et al determined tl1at disruption of the P-2 microglobulin gene, which results in a specific loss of CD8 + T-cells, inhibits both insulitis and diabetes in NOD nuce (92) However in prediabetic mice, co-tra11sfer of botl1 CD4 + and CD8 + populations is necessary to initiate diabetes (85) Therefore, while an aggressive CD4 + or CDS + T cell clone may be able to cause diabetes, both cell types appear essential for the initiation of disease.

PAGE 32

22 Similarly, despite the fact tl1at autoantibodies are detected on the surface of islet cells prior to leukocytic infiltration, the role of B-cells has been largely downplayed in tl1e literature (93 ) Re11ewed interest in tl1e B cell component is bei11g generated witl1 tl1e discovery that NOD n1ice containing a -heavy chain knockout which halts tl1e generation of 1nature B lymphocytes do not develop in s ulitis or diabetes (94) Wl1ile exocrine dysfunction l1as yet to be evaluated in these mice, several studies have indicated that the B cell component may be important for exocrine dy s function in NOD mice 111 addition to the presence of antibodies directed against islet cell components, NOD sera contains both anti-nuclear antibodies and antibodies directed agai11st sali v ary gland cells (95 96) In an analysis of the signal transduction pathways responsible for secretion from salivary a11d lacrin1al gland s, Hu111phreys Beher et al identified a subclass of autoantibodies directed against the muscarini c and P-adrenergic cell surface r e ceptors that initiate this process (97 98) The density of these cell surfa c e recept o rs is reduced on both the parotid and subn1ru1dibular gla11ds along with s ecretory re s pon s e to P-adrenergic, muscarinic / cholinergic and n e urop e ptide agonists during disease Thus it is po s sible that soluble factors such as autoantibodies may ulti1nately be re s p o n sible for the loss of se c retory function in NOD mice This may also explain how parotid gland function is inlubited despite tl1e lack of focal lymphocytic i11filtration Together, these findings suggest that the initiation of autoimmunity in the NOD mouse involves multiple immune components and nun1erous cell types

PAGE 33

23 Pote11tial Dichotomy of Diabetes and Sialoade11itis Approximately 30/o of autoimmune diabetic patients suffer conconutantly fro111 xerostomia a11d xerophthal1nia due to insufficient glandular secretions ( 13 ) Tl1ese clinical syn1ptoms are th o ught to result from poorly mai11tained blood glucose regulation lack of insuljn secretion, or neuropathy (99) Expan s ion of the autoimmune processes into the exocrine gla11ds of diabetic patients is rarely encountered and generally not considered to be the causative factor for exocrine dysfunction in diabetics (12) In sup port of this, ti g ht glucose control generally alleviates the feeling of d ryness in rno s t patie11ts. Tl1erefore, is it pos si ble that the lo ss of exocrine secretion in NOD mice is due to the lo ss of blood glucose regulation a11d not anti-exocrine autoaggression? In addition to tl1e immunogen e tic studies discussed above, several studies have illustrated a di c hot on1y between autoimmune diabetes and autoimmune sialadenitis in tl1e NOD mou se Loss of secretory fun c tio11 in NOD mice, wliile more severe after diabetes onset, is pre se11 t in both male and female predi ab etic 1nice, which l os e 5070% of s tin1ulated salivary flow b etw een 8 and 20 weeks of age (6 7) Similarly injection of diabetic mice with daily insulin injections does not re s tore secretory function Recent findings u s ing NOD.BI O-H-2b nlice have shown that the loss of secretory function occurs in the absence of eitl1er insulitis or diabetes ( unpublished observations). Treatment of NOD mice with antibodies against alpha 4-integrin and Lse lectin w as able to signifi can tly inhibit b o th in s ulitis and diabetes, however, si aladenitis was unaffected (100) In humans, increas e d expression of ICAM-1 and E-selectin is detected on salivary epithelial cells ( 43) From the s tandpoint of tissue tolerance, intrathytnic injection of islet cell homogenates or

PAGE 34

24 trn11sgenic expression of proinsulin II in NOD mice prevents diabetes but fails to protect against sialadenitis (101,102). Tl1ese studies su g gest tl1at the ai1tige11s involved in the a11ti exocrine response are distinct from tl1ose in the islet, and tl1at loss of exocrine gla11d tolerance is not secondary to P cell autoin1n1unity Cl1anges in Saliva Proteins in the NOD n1ouse In additio11 to loss of secretory function in NOD n1ice, the protein constituents of NOD saliva c hange over time This is reflected by reductions i11 amylase activity ( > 50%) and loss of ductal cell secretion of EGF (67). In newly diabetic male NOD mice, over 97% of EGF production is lost. In this dissertation, specific changes in both parotid secretory protein (PSP) and proline rich proteins (PRP) are observed in both NOD and NOD-scid anirnals wluch parallel changes in EGF and amylase TJ1e proline rich protei11s are latent in nom1al mice ~ however they comprise > 70o/o of the protein in human saliva, where they serve to bind both calcium and hydroxyapatite ( 103 ) Expression of the PRPs can be induced in both the 1nurine parotid and submandibular glands by chronic adrenergic stin1ulation or the introduction of high levels of tannic acid in the diet ( 104) In these studies we demonstrate the abnormal presence of PRPs in both submandibular and parotid glands of aging NOD mice In normal mice, PSP is described as a 20 k.Da, leucine-rich glycoprotein ( ~ 23% leucine; 235 amino acids) of unknown function that is secreted predominately by amylase producing acinar cells of the parotid gland (105) Studies have shown a develop1nental coordination of murine PSP and salivary amylase expression in tl1e parotid gland of adult

PAGE 35

25 111ice wl1ere the two protei11s appear i11 consta11t ratios ( I 06) Coordinate expression, however, is 11ot deterr11ined by tl1e rate of gene expression, s ince developrnental expres s ion of the PSP gene occurs before an1ylase expression ( I 07) Altl1ough PSP is specific to tl1e parotid and sublingual glands of adult n1ice, PSP is expressed in the developing subrnandibular gland up to 5 days of age ( 108) This i s ir11portant in that abnorn1al re expression of PSP in the submandibular gla11d is detected in agi11g NOD mice and n1ay indicate d e ditferentiation of the glandular acini While the fu11ction of PSP is u11known, our fi11dings suggest tl1at it n1ay play a role in anti-microbial binding To date PSP l1as not been detected in human saliva ( our anti-mouse PSP antibody does not crossreact), l1owever a single copy of tl1e PSP gene is present in human cells and PSP mRNA has been detected in human parotid tumors (109) Results in this dissertatio11 show that PSP is enzymatically cleaved in older NOD saliva The c leavage occurs between leucine and a spa ra g ine at tl1e 26 th and 27 th amino acids of the protein Database searches uncovered no known enzymes which may be responsible for this cleavage However si nce PSP cleavage corresponds to the ti1ne of dra1natic acinar cell lo ss in the NOD mouse, we investigated the potential of both matrix metaUoproteinases and apoptotic cysteine proteases to c leave PSP through bystander activity These proteins, involved in glandular restructuring and apoptotic cell cleat~ respectively, may play a key role in the loss of submandibular acini in NOD and NOD-scid nuce.

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26 Matrix ietalloproteinases and Cysteine Proteases Cellular ho1n eos tasis depe11ds on re g ulated ce ll proliferation coupled to cell death ( 110) The matrix n1etalloprotein ase s ("M11Ps) are a class of zinc-dependent enzyn1es wl1ich in conjunction with their specific inhibitors (TllvfPS), are respon s ible for restructuring and 1naintenance of the extracellular rnatrix Over 12 MMPs have been describ e d and include the collagenases, gelatinases ( collagen type IV), s tromaly si ns (laminases ) and el a s tins \vhi c l1 are able to cleave virtually all comp o nents of the extracellular matrix (111 ) Expression of spec ific MMPs during the developmental process is re s pons i ble for sculpturing the extracellular en v ir onn 1ent and th er eby dictating cellular turnover and differentiation While much re se arcl1 l1a s foct1sed on their association with tissue homeo s ta s is and tun1or metastasis, a prominent r ol e of "M11?s in autoimn1une processes has rece11tly been described (111 ). Monocyte pr oduc tion of "M11Ps is involved i11 dictatin g the extent of ti ss ue d amage caused by inflammat io n and may additionally be critical for n1atrix degradat io n necessary for lymphocyte cl1en1otaxis Inhibitor s of MMPs have been shown to significantly reduce the appearance and severity of EAE in anin1al models (112) This effect was attributed to the inhibition of monocyte-derived gelatinases responsible for the degradation of the blood-brain barrier ba se ment 1nembrane Similarly MMP inhibitors significantly reduce joint inflamn 1a tion and ti ss ue destruction in collagen i11duced murine model of rheumatoid arthritis (113) Studies have further d emonstra ted that MMP activity is connected to the apoptotic patl1~ay through the ability to cleave cell surface FAS on apoptotic target cells (111) In humans, Sjogren s patients have elevated levels of collagenase (MMP-1) in their saliva, and supematants from excised biop s ies

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27 cor1tain elevated MMP activities ( 114) Wl1ether elevated or aberrant MMP production precedes or is a result of tissue inflamn1ation in autoi1111nune target tissues is still unknow11, however the exte11sive changes in glandular arcl1itecture s een in tissue biopsies may, in part, be attributed to MMP activity Extracellular signaling molecules are capable of regulating glandular cell populations through a series of intracellular events termed programmed cell deatl1 or apoptosis ( 110) Apoptosis is distinct from cell lysis in that the target cell actively and efficie11tly mediates its own d e atl1 This process involves intracellular activation, reduction of cell volume, c l1romatin cond e nsatio~ and endonuclease cleavage of DNA within the cell membrane ( 115). By tins process the apoptotic cell contents are kept safely inside the cell membrane until ph agoc yti ze d by ma c rophages or neighboring cells Immune system molecules capable of medi a ting apoptosis include the cytokine tumor necrosis factor (INF) and the related protein CD95 (FAS) ( 115) Mice tl1at lack functional FAS surface protein develop a lyn1phoproliferative disorder and lupus-like patl1ology associated witl1 the inability to delete lymphocytes during the immu 11o logical education process (116) In Sjogren's patients, it is hypotl1esized that dy s function in apoptosis results in abnormal longevity of both T and B lymphocytes and may underlie polyclonal B-cell activation Elevated levels of bcl-2, wruch is able to rescue target cells from apoptotic death, are detected in salivary gland biopsies ( 117) Interestingly, 2-fold increases in surface FAS expression are detected in peripheral blood T cells from both Sjogren's Syndrome and SLE patients than controls which correlated with accelerated apoptosis of these cells in vitro ( 118). Therefore, it is unlikely tl1at defective FAS molecule is directly responsible for Sjogren's Syndrome or SLE in most diseased patients.

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28 Activation of tl1e FAS or TNF receptors leads to a proteolytic cascade involving n1embers of the cysteine protease family ( 110) Tl1ese proteases cleave important cellular protei11s including pro-enzymes of other men1bers of tl1is class of proteases a1 1 d endonucleases responsible for DNA cleavage A prototype protein of the cysteine proteases is interleukjn-1 P-converting enzyme (ICE) v l1ich has been characterized as tl1e activator of the cytokine i11terleukinl P through cleavage of its precursor at Asp 116 / Ala 117 ( 119). All members of the I CE-related cy s tei11e protease farojly, including Nedd-2 and apopain/cpp32, cleave their substrates after an aspartate residue followed by a small an1ino acid residue wlu c h is important for substrate conse11sus recogrution (120) Expression of ICE in 11euroblasto1na or fibroblast cell lines leads to apoptotic death ( 110) Therefore, l1igh levels of cysteine protease activity may be indicative of both the activation of apoptotic mecl1amsms as well as the proces s ing of proinflammatory cytokine precursors Sin c e both MMPs ru1d cysteine proteases appear involved in regulated cell pro I if eration and cell deatl1, it is possible that upregulation of tl1ese enzymes in the NOD mouse are directly involved in a cinar cell death and tl1e aberrant cleavage of additional salivary proteins Further Development of the NOD Mouse Model of Sjogren's Syndrome The physiological loss of secretory function makes the NOD mouse an ideal animal model for tl1e study of human Sjogren's Syndrome Despite the linuted amount of exocrine gland research, the most exciting aspect of the NOD mouse is the ability to apply

PAGE 39

29 tl1e findings of over 15 years of diabetes research to the u11derstandi11g of autoimmune sicca synd ro n1e Additionally, the seerning dichoton1y bet\veen sialadenitis and diabetes suggest s that tl1e study of congenic NOD mice, sucl1 as the NOD Bl OH-2b, will lead to tl1e development of a primary model to study anti-exocrine autoimn1unity To begi11 tl1ese studies however it is necessary to determine tl1e imn1unological and physiological processes underlying exocrine dysfunction in the NOD mice Therefore, the studies of this dissertation explore tl1e following specific aims : 1. To deterrnine tl1e ten1poral changes in exocrine gland histology, infiltrati11g lympl1ocyte populations, and cytokine production spanning the initiation of tl1e autoimmune process to the final stages of glandular destruction 2 To investigate exocrine gland abnom1alities in immunodeficient NOD-scid mice in order to separate immune and non-immune components of the disease process 3 To investigate the tissue specificity and function of parotid secretory protein, which is found to be aberrantly expressed and processed in aging NOD mice 4 To evaluate the proteolytic activity in NOD saliva responsible for the aberrant cleavage of PSP and investigate candidate enzymes for PSP cleavage

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CHAPTER2 CHARACTERIZATION OF THE CHANGING LYMPHOCYTE POPULATIONS AND CYTOKINE EXPRESSION IN THE EXOCRINE TISSUES OF AUTO NOD MICE Introduction During tl1e past several years significant i11terest l1as developed in detailing tl1e autoimmune destruction of tl1e salivary and lacrimal tissues in the NOD mouse Pioneering studies have den1onstrated that tl1e lympl1ocytic infiltration of the salivary and lacrimal glands correlates with a functional decline in saliva flow and tear production indepe11der1t of the loss of blood glucose regulation observed in NOD mice (67,71). Lymphocyte transfer studies l1ave now sho,vn that the induction of thymic tolerance to the pancreatic islets does not confer immunologic tolerance to the salivary tissues, s uggesti11g a pote11tial dichoto1ny of disease states between these target tissues ( 10 I) This is supported as well, by immunogenetic studies linking sialoadenitis, hyper-IgG production, and peri-insulitis to a centromeric loci on Chro111osome 1, while insulitis is linked to a telomeric loci on the same chromosome (8 I). In addition to anti-pancreatic and anti insulin autoantibodies, NOD sera has been sl1own to contain autoantibodies targeting the acinar and ductal cells of the subn1andibular and parotid glands, including those directed against the muscarinic and P-adrenergic receptors responsible for the generation of saliva flow (97,98). The decline in saliva output is accompruued by changes in salivary protein content as well. Both alterations appear to be the result of down-regulation of signal 30

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31 tra11 s duction con1p o ne11ts of the salivary glands ( 9 7) Abr1ormalities i11 gene trans c ription and protein proce s s ing i11 tl1e s alivary glands of immunod e ficient NOD-s c id 111ice suggest tl1at gla11dular dy s fun c tion in NOD mice n1ay pre ce de lympl1ocytic infiltration and provide a potential trigger for tl1e autoinunune targ e ting of the sa livary glands ( 121 ) The NOD m o u s e r e pre s ents tl1e first-de s cribed anin1al model for tl1e spontane o us autoimmune-induced loss of both saliva flow and tear production, and, as such is emerging as an excellent n1 o del for the s tudy of Sjo g r e n's Syndrome in humans Sjogren's Syndrome is an a ut o in1mune di s ea s e characterized by dry e y e ru1d dry n1outh syndromes due to the destructi o n of e xo c rine tissue. Clini c al patients develop chronic l y mp h ocytic infiltrati o n of tl1e sa livary and lacrimal gl a 11ds as well as a cell-mediated and autoantib o dy re s p o n s e agai11 s t t he exocrine ti ss ue (11 ) D i agno s is of Sj o gren's Syndrome often reli e s on the detection of lymphocytic infiltration in labial lip biopsies excised from patients whi c h h av e revealed a predominance of CD4 + T ly1nphocytes with an oligoclonality of the T-ceU recept o r repertoire (38) In a maj o rity of p a tients, autoantibodies to rib o nuclear protein antigens are al s o pre s ent (20) In addition, incre a ses in IFNy, TNFa., IL-2, IL-6 and ILIO cytokine production l1ave been d esc ribed in biopsy tissues ( 11 ) Howe v er since patient samples are from a late stage, the initial events surrounding the development of autoimmune infiltration and de s truction of tissue remain unknown In this study I have characterized the infiltrating lymphocyte repertoire and cytoki n e mRNA pr o file of the lacrimal, p a rotid and submandibular glands of NOD mice during the cour s e of immunopatho g enesis Sple e n and islet-infiltrating cells were analyzed as control lymphocyte populations throughout the time course of disease progression This has allowed for a qualitative comparison of lymphocyte populations infiltrating the

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32 pancreas, subn1andibular and lacrimal tissues of NOD mice, as well as provide a detailed accour1t of tl1e a11ti-exocrine autoimmune response wl1icl1 can be co111pared to tl1at s ee11 in Sjogren's Syndrome Materials a11d Methods Atiirnals Female NOD mice (7 wks of age) were purchased from Jackson Laboratories (Bar Harbor, ME) and maintained t hroughout the cour s e of tl1e study i11 th e a r1imal facility at the Health Scie11ce Center at the University of Florida (Gainesville FL) Diabetes was diagnosed by elevated blood glucose levels using Chemstrip bG reagent strips (Boehringer Mannheim, Indi a napolis IN) Mice with blood glucose > 200 mg/dL were given insulin injections (IU / mou s e/day ip) (67) Evidence of diabetes was first noted at 14 weeks of age At 16 we e ks, 3 of 5 mice were diab e tic~ at 18 weeks 1 of 5, and at 20 w e eks 2 of 5 mice were diabetic. Antibodies Monoclonal antibodies used in this study were purchased from PharMin g en (San Diego, CA) and are as follows; CD3e (clone 145-2cl 1), CD4 (RM4-5), CD8a. (53-6 7) CD45RB/B220 (RA3-6B2), CD45RB (23G2), TCR VP3 (KJ25), TCR VP6 (RR47) TCR VP8 1, 8 2 (MRS-2), TCR VP9 (MR 10-2) TCR vp l 1 (RR3-15), and TCR vp I 7a (KJ-23)

PAGE 43

33 Tisst1e Preparation Spleen pancreas, lacrimal parotid and submandibular glands were removed at each l1arvest A small piece was cut fron1 each tissue, placed in 1 Oo/o buffered f or1nalin and submitted to the Diagnostic Referral Laboratory at the University of Florida (Gai11esville, FL) for hj s tologic sectioning ar1d stai11i11g Tl1e re n1ainder was processed as a pool from the 5 mice per group A small aliquot of eacl1 pool was removed for RNA isolation and all ren1aining tissue prepared for flow cytometric analyses Single cell suspensions of splenic leukocytes were obtained by gently pressing spleens through wire mesh screens and wasrung in PBS (68) Red blood cells were lysed witl1 0 84% ammonium cliloride. After \lt-asrung, the remai11ing leukocytes were aliquoted at I x 10 6 / tube and washed with FACS buffer (PBS with 0 1% NaN 3 (FISHER Scientific, Orla11do, FL) and O So/o BSA (SIGMA Chemical Co, St Louis, MO) prior to antib o dy staining Tissue Digestion Except for spleen, all pooled tis s ues were dissociated by gentle mincing folJowed by digestion at 3 7 C in a shakj11g water bath for 15 minutes in a mixture of 4 mg/ml collagenase Type V (SIGMA) + 100 U / mJ DNase Type II (SIGMA) Digested tissue was further dissociated with vigorous pipetting, ren1oved (after allowing large, undigested pieces to settle) and placed in ice-cold HBSS with 2% FBS (GIBCO/BRL, Grand Island, NY) Digestion was continued u s ing a mixtt i re of 2 n1g/mJ collagenase + I 00 U / ml DNase in 5 min incubations at 3 7C in a shaking water bath and repeated until completion.

PAGE 44

mIL 1~ mIL 2 mIL4 mIL 5 mIL-6 mlL 7 mIL 10 mIL 12 (p40) mIF N y mT NF a mi NOS G 3PDH 5 primer Table I Murine primer and probe sequences 3 prim e r 5' TGGCAAC T GTTCC T GAAC T C AA CT 5'ATGTA CAGC.-'\.TGCAGCTCGCATC 5 1 ATGGGTC T CAACCCCCAGCTAGT 5'A T GAGAAGGATGCT T CTGCACT TGA 5'ATGAAGTTCCTC T CTGCAAGAGAC T 5 ATGTTCCATGT TT C TTTTAGATA 5' GCAGGGGCCAGT ACAGCCGGGAA 5'ATGTGGGAGCTGGAGAAAGACG 5'TGAACGCTACACACTGCATCTTGG 5'ATGAGCACAGAAAGCATG .\. T CCGC 5' CCC TT CCGAAGT T TC T GGCAGCAGC 5' TGAAGGTCGGTGTGAACGGA T TTGGC 5'CAGGACAGGTA T .\.GAT T CT TT CCT TT 5' GGCTTGTTGAGATGA T GCTTTGACA 5' GCTCTT T AGGCTTTCCAGGAAGTC 5 GTCACCATGGAGCAGC T CAGCC 5'CACTAGGTT T GCCGAG T AGATC T C 5 G AAACATGCATCATTCTTTTTCT 5'GCTTTTCATTTTGATCA T CA T G T 5' T CACGACGCGGG T GC T GAAGGCGTG 5'CGAC T CC TTTT CCGCTTCC TGAG 5' CCAAAGTAGACCTGCCCGGAC T C 5'GGC T GTCAGAGCCTC GTGG C TT TGG 5'CA T GTAGGCCATGAGG T CC A CCAC Int e rnal probe 5 1 CCGTGGACC TT CC AGGATGAGGA CATG AG C 5' GCCACAGAATTGAAAGATCTTCAG TG CC T A 5'AGGG C TT CCAAGG TG C TT CGCATATT T TAT 5'ATAAAAATCACCAGC T ATGCATTGGAGAAA 5'TCTGGT CT T C T GGAGT ACCATAGCT,<\.CCTG 5 G AAGC T GC TT TTCT AAA T CGTGCTGCTCGC 5' T C AGGAT GCGGCTGAGGCGCTGTCATCGAT 5' ACTCCGGACGG TTCA CGTGCTCATGGC 5'GACATGAAAATCC T GCAGAGCCAGATTATC 5' C TC C T GGCCAACGGCATGGATC T CAAAGAC 5'TGCCGGCACCCGGAGGAGGAC 5' CC T TCCGTGT T CC T ACCCCCAATGTGTCCG w +:a

PAGE 45

3 5 Islets were picked n1anually fron1 digested pancreatic tissue under a di sse cting 111icroscope and collected in ice-cold IMDM (GlBCO / BRL) witl1 I% NMS (68) Islets were collected by centrifugatio11, supernatant discarded and the cells di s per s ed by conti11uous pipetting i11 10 ml of a solution co11taining trypsin/EDT A (GIBCO /B RL) + 2000 U DNase (SIGMA) at 37 C for l O 111in. Digestion was stopped by adding i c e-cold IMDM with 1 % NMS. Dige s ted lacrimal, parotid and submandibular tissue and dispersed islets were wa s hed and tl1en separated by centrifugation tl1rougl1 a SSo/o Percoll (SIGMA) gradient. Tl1e i1tliltrating cells were collected in the pellet and contamu1ating red blood cells ly s ed witl1 0 84% ammonium chloride After wa s hing cells were divided into aliquots containing approximately 1 x I 0 6 cells and washed in F ACS buffer prior to antibody staining Flow Cytometry Aliquots of all cell populations were re s u s p e nded in 100 l FACS buffer and stained with antibody at l g/10 6 cells Cells were stained first with anti-CD3 for 40 min at 4C, washed with F ACS buffer and then stained with the appropriate second antibody in a 40-tnin incubation at 4 C. After a final wash, cells were suspended in F ACS buffer for analysis Flow cytometric analyses were performed usi11g a F AC Scan flow cytometer (Becton Dickinson, Mountain View, CA) equipped witl1 a 15 milliwatt, 488nm air-cooled argon-ion laser and using LYSYS II software (68) Ten thousand events were coll ec ted

PAGE 46

36 per sample fro111 a population gated on a window e11compassing the splenic lymphocyte population RNA Isolation and RT-PCR Detection of Cytok.ine nlRNA Pooled tissues were mi11ced in PBS, placed in lysis buffer and mRNA isolated using a Micro-FastTrack Kit (lnvitrogen) Isolated mRNA was stored at -70 C in ethanol until all san1ples were collected. mRNA was pelleted by centrifugation and cDNA prepared by reverse transcription using Superscript n Reverse Transcriptase (GIBCO/BRL) cDNA was quantified using a DNA Dipstick Kit (lnvitroge~ San Diego CA). Equal quantities of cDNA from eac11 sample (50 ng per reaction) were amplified by PCR for 40 cycles at 60C annealing ( I min) and 72C elongation (2 min) using cytokine primer pairs shown in Table 1 PCR products were separated on 1 2% agarose gels and transferred to positively charged nylon metnbranes (Boehringer Mannheim) by Southern blotting (122) Detection of Cytokir1e nlRNA Specific PCR products were identified using the Genius system of nonradioactive DNA labeling and detection (Boeliringer Mannheim) according to the manufacturer's protocols Briefly, internal oligonucleotide probes specific for each cytokine (Table I) were labeled by random primed incorporation of digoxige1un-Jabeled deoxyuridine-triphosphate. After overnight hybridization at 65C, the PCR products were detected colorimetrically using an anti-digoxigenin alkaline phosphatase conjugate in an enzyme-linked immunoassay. Concanavalin A (SIGMA)-stimulated mouse splenocytes

PAGE 47

37 were used as a positive control for tl1e prin1ers a11d probes a11d G3 PDH was u s ed as a positive control for tl1e isolation of 1 1 11tN A ( 123 ) All nucleotide prin1ers and probes were syt1tl1esized ir1 the l11terdisciplinary C e nter for Biotechnology Research DNA Synth es is Core Laboratory at tl1e U11iversity of Florida (Gainesville, FL) Densitometric Analysis Semi-quantitative analy ses of cytokine PCR products was done using densitometry and One-D sca n software San1ple calculations w er e standardized using G3PDH values to ensure tl1at equal a m o unts of niRNA were pre se nt in each sa mple thus allowing for semi quantitative compariso11 between sample b a nds (G3PDH values for 8 wk lacrimal and 10 wk sub1nandibular samples are reported as raw data since tl1ey did not appear to be consistent with experimental samples). Lanes wtlich did not exceed background were given a value of ze ro and s ub se quent s ample values were deter1nined using auto ba c k g round cal c ulations Results Tissue Histology Lacrimal, submandibular, parotid and pancreatic tis s ues were surgically removed from groups of 5 NOD nlice at 2 wk inteIVals from 8 through 20 weeks of age A s malJ piece of each ti ss ue was s tained with h e matoxylin/eosin(H&E) and e xami ned for leukocyte infiltration As expected, leukocytic infiltration was ob serve d in the pancreatic islets of 8 wk old mice and increased in severity over time By 18 wks, few islets remained due to

PAGE 48

38 I .. . .. ,. Figure 1 Histological profile of tissues s l 1owing lyinphocytic infiltrates of the exocrine tissues and insu l itis in the NOD mouse T i ssue sections were stained with hematoxylin/eosin (A) 10 wk pancreas ; (B) 12 wk submandibu l ar g l and ; (C) 12 wk Jacrimal gland ; (D) 16 wk parotid gland

PAGE 49

M Q u SPLEEN 2 ------------t o-. CD4 !? y------t: r"I l!. 0 u . .... ,.,. .,. ,u CD8 1l '2> -. ~ oSUB ot CD 4 10 10' !>y-----------, ,.,.. 10 C D R LAC ~ ------:---1 -, 71, 1 .. . ~,,. . . ..,,,o~.-: . .. -~ -. ~ #:.:j:.. :., i_.-, r ., ,., .._. -4 1.,-r. .. ,-,. -of "'i \ I .: ; ~ :, ~.; :1~. '. .1 g ... ,, C' ... .. .J ... .. .._ ..... : .. t ~ .... .. . . CD4 o ,, --------------------~ \!, l :,. ~ .__c'~c ,. .,_ "' ,.".: ; .. J 3':' . 't > ,. .... --_/ .. ~ 1:>; .. . \ .,;a~ ,~r ,.,. I ..... >. ~l:'7 7: Ill' tO' rk.' -,-.. ...,* :/ V -' ": . ,n, C D 8 I PAR h .-----------0 ~ , b . . . . .. . . .. 0 . ,. I -~ ,;. ~ !. ,c,o ,.,. ... CD4 . ... ---------~ .... j :~ 11 > ~g t.. 1..z > !\ ): ... ..-n :.~~ ;. ... -tot.'... , t\,: o< CD8 Figure 2 Flow cytometric analysis of CD4 + and CD8 + T-cell populations in lymphoc ytic infiltrates in tissues from 12 wk NOD mice Spleen (SPL) : CD3 44%, CD4 27% CD8 11 % ; Submandibular gland (SMG): CD3 62% CD4 45% CD8 11 % ; Lacrimal gland (LAC) : CD3 47% CD4 26% CD8 10% ; Parotid gland (PAR) : CD3 3 1 % CD4 13% CD8 3% Result s are expressed as percentages of the total infiltrating populations w

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CD4 100 --------------------, 90 80 70 Cl) ::l 80 w 0 ,., 0 0 "" 0 40 '# SPL S M G O N G 0 0 N C G 0 N N CDS 50 -------45 30 ...J w 0 8 26 0 "" 0 20 ';I. 15 10 5 0 SPL S M G ., N~ ~ m_ 0 0 N G 0 N N AGE IN WEEKS LAC PAR LAC PAR 40 Figure 3 Histogram of the temporal expression of CD4 + and CD8 + T-cells in lympl1ocytic infiltrates of submandibular (SMG) lacrimal (LAC), parotid (PAR) glands and spleen (SPL) from NOD 1nice Data was obtained from flow cytometric analyses of infiltrating cells using FITC-conjugated CD3 and PE-conjugated CD4 or CD8 All values are expressed as a percent of CD3 + cells

PAGE 51

41 autoimn1t111e destruction At 8 wks of age the subn1andibular gla11ds of 2 of the S 1nice showed sn 1all focal areas of infiltrati o n wl1ile tl1e la cr in1al and parotid glands remained normal By 12 wks, the lacrimal glands sl 1owed infiltration but their glands had fewer ar1d sn1aller l ymphocyt ic foci than tl1e submandibular glands which were heavily irililtrated in all mice by 14 wks In contrast, the parotid glands s l1owed no focal ly1nphocytic infiltrati on up to 20 wks of age, although a few mononu c lear cells were occasionally seen in some n1ice from 14 to 20 wks of age Represe11tative l1i s tological profiles of glandular infiltrates are shown in Fig 1 Flow cytometr ic analysis of glandular infiltrating lyn1ph ocytes Monoclonal antibodies to cell s urfa c e molecules have been widely u se d as pl1 enotypic 1narkers corresponding to functionally distinct subsets of lymphocyte populati ons. We have used flow cytometric analysis to determine the cellular ph enotypes of lymphocytes infiltrating the pancreas lacrimal parotid and submandibular glands of the NOD mouse fron1 the first appearance of lyn1phocytes at 8 wks tl1rough 20 wks, wluch is 4 wk beyond the d etec tion of exocrine gla11d dysfunction Gates set on the NOD sp leen lymphocyte populations were used to select the infiltrating populations of the otl1er tissue san1ples. The percentage of the cell populations falling in tlus gated window remained fairly co11stant t lrroughout the tin1e course of tl1e study averaging approxitnately 73% for splee11, 47% for islets, 59% for submandibular glan d, 24% for lacrimal gland and 17% for parotid gland Within these gated populations the percentage of CD3 + cells also ren1ained relatively constant over the time course of the study at 48% for spleens, 41 o/o for islets,

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42 3 7% for lacri111al glands and 39o/o for parotid glar1ds Howevert in tl1e submandibl1lar gland, tl1ere was an in c rease in CD3 + cells from 41 o/ o at S wks to 70 3/ o at 2 0 wks Double labeling of tl1e infiltrating populations with CD3 / CD4 or CD3 / CD8 is shown in Fig 2 for the 12 wk ti1ne point Similar profiles were o btained for eacl1 ti111e period to determi11e the perce11t of CD4 or CDS c e lls (Fig 3) The CD4 + popt1lation in tl1e submandibular gland approximated that in the spleen : 68 o/ o and 62o/o respectively In the lacrimal gland, the CD4 + population was about half that of the spleen at 8 wks ( ~ 30 o/ o) and increased over time to the level of the spleen Tl1e parotid gland sl1owed a decr eas e fron1 84% CD4 + cells at 8 wks to ~ 50% at 20 wks However, tl1e low total number of lyn1phocytes dete c ted in tl1e parotid glands as well as their similarity to splenic profiles may be indicative of co11tamination by lymphatic vessels wl1 i ch are i11ter s persed throughout the gland Tl 1 ere was greater variability m the infiltrating CD8 + populations of the exocrine gla11d s than seen in CD4 + cells (Fig 3) Generally fewer than 1 Oo/o of the CD3 + population were CDS + in these glands at 8 wks -co11 s iderably lower than the 27% found in th e spleen As the disease progressed tl1e percentage o f CD8 + cells increased in all 3 glands, but it is unclear if tl1e apparent decrease at 20 wks i s significant As indicated by the spleen population, the ratio of peripheral CD4 + to CD8 + cells remained relatively constant at ~ 2 5 : 1. In the submandibular gland, the CD4 : CD8 ratio was 6 : 1 at 8 wks, and decreased to an average of 3 5 : l from 12 wks on Both the lacrimal and parotid glands also l1ad higher ratios in early infiltrates, but decreased to spleen levels at 1 2 wks. The CD4 + and CD8 + populations did not account for all of the CD3 + cells The CD3 + CD4 CD8population accounted for 5% of the spleen cells, 8% of the

PAGE 53

T,1b l c 2. Percentage of CD3 a11d B220 ce ll s in tl1e l yt1 1pho cytic irtfiltrat es o f s ubmandjbular (S MG ) a nd I ac rin 1a l (LAC) g land s of NOD 1 11i ce TISSUE ANTIBODY AGE IN WEEKS ga ) I oa > 1 2b) 14 a ) 1 6b) } 8b 1 2Qb ) SMG CD3 3 1 7 49.3 59 1 5 4 .0 60. 7 63.5 60.6 C D 3+ B22 0 + 0 5 0.6 3 4 0 6 8.4 3 9 6 9 CD3 + B 2203 1 .2 48 7 55 7 53 5 52 2 59 6 53 8 C D 3 -B 220 + 1 .3 1 4 6 I 3 9 1 2 3 1 5. 7 23 1 l 6 5 LAC CD3 ?1 _) 36 1 39 5 28 7 2 4 6 28 5 32.7 CD 3+ B 220 + 0 4 0 4 2 7 0 6 3 7 4 3 2.6 C D 3+B2202 4 7 35.7 36 8 28 2 21.0 9 3 13 4 C D 3-B220 + 0 9 4 4 33.5 3 5 40 .9 27 5 30.7 a) Flo,v cy t o 111 e tri c d ata vverc co ll ec t e d t 1 s in g FITCco njt1 gate d CD3 and PE co nju ga t e d B2 20 b) PEc onjug a ted CD 3 and FTT C-co nju ga ted 8220 ,, ere lt se d .... w

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T ~ tbl e 3. CD ..i 5 RB expressio 1 1 in ly n1pl1oc)rtic infillratir1 g ce ll s of s ubn1andibt1 l ar (SMG) a 1 1d l ac rirnal ( LAC) g l a n ds o f NOD 1 1lice TI SSUE ANT IBODY AGE IN WEEK S 8 10 12 14 1 6 18 S MG :i) C D 3 45 6 52 9 68 0 61.3 65.7 72 8 CD3+CD45RB/h + 3 l 13 5 14 7 1 5.0 18 9 23. 4 C D 3+c D 45 RB / I + 1 4 6 18 7 27 5 26 0 24 7 30.4 C D 3 CD45RB / l1 + 1 6 1 28 4 24 7 25 8 26 0 22 6 L AC 1 l CD3 ?9 . 6 43 2 47 9 38 1 30 3 49 3 C D 3 + C D4 5RB /1 1 + 1 5 7 2 12 0 5.6 l 0 0 l 0 6 C D 3 + CD4SRB / l + 9 9 1 6 6 1 6 9 21 3 8 6 2 1 6 C D 3 -CD4 5 RB/l1 + 6 7 18 8 41 6 1 7 I 50.2 1 2.2 a) Flo,v C y ton1elri c d a ta \ vc r e co ll ec t e d usi1 1 g FITC-conju ga t ed CD3 a 1 1 d P E-co nju ga t ed 2 0 67.3 1 7 2 27.8 23 6 43 8 7 2 1 9.4 1 8.7

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30 r------------------(") 0 0 2 5 20 LL 15 0 ,ft. 10 5 0 3 6 8 9 11 17 TCRV BETA 45 Figure 4 Histo g ran1 of selective TCR VP distribution in infiltrating lymphocytes isolated from spleen pancreatic islet, subn1andibu l ar, and l acrimal g l ands of NOD mice Data frotn flow cytometric analy s es of 8 through 20 wk sru11p les were averaged for each tissue. Spleen-solid bar ; Pancreatic islet-l1orizontal striped bar Submandibular gland-clear bar ; and Lacrimal gland-diagona l striped bar Data are preset1ted as percentage of CD3 + cells SE of the n1ean

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46 sub111andibular cells, l 5% of tl1e lacrimal cells and 9% of tl1e parotid cells Tl1is indicates a11 i11crease in the double negative population in tl1e exocrine tissues over that of tl1e peri p l1ery The infiltrating populations of the stibmandibular and lacrimal glands were further ct1aracterized using a11tibodies against tl1e B-cell rnarker 8220 (63) and tl1e activation/n1emory marker CD45RB (123) As shown in Table 2, the CDJ-B220 + 8-cell population in the submandibular gland i11creased fron1 ~ 1 o/o at 8 wks to ~ 15% at IO wks and maintai11ed that level througl1out the rest of tl1e s tudy In the lacrimal gland, co3 8220 + cells al s o started at 1 % at 8 wks and increased to ~ 33% at 12 weeks. Whetl1er the fluctuation seen between 10 and 14 weeks is n1eaningful is unknown at this time In addition, the presence of a CD3 4B220+ cell population was detected in both of these tissues This phenotype did not appear in significant numbers until 16 wks of age in eitl1er gland and never exceeded 10% in the subma11dibular gland or 5% i11 the lacrimal gland The CD3 ~220 + phenotype has been reported as a double negativ e population in MRL / lpr 1nice (63) and as a lymphokine activated killer phenotype (LAK cell) in other studies (124) CD45RB staining presented a much n1ore con1plex picture (Table 3) In submandibular glands at 8 wks, ~ 3% of the CD3 + cells were CD45RBhi + (naive T-celJ) and from 10 wk on, this CD45RBru + population remained relatively stable at 16% The percentage ofCD3 + CD45RB 10 4 (memory T-cells) population was 15/o at 8 wks, showed a 1.8-fold increase to 27% at 12 wks and remained at that level through 20 wks. The CD3CD45RB1" + population in the subma11dibular gland remained constant at ~ 2So/o from 10 20 wks In the lacrimal gland, the picture was less clear due to a smaller sample size

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47 (Table 3) In ge11eral, tl1e CD45RBhi + populatio11 (botl1 CD3 and CD3") i11creased to a 111axirnu1n of ~ 55o/o at 12 wks plateaued and then decreased at 18 and 20 wks to ~ 24% Tl1e CDJ + CD45RB 10 + population remruned fairly constru1t at ~ 200/o from 10 wks on Analysis of TCR VP usage was done using the monoclonal antibodies listed in the Materials sectior1 It has been previously reported tl1at the majority of tt1e infiltrating populations of the submandibular and lacrimal glands are TCRa.P (64) 1n the vps tested, tl1ere was no appreciable time-rel a ted variation in response, therefore, the data from all time points we re averaged for VP distribution (Fig 4) The sub1na11dibular a11d lacrimal glands showed a similar distribution patter11 to that seen in tl1e splee11 Botl1 VP6 at1d vps were sig1uficantly increased over background as represented by VP3, wbjle, to a lesser extent, VP9 and V~ 17 were also increased These increases were 111uch more drat11atic in tl1e sub1nandibular gland tha11 in the lacrimal gland whicl1 paralleled tl1e response in the periphery as represented by the spleen Cytokine mRNA expression in salivary and lacrimal tissues Temporal expression of mRNA tra11scripts for selected cytolcines expressed within tl1e infiltrates of lacrin1al and submandibular tissues from each experimental age group was determined through the use of semi-quantitative RT-PCR PCR bands were quantified using de11sitometry and compared against PCR bands of G3PDH to provide a measurement of ten1poral changes in the production of mRNA transcripts a11d provide a detailed analysis of cytokine expression throughout the progression of the autoimmune activity. As presented in Fig 5, mRNA transcripts for a number of interleukins were detected generally at both an earlier age and with greater intensity in the lacrimal glat1ds

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48 LA C RIMAL SUB MAN DIBULAR "'JOO C lOOOO :,0000 IL 1 P I 10000 20000 ,... ICOOI) I 1:! I 0 10 ti ,. ,, 16 10 11 1 16 16 4 0EI W-. J A OE W .... I 20000 1COOO 1 ,_, _, IL-2 1 100)0 10000 :I ,000 ,000 1:! ~ .... 10 ll .. ,. "' in ., .. 11 , .t.0" CW tillQ I AGEi i 20000 20000 0 z I"""" """" Z 10000 DOM C: :I J; ,000 ""' .. "' 0 1 0 .. ,, ,, /0 10 ,, ,. .. ,0 4 QEf W MUl t AG< ) >OOOl1 ~Of...o Q 1 ,_, ',000 IL-5 z ,0000 '~ \. C: .. ~! ,000 1:! 0 '" ,; .. ,. 10 ,, ,. ,, "' aErw .-. 1 ~ w-.u. 1 0 0000 JOOO >0000 J UOOI'., IL-6 0000 ,!~ >0000 :\ 10000 I"""' I 1:! ,, u ,, ,. , .'C ,, 1 ,, A Q[ fW .. h J A OE ( W..-.. 1 0 '""' t JOOOO >)( Z j i \000) .. "' 0 ., " ., ,. 10 AOf ( W.. ll a l AG E CW'"-" 1 Figure 5 Interleukin mRNA expression of l acrimal determi11ed by RT-PCR and Southern blotting g land s as were scanned and a 11 a l yzed by densito1netric comparison u s ing G3PDH as a s tandard Values were plotted by arbitrary Blot s and sub mandibular poJynotJtial or exponential trend)ine density units and ana l yzed u s in g a best-fit

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49 LA C RIMAL SUBMANOIBULAR 0 4 0000 4 0000 z )0000 30000 IFN -y ill ;? z 20000 20000 3 10000 1 0000 .., "' 0 0 8 10 12 .. 16 a 20 8 1 0 12 1 4 16 18 20 A GE (WJ II GE( W1 40000 40000 0 z 30000 < 30000 TNF -a (I) 20000 20000 j::: !. 1 0000 '" 1 0000 .., "' o 0 8 C 12 \4 16 lb ~o ij 10 1: 1 16 18 20 A GE ( WNk9 1 AGE ( W M I 20000 20000 0 z 1!>000 ,sooo i NOS (I) !;:: i 10000 10000 3 5000 ~000 .., "' 0 . B 10 17 ,. 16 ,~ 20 ij 10 1.' ,. 16 18 ~o AGE (W N k s l 4 G E ( W ee u 1 Figure 6 Proi11tlammatory nlRNA expression of l acri111a l and submandibular gla11ds as determined by RT-PCR and Southern b l otting B l ots were s crumed a11d analyzed by densitometric comparison using G3PDH as a standard Values were p l otted by arbitrary density units and analyzed using a best-fit polynomial or exponential trendline.

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50 tl1ar1 submar1dibt1lar glands In tl1e lacri1nal ti ss ue, IL IP, IL-5, JL-6, IL7, and ILI 0 111RNA tran sc ripts were detected as ear l y as tl1e 8 wk as well as at 1n os t later tim e poi11ts In the sub mandibular gla11d i11filtrates, detectable int er l euki n nlRNA transcripts were usually not observed before 14 wk of age Of note, mRNA tr a nscripts for IL 4 were absent fron1 Iacrimal glands, wl1ile IL-4 and IL-5 were absent from tl1e s ubmandibular glands Both interleukin tran sc ri p ts were detected in control n~A obtained from Corl.A st imulated NOD splenocytes, while IL-4 is cotnmonly seen in islet infiltr ates ( 122) mRNA tr anscri pts for tl1ree effector cytokines, IFNy, TNFa. and iNOS were detected in botl1 lacrin1al and s ubn1andibular gland infiltrates of 111i ce aged 8 a11d 12 wks, respectively (Fig 6). All tl1ree cytokines exlubited in c re ased levels of nlRNA tra11 sc ription through 16-18 wks. Cytokine mRNA trans cr ipt s were rarely detected in con trol parotid ti ssues Considered i11 toto the se results indicate tl1at major temporal c l1an ges in the cytoki ne profiles occur in tl1e la crima l a11d submandibu lar gla 1 1ds of NOD nuce between 12 and 16 wks of age. In the case of the subma 11 d ibular gla1 1d s, thi s is several weeks following tl1e first appearance of lympl1oc y tic infiltratio11 Di sc u ssio n Ov e rt di abe tes in our NOD mouse colony generally begins at 12 wks of age, while the first appea r a nce of focal lymphocytic l esio n s it1 the subn1an dibular and la c rimal glands begins at 8-10 wks and 10-12 wks of age r espec tively Despite the early appearance of lymphocytic infiltrates within the s ubmandibular and lacrimal glru1ds, loss of saliva flow and tear production does not occur until 1416 wks of age (71 ) For thls reason, the

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51 argu111e11t has been advanced that the autoimmune attack agai11st the sa livary and lacr ima l ti ss u es i s n1 e rel y se condary to the lo ss of immun o l og i ca l t o l era nce for a pancreatic p ce ll antigen al so expressed o n the salivary and lacrimal g la11ds A seco nd argument s ug ges ts that tl1e decreased sa liva and tear flow is a conseque11ce of the loss of blood g luco se re g ulation Several r ece nt studies have raised serious que s tio11s with both arguments First, Leiter et al (IOI) have reported that de ve lop1n e nt of in1munological tol era nce a ga in s t the p cell f o llowing intratl1ymic inj ectio n of i s let ce ll l1 omogenates into n eo natal NOD n1 i ce prevented the d e velopment of diab etes, but not the autoimn1une a tt ac k against the sa livary g)ands Second Gar c hon et al (81) l1ave linked s ial oa denitis h y per-IgG pro d uction and peri-i11sulitis to a tel ome tric l oc us but insulitis and diabetes to a ce ntrorneric l oc us on chromosome I Third, I ha ve ob s erved (unpubli s hed data) tl1at lo ss of sec retory function in tl1 e salivary g land s pre ceeds tl1 e onse t of chan g es in blood g lu cos e lev e ls and di abetes in N OR/cl1r 9 nuce a s trai11 w hi c h ha s d e la ye d 011 se t diabetes (85). The s e st udies i ndi cate diabetes and sa li vary/lacrin 1al g l an d complicati o n s in NOD mice are most l ik el y di s tinct ent 1t1es ln the pre se nt study, I h ave atten1pted to defme the t em poral development of the aut oimm une attack agai11st the s alivary and la c rimal g lands of N OD mice A unique feature of thi s pathogenesis and com p arab le to Sjo gre n s Syndrome in humans is the loss of sec retory function resulting in c linical pre se ntations of xe ro s t omi a and xerophthalnlia (71 11) As stic h, I believe the s e d a ta will allow for a com p ar i so n of the autoimmune proc esses, e g lymphocyte ph enotype s activation states and cyto k:ine profiles within

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52 several organs of the san1e host as well as a comparison between tl1is ani1nal n1odel and Sjo g ren' s Syndrome Analyses of tl1e ly1npl1ocytic i11fil t rates of tl1e salivary and lacrimal glands uncovered a relatively l1igh percentage of CD4 .. T-cells, as might be expected from previous studies on the islet infiltrating lymphocyte populations (68) In contrast 011ly 10% of the T-cells detected were CD8+, and even fewer were CD3 + /CD4"CD8double negative T-cells. While generally accepted that CD4 + T-lymphocytes are necessary for the in1111unopatl1ogenesis of diabetes in the NOD mouse, the role of the CDS+ population, particularly in the initiation of the disease, has remained n1ore elusive (92) T-cell transfer studies in NOD mice l1ave c o nfim1ed the necessity of CD4 + I-cell populations to transfer autoi1nmunit y, wl1ereas neither purified CD8 + T nor B-cells were capable (85) It is becoming increasingly apparent that the activati o n state of transferred lymphocyte populations is critical Usi11g the CD45RB as a marker of lymphocyte activation, I observed that the majority of T-cells present in the infiltrates of both submandibular and lacrirnaJ glands were either of a n1emory (CD45RB 10 ) phenotype or a pl1e11otype (CD45RB 11 j) suggesting transition to an activated stage The presence of a distinct CD45RBru population throughout the time course of this study suggests that a population of naive T-cel]s are being actively recruited to tl1e salivary and lacrima] glands Interestingly a recent study has shown that the ability to tra11sf er diabetes in NOD mice is associated with a CD45RB 10 memory T-cell population and a concomitant increase in the IFNy to IL-4 ratio (87) I-cells that produce IL-4 have consistently failed to transfer disease Markedly increased levels of IFNy mRNA transcripts were observed in both the

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53 sub111a11dibular and lacri111al glands possibly i11dicati11g a switcl1 in tl1e cytokine productio11 by activated CD4SRB 1 0 T-cells In addition to exploring the lympl1ocytic activation states, I l1ave also investigated selected VP TCR ph e notypes of the infiltrating cells Wl1ile most Vj3 TCR p o pulations were detected in the salivary and lacrirnal gland infiltrates (as expected in the NOD n1ouse), an increased percentage of Vj36 ar1d Vj38 expression was present, consistent witl1 previous observatior1s of preferential TCR usage observed im111unohistocl1enucally (89) The importance of Vj38 and Vj36 lympl1ocytes in tl1e development of autoi1n1nunity i11 botl1 NOD and MRL/lpr mice l1as been demonstrated through tl1e use of both T-cell transfer and specific anti-VP therapy (64,65, 125) Differences in the numbers of B ly1nphocytes infiltrating tl1e salivary and lacrin1al glands were also ob s erved : approximately 15% in the submandibular gland, but 33% in the lacrir11al tissue Whether the in c reased percenta ge of B lymphocytes witlu11 the lacrimal glands is due to increased B-cell proliferation or active recruitment remains unknown However, detection of CD23 (blast-2 antigen) in the lacrimal infiltrates of Sjogren's Syndrome patients support the po ss ibility of ly1nphoproliferation (34) In this study, I have also documented several age-dependent increases in cytokine nlRNA expression in the salivary and lacrimal glands. Of particular interest is the increased expression of IFNy, TNFa., and iNOS in both salivary and lacrima.l tissues starting at 12-14 wks of age and increasing to maximally detected levels by 20 wks of age By 16 wks, high levels of IL-10 rnRN A production was detected as well. The dramatic rise of each of these transcripts coincides with the tin1e of the first evidence of the loss of saliva flow as reported in previous studies (67) Whether these cytokines are involved in

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54 etTectir1g loss of exocrine function directly via a cytotoxic pathway or their pre s ence is necessary to il1itiate lyn1pl1ocyte-n 1ediated cell killing is not yet known Many recent studies have focused on the potential roles of cytolcines as cytotoxic effe c tors of exocrine tissue destruction Evidence indicates that lL-1 IFNy, TNFa. and 11itric oxide n1ay be key mediators in the patl1ogeni c proce s s of islet cell destruction in botl1 hun1ans and NOD mice (126-129) Of particular interest production of high levels of lFNy by islet il1filtrating cells has been repeatedly de111onstrated in NOD mice and appears tigl1tly linked to P cell de s truction (127 130) Recent studies by Mathis et al de1nonstrated that an islet-specific Th 1 T-cell c lone producing higl1 levels of IFNy is able to rapidly transfer diabetes to young NOD recipients (88) However, when shifted in vitro to at1 IL-4 producing pl1e11otype, the sa1ne T-cell clo11e was unable to transfer diabetes Furthermore IFNy has been noted to induce cell death of both islet and sali v ary gland cells in vitro and may repre s ent a nor1s pecif1c mediator of ex oc rine cell de s truction in vivo (45) However, a caveat to this argument is the detection of hlgh levels of IFNy and TNFa. in the salivary glands of MRL/lpr mice whlch do not lose secretory function (66 54) U11expectedly, cytokine tnRNA expression in the lacrimal glands typically appeared at an earlier age and in larger quantities tl1an in the subn1andibular gland This occurred despite the fact tl1at both fewer and smaller f ocaJ lymphocytic le s ions were present in the lacrimal glands in tl1e earlier age groups thus suggesting that lymphocytes infiltrating tl1e lacrimal tissue are activated at an earlier stage than those in the submandibular gland Alternatively, a lag time between the detection of focal lymphocytic lesions in the submandibular gland (8-10 wks) and the detection of increased cytokine mRNA

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55 expression ( 12-16 wks) may indicate tl1at the submar1dibular gland-infiltrates remair1 f unctior1ally quie sc e11t and require a signal ot' activation In cor1 c lu s ion this data demonstrate nu111erous s imil a rities in botl1 lympl1ocyte phe11otypes (CD4 / CD8 ratios, VP TCR restriction, and B220 populations) and cytokine expres s ion (i11crea s ed IL-1 p, IL-2 IL-10, TNFa ar1d lFNy) b e tween the NOD mouse and other animal models for autoimmune sialoadenitis as well as Sjogren's Syndrome Although the initiating agent for Sjogren's Syndrome ren1ains unknown, it is often believed that extrinsic factors, i e ~ viral agents may be responsible for the breakdown of tolerance in immunologically susceptible individuals ln NOD n1ice, l1owever, the necessary intrin s ic elements for the breakdown of salivary gland tolerance exist in their genetic background Using tl1e NOD-scid mice I have recently described 1nultiple salivary gland ab11ormalities of NOD-scid mice that do not appear to be immunologically related ( 121 ) These alterations are detectable starting at 8-10 wks of age and include n1orphological a b normalities aberrant gene expre s sion, and increa s ed proteolytic acti vit y Wl1ile any number of immunological cl1 a nges can re s ult in aggregation of lymphocytes i11 e x ocrine tissues such as the lpr / gld mutation of MRL nu c e, graft vs host models ( 131 ), TGF-P knockouts (6 I) and bcl-2 overexpression (18) only with the appearance of developmental defects or extrinsic destruction of the exocrine tissue leads to the development of secretory dysfunction through loss of imn1une regulation

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CHAPTER3 GENETICALLY PROGRAM1v1ED DEVELOPMENT OF SALIVARY GLAND ABNORMALITIES TN THE NOD (NON-OBESE DIABETIC)-SC/D MOUSE IN THE ABSENCE OF DETECT ABLE L YMPHOCYTIC INFILTRATION : A POTENTIAL TRJGGER FOR SIALOADENlTIS OF NOD MICE Introduction Tl1e recent development of the NOD-scid congenic strain provides a unique 1nodel to investigate tl1e role of the immune response in tl1e pathoge11esis of Sjogren's Syndrome (82) The NOD-scid mouse is homozygous at the scid (severe combined inununodeficiency) locus and thus lacks functio11al T and B lympl1ocytes. The scid n1utation prevents the spontaneous deve)opn1ent of sialoadenitis, insulitis, and diabetes in tl1ese mice; however, the transfer of T lymphocytes from diabetic NOD mice to recipient NOD-scid mice can restore an a utoimmune phenotype (85) In addition, because of its NOD background, tl1e NOD-scid has i1npaired NK (natural killer) cell and reduced complement activity (82). Since NOD-scid mice share the same NOD genetic background, this model is ideal for studying the non-immu11e genetic factors that contribute to the developn1ent of autoimmunity. Previously, the dramatic changes in exocrine gland histology, protein sy11thesis, and secretory dysfunction are tl1ought to be a direct result of the autoimmune lyn1phocytic component (67). By investigating exocrine g]and function in the absence of functional lymphocytes, this specific aim demonstrates that temporal changes in salivary gland function occur in the absence of overt 56

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57 autoir11111unity Tl1is indicates that tl1e re s ulting in1111une response 111ay actually be trig g ered by aberrar1t phy s i o l o gical cha11ges in ex o crine gland homeo s tasis and fun c tion Material s and Metl1ods Animals BALB / cJ, C3H CBA, and NOD/Uf mice were bred a11d n1aintained in tl1e Departn1ent of Pathology's mou s e facility (University of Florida, Gai11esville FL) NOD-scid mice were bred and hou s ed in the Depa.rtme11t of Pathology's transgenic mouse breeding colony Both male and female n1ice ranging in age fro1n 3 to 30 weeks were u sed Maintenance of the s c id n1utation was a ss e s sed in e x peru11ental a11imals by flow cyton1etry of spleen cells and RT-PCR analysis of CD4 CD8, and TCR V~ repertoire fron1 isolated s ubmandibular gland total RNA NOD 1njce were routinely tested for blood glucose levels using Ch e m s trip bG rea g ent strip s (Boehringer Mannheim Indianapolis, IN) Consecutive elevated fasting blood glucose l e vels > 240 mg/dl were considered onset of diabetes, after which time, tl1e mice were mait1 tai ned on daily insulin injections Saliva Collection and Flow Rate Saliva was collected from control and experimental groups of male and female mice following stimulation of secretion using isoproterenol (0 20 n1g/lOO g body weigl1t) and pilocarpine (0 05 mg/100 g body weight) (Sign1a Chemicals St Louis, ~O) djssolved in saline The secretogogue cocktail was injected (0 1 ml volume) intraperitoneally and saliva was collected, starting I min post-injection, for l O min from the oral cavity by

PAGE 68

58 micropipette and placed into cl1illed 1 5 n1I microcer1trifuge tubes (67) Volume was determined by n1easurement with 200 I n1icropipettes Saliva san1ples were collected frorn groups of 7 n1ale or 5 fen1ale mice tt1e11 frozen at 70 C until ar1aly z ed for te1nporal protein changes by e11zyme assay, radio-receptor assay SOS polyacrylan1ide gels and Western blotting Total Salivary Protein and a-Amylase Analysis Saliva s a n1ples were analyzed for total protein u s i11g bovine serum albumjn as the s tandard (132) Amylase was determined by its ability to hydrolyze starch according to published protocols (133) In brief 500-1000 fold dilutions of saliva in phosphate-buffered saline (PBS) were added to a solution containing 0 4 g soluble starch in 60 mM tris(hydroxymethyl)a111ino1nethane (Tris)-HCI, 0 1 S M NaCl and 3 mM CaCl 2. Tl1e reaction was stopped after 5 or 10 min by the addition of0 045% 1 2 0 045 % KI and 0 03 N HCl Absorbance was mea s ured at wavelength 620 run One unit of amylase was defined as tl1e an1ount tl1at hydroly z ed l n1g starcllf 1nin/mg protein at 3 7 C EGF Analysis Salivary epidermal growth factor (EGF) was e s tin1ated by the procedure of Bootl1 et al (134) Saliva was diluted 10 fold in PBS containing 0 2 n1g/ml bovine serum albumin (BSA) Reactions consisting of 100 l diluted saliva, l 00 human place11tal microvilli 1nembranes a11d I 00 I 1251_1abeled human EGF were incubated for 24 hr at 4C (67) Each reaction was then diluted with 3 5 ml ice-cold 0 1% BSA in PBS solution, centrifuged for 20 min at 7,000 g in an RC-3B SorvaU centrifuge and the quantity of

PAGE 69

59 radiolabel associated with the membrar1e pellet deter111ined u s ing a Beck111an gan1n1a cour1ter Tl1e cor1ce11tration of EGF was con1pared to a star1dard curve generated witl1 dilutions of k11own quantities of human re c on1binant EGF Merr1brane binding con1petition is i11depende11t of species origin for the source of EGF (135) Isolation of Salivary Gla11d Ti ss ues Parotid a11d subma11dibular glands were excised from mice killed by cervical dislocation Each gland was freed of cormective tis s ue fat and any lymph nodes, tl1en l1omo g enized in 10 mM Tris buffer (Ph 7 4) containjng 100 M phenyl1nethyl s ulfonyl fluoride, 1 M leupeptin, and I 0 0 M benzamidine The slurry was tl1en centrifuged at 100,000 g for 30 1nin to reco v er total membrane (67) Tl1e supernatant was saved and frozen until analysis for a.-amylase activity Polyacryla11lide gel electrophore s i s and Western bl o t analysis Total saliva proteins (5 g of total protein or 5 l of total saliva volume) were subjected to ele c tropl1oretic separation on a 1 5 nun tluck I O o/ o SDS-polyacrylarrlide gel (12% SDS for Western blots) using a modified Tris-Glycine system of Pugsley and Schnaitrnan (136) Western bl o ts of gland lysates contained 20 g protein per lane Gels were fixed and stained using Coomassie Brilliat1t Blue R-250 or transferred to Immobilon-P n1embranes (Millipore B os ton MA) for 2 hr at 70v for We s tern blotting (137,138) The blocking buffer con s isted of 3o/o nonfat dry milk and 3% BSA in Tris-buffered saline Polyclonal rabbit anti-mouse parotid secretory protein (mPSP) IgG antibody ( 139), kindly provided by Dr William Ball (Dept of Anatomy, Howard

PAGE 70

60 U11iversity) or polyclonal rabbit a11ti-rat proli11e rich protein ( 140), kindly provided by Dr David Ca s tle (Dept of Anatomy and Cell Biology Un ive r si ty of Virginia), was in c ubated with each n1embrane for 2 l1r at 25 C. Followir1g three IO min washes the 111embra11es were incubated in alkaline phosphatase conjugated goat anti-rabbit immunoglobulin (Sigma Chen1ical Co ) and exposed to sub s trate a s previou s ly described (95) Protei11 Sequencing Salivary proteins we re sub jected to electrophoresis on l O o/ o SDS-polyacrylamide gels as described above and transferred to Imn1obilon-P 111embra11es Selected protein bands were carefully cut from tl1e membranes and subjected to N-ter1ninal seq uencing using Applied Bio sys tems Model 470A Gas Pha se Protein Sequencer with Model 120A on-line PTH analyzer (University of Florida ICBR Prot ei n Sequencing Core Laboratory) Pr o tein seque nces for tl1e first 12 amino acids of ea c h protein were entered i11to a protein database fo r comparison with known pr otei n seque nces Histology Freshly excised submandibular glands were pla ce d immediately into I 0% PBSbuffered f or1naldehyde (pH 7 2) Each tissue was en1bedded in paraffin, sectioned, then stained with hematoxylin/eosin dyes (UF Diagnostics Referral Laboratory, Gainesville, FL) Tl1e stained sections were viewed u sing light microscopy

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61 Statistical Analyses All ,neasures of varia11ce are given as sta11dard deviations of the mean Tests of significance for differe11ces between indep e1 1dent n1eans were perfor111ed with the unpaired Student I-test Results i11 which p < 0 05 were co11sidered significa nt R es ults Analysis of NOD-scid Saliva To detertnine the impact o n salivary fu11 ction of the scid n1utatio n in the NOD m o use, I analyzed whole sa liva samples from indi vi dual NOD-scid nuce for total volume, total protein, amylase activity and EGF concentration Saliva from 10-12 wk old NOD-scid mice, an age at which sa livary g l ands in N OD nlice are de voi d of detectable l ym ph ocytic infiltrati on, was compared to that of >20 wk old NOD-s c id mice, an age wl1en l ymp h ocyt ic infiltration is present in th e sa li vary g lands of N OD mice (Table 4) While there was no significa nt age or sex diffe re n ces observed for salivary flow rates (P > 0 05) significant sex -rel ate d differen ces were observed for protein content (P <0 05) Saliva from fen1ale N ODsci d n1ice contained approximately 40 o/ o less total protein than that of tnale mice in both experimental groups Saliva fr o m NOD-scid mice were analy ze d for two proteins, EGF ( a product of sub1 11andibular gland duct al cells) and an1ylase ( a product of parotid gland acinar cells), non11ally secreted in high quantities. A s presented in Table 4, the quantity of EG F in the saliva from > 20 wk old male NOD-scid mice showed a 21 % decline compared to the amount present in 10 12 wk old male mice (P < 0 002) A comparison of amylase activity

PAGE 72

Age of Mice 10-12 weeks ( n=l2 ) Mal e ( n=7 ) Female ( n=5 ) >20 week s ( n=12 ) Male ( n=8 ) Female ( n=4) Total Salivary Volume ( / 10 min) 271 b 240 98 329 t o 321 345Q d t o a n = 6 mal es and n = 6 f e male s f or EGF d e t e rmination s bVal u es are given as th e mean : s t andard deviation e nd not determined Table 4 Analysis of NOD-scid Saliva Tota l Protein ( mg / ml ) 1 9 7 2.3.1 1 8 I 5 1 .9 7 e, t 0 2.3 .8 l.ll.4 *** EGP ( ng / ml ) nd 563 nd nd 443 ** nd Amylase Activity ( mg s tarch/min/ml ) 1140 1099 1181 d 635 *** 5 90 162 680 d t 0 dSta t isti c al co mpari so n o f female to mal e val u es by the u npaired Student t test : (*P <0 05, **P< 0.002, ***P < 0 0002 t OP = NS). tati s ti ca l compariso n of> 20 week NOD-sc id value s to 10to 12 week val u es bythe unpaired Stude nt t te s t ( P <0.05 **P < 0.00 2 ***p < 0.000 2 P = N S ). N

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a ... ct,~ 1 oo > "tr,,. a Parotid 0 10 wk NOD-scid 0 20 wk NOD-scid 20 wk BALB/c Submandibular 63 Figure 7 Cytop l asrnic amylase activity of parotid and subn1andibu l ar glands Homogenates of parotid and submandibu l ar g l ands were centrifuged at 1 00,000 x g Lysates prepa r ed fro1n the gla11ds of 10-12 wk old NOD-scid ( open bars) > 20 wk o l d NOD scid (hatcl1ed bars) and 20 wk o l d BALB / cJ 1 11ice (solid bars) were tested for amy l ase activity in a starch hydro l ys i s assay. Va lu es are expressed as means of 4 experimenta l animals performed in triplicate the standard deviations

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64 96 68 AMY 45 32 24 STD 6wks 10wks 15 wks 20wks Figure 8 Temporal changes in the protein profiles of saliva from NOD scid mice Saliva was collected from 6 wk old NOD-scid (Lanes 1-3 ) l O wk old NOD-scid (Lanes 4 and 5), 15 wk old NOD-scid (Lanes 6 and 7) a11d 20 wk o ld NOD-.s cid mice (Lanes 8 and 9) foUowing isoproterenol/pilocarpine stimulation Salivary protein (5 g protein in 35 I SDS-PAGE sample buffer containing P-mercaptoethanol) was loaded on each lane and separated by electropl1oresis througl1 a I 0/o polyacrylamide ge l The gel was stained with Coomassie Brilliant Blue R-250 Prestained molecular weight markers (STD) were : 96 kDa Pl1osphorylase B ; 68 k.Da~ bovine serum a lbumin ; 45 kDa ovalbumin ; 32 kDa, carbonic anhydrase; 24 kDa soybean trypsin i11l1ibitor

PAGE 75

mPSP 32 k D 20 kD 2 7 k D -2 0 +1 +27 MF QLGS LVVL CGL LI G N S ESLLGELG S AVNNLKILNPPSEAVPQNLNLDVELLQQATSWPL ...... LLGELG S AVNNL ...... LL GELGSAV NNL ....... NLDVEL LQQA T S ...... Figure 9 N-terminal amino acid residue sequences of the 32 k.Da 27 kDa and 20 kDa protein bands ( shown on Fig. 8) and their alignment with the published sequence of murine parotid secretory protein (PSP) C\ Ul

PAGE 76

66 in the saliva of o ld versus young 1naJe and female NOD. ~ l i,I mice (Table 4) also revealed significant de c r eases over ti1ne Again n o sex -related diffe re nces i11 amylase activity were observed, witl1 botl1 1nale and female N ODsc id mice st1owing approximately 50% declines in activity between the two a g e groups (P < 0 0002) Analysis of Cytoplasmic Alnylase The temporal decline in a111ylase activity present in saliva of NOD-scid mice was also reflected in the amyla s e activity present in parotid and subn1andibular gland lysates (Fig 7) Cyt o pl as mic fractions frotn par o tid gla11d ly sa tes s howed a 50% reduction in amyla se activity in the > 20 wk old N ODsc id mice wl1en compared to those of I 0-12 wk NOD-s c id mice and a 90o/o reduction when con1pared to levels obtained from parotid gland lysates of BALB / c mice As expected submandibular gland lysates exhibited low lev e ls of amyla se ac tivity in all e x perimental g roups Tetnporal Changes in NOD-scid Salivary Proteins by SOS PAGE To evaluate te1nporal cl1anges of salivary prote i ns in N ODsc id mice we compared electrophoretic separations in SDS-polyacryla111ide gels of three individual saliva samples collected from 6 wk old female NOD-scid mice plus two samples collected from each of IO wk 15 wk and 20 wk old fen1ale animals (Fig 8) Although the total protein concentration in each s aliva sample is con s tant the gel profil es revealed substantial differences in protein composition between animals of the various age groups. These protein profiles were similar whether con s tant protein (5 g) or constant volumes (5 l) were applied to tl1e SDS-PAGE The temporal decline (then precipitous disappearance

PAGE 77

( ,7 from tl 1 e saliva sa111ples of 20 wk old NOD. 'i<. : id n1ice) of tl1e protein observed at approximately 55 kDa, s l1own to be an1yla s e ( I 05), is consistent with reported changes observed fo r sa livary amyla se in NOD 111i c e when analyzed on SOS-PAGE gels Two additional protein bands one at 32 kDa and one at 20 kDa (i11dicated by arrows in Fig 8) pre se 11t in relatively high amou11ts in s aliva of NOD-scid 1nice of l O wks of ag e or less, virtually disappear from saliva of NOD-scid mice 15 wks of age a11d older Interestingly, the decline of these two proteins appeared to correlate with the emer g ence of a third protein band at appro xi 111ately 27 kDa (indicated by an as teri s k in Fig 8) By 20 wks of age, this 27 kDa protein band appears to be 011e o f the n1o s t abunda11t proteins in the salivary samples. Sequence Homology of the 20 kDa, 27 k.Da and 32 kDa Protein s with Parotid Se cr etory Protein N-terminal anuno acid sequence anal ys e s of the 20 k.Da 27 k.Da and 32 kDa protein bands indicated that all three were homologou s to parotid secretory protein (PSP) (Fig 9) Interestingly, the N-terminal amino acid residues of both the 32 kDa and 20 kDa proteins, the two i so for1ns observed prior to the time NOD nlice present with sialoadenitis were not only identical to each other but were also identical to the published murine PSP sequence beginnit1g at the signal cleava g e site of the s e cretory forn1 (108 ~ 141142) In c o ntrast, the N-tem1inaJ sequence of the 27 kDa protein, appearing at a time NOD mice begin exhibiting s ialoadenitis, matched an internal portion of the PSP s tarting at the 27th amino acid after the signal cleavage site The sec retory fo1 rn of PSP l1as been

PAGE 78

68 reported to be a 20 k.Da, leucine-rich glycoprotein tl1at is abundant in the secretory fluids of tl1e parotid gla11d ( 105) Antigenic Cross-reactivity of Salivary PSP from NOD-scid, NOD and BALB/c Mice To confirm the ide11tity of the 20 kDa, 27 kDa and 32 kDa protein bands as PSP, Western blots of SOS-PAGE separated saliva frotn 10 wk old NOD-scid, 20 wk old NOD-scid, diabetic NOD and normal BALB / c mice were treated with anti-PSP polyclonal antibody As sl1own in Fig 10, anti-PSP antibody detect e d each of the tl1ree isoforms Saliva from BALB/c and 10 wk old NOD-scid n1ice contai11ed predorninately the 20 and 3 2 k.Da isoforms, while saliva from diabetic NOD contained the 27 and 20 k.Da isoforms and lacked the 32 kDa isoform Saliva from 20 wk old NOD-scid 1nice contained each of tl1e isoforms, but had greatly reduced levels of both tl1e 20 and 32 kDa isoforms compared to the BALB / c and 10 wk NOD-scid nuce PSP Detected in tl1e Cytoplasmic Fractions of Salivary Glands of NOD-scid Mice Cytoplasmic lysates of the submandibular and parotid gla11ds were analyzed on Western blots for the presence of PSP using anti-PSP antibody (Fig 11 ). Lysates of submandibular glands from 10 wk old NOD-scid mice revealed the presence of all three PSP isofor111s, and each isoform increased in quantity by 20 wks of age (Lane 2 versus Lane 3). No PSP was pre s ent in the lysates ofBALB / c submandibular glands {Lane I). Lysates of parotid glands from 10 wk old NOD-scid mice contained the 3~ kDa protein band plus a con1paratively low level of the 20 kDa protein (lane 5). In contrast, by 26 wks of age, parotid gland lysates from NOD-scid mice exhibited all three isoforms

PAGE 79

69 3 3 3 0 0 N 0 N 'C 'C 3 0 (.) (.) (.) N ...... fl) fl) al I I C'0 C .J C C C 0 < 0 0 z al z z 45 32 24 Figure 10 Identification of parotid secretory protei 1 1 in saliva using polyclonal a11ti-PSP antibody on Western blots Protein (5 g/lane) in whole saliva from 20 wk o l d diabetic NOD (La11e 1) 20 wk old BALB / c (Lane 2), 10 wk old NOD-scid (Lane 3 and 20 wk old NOD-scid mice (La11e 4) was separated usi11g SDS-PAG E electroblotted to lmmobilon-P membranes ru1d reacted with rabbit anti-mouse PSP antibody The Western blots were then developed using alkaline pl1osphatase-conjugated anti-rabbit IgG plus substrate The 32 kDa and 20 k.Da bands are indicated witl1 arrows and the 27 kDa protein with a star Prestai11ed r11o l ecu l ar weig l 1t markers are 45 kDa ova l bumin ; 32 kDa carbonic anhydrase ; and 24 k.Da soybean trypsin inhibitor (La11e S)

PAGE 80

120 96 68 45 32 24 (ts C Submandibular .:.:: 3 3 3 0 CD CD TN "C "C N (.) (.) "' "' al I I ..J C C < 0 0 m z z 70 Parot i d 3 3 3 0 CD CD TN "C "C N (.) (.) (.) ...... "' "' al I I ..J C C < 0 0 al z z .._ I Figure 11 Representative Western blot a11a l ysis of cytop l asmic fractions of submandibular and parotid ce l l lysates using anti-PSP po l yc l ona l antibody The cytoplasmic proteins (20 g/lane) of either submandibular g l and l ysates prepared from 26 wk old BALB / c (Lane 1) 10 wk old NOD scid (Lane 2), and 26 wk old NOD scid 1nice (Lane 3) or parotid gland l ysates prepared from 26 wk old BALB / c (Lai1e 4) IO wk old NOD-scid (Lane 5) and 26 wk old NOD sc id mice (Lane 6) were separated 011 a 12% po l yacrylamide gel and transferred to Immobi l on P membranes Membranes were treated witl1 alka l ine pl1osphatase-conj u gated rabbit anti-mouse PSP and substrate The 32 kDa and 20 kDa protein bands are ind i cated witl1 arrows while the 2 7 kDa protein band is shown witl1 a star Prestained molecular weight markers are the same as for Fig 9

PAGE 81

A 53.2 34.9 28 7 20.5 kDa B 112 84 0 53 2 34.9 28.7 20.5 C .... en a: <( a. 0 .._ m C ..J .... <( en m C 0 0 z .._ m :E ..J ::c C <( M I 0 m a. a: <( :E :E a. en en C C 0 0 0 .._ z z m :E :E ..J C C <( I m a. a. 71 C) It) '"0 C u u 0 (I) (I) z I I <( C C :E m 0 0 C 0 z z ... .. a: a: <( :E <( :E a. en a. en '"0 1::1 C C 0 u u 0 (I) (I) z z I I C C :E :E 0 0 C C z z Figure 12 Western b l ot ana l ysis of sa l iva and cytop l asmic fractions of parotid and s u bn1andib ul ar ce l l l ysates u sing anti-Pro l i n e-Ricl1 Protein po l yc l ona l antibody Pane l A, 10% SDS-PAGE of constant saliva protein of 5 g from mouse strains C3H, BALB / c CBA, NOD and NOD-.s c id P-DM prediabetic NOD ; DM, diabetic NOD Pane l B represents the ant i body staining ot PRP i1 1 p arotid (PAR) an d subman d ibu l ar (SM) g l and l y sates fi om BALB / c P DM and DM NOD a 1 1d NOD-s c id mice Eac h l ane co n tained 20 g of protein frotn tota l ce U lysate

PAGE 82

I f t ~ l A \ ,.-.,'C 'I' ,:, It ., ... ........ .. ~' ~ .. 'C' ..,. .. ., . ..... c..""--1 ---.A'' . ,,, . \ f ?~ ,, ... .,..,. ,... ...... .... r_.,_, ,, ~ ~ "' > .; ~ . ~ ", ~ -r-"' ~ I, .. :, ,, ., .. r-,r., .... , -., .,,,, .. ~ .Jill...,~ .. .,. .. h .. .., .,_. l,,..... ,: A i I ~ \ :.:Ji l t i II' r ... 1 ,. ... ,,.,. t:.,. \,: I > '~ : ~~ ~~ >~ ~ : t ,., I .-4' ... "' .. ;,J ., \. 'I ,,, ... ,., .. . , . ,.. .-.; a.> . ~ ~ .. . .-. .... .. .. .. ~ ~ ,.,J ,. ., ,~ -. : .:; i .. ~" . ;, 1 ~, . f. ~ .,. , -: . .. : .,,, .... !jia '... e:\"" ,. .. J~,~ ... .. It> A .,. tll -~ ,.\ t' 't.,_ 1 ,. _; ~ .._ -r, lr-' :; r ~":',~ ... :itQ.::1 : .. r:';., .1 -: ~ ;. ir .:.. 1 .. .;,; . ...... ';f. s vV 1 # I r ., JO ,. "v ".'~ 1;:~ : I ~ .. .:. r 7'! .,;,J, ~ ... ' -=r~ .:: ~~ ~ : ~ ~ -' (~~\~k,,~ : : . ~,.I : _..-.,,.. ,,.,'f (t _; ..~ft: ~. 1 ~';, "' -. ,. ,, : .... ,,. ~. ., .. :,. ,., -rfi# .. ~, l .. : ,. . . .. .. -~ ~., ..... ~., "l& ~ :\~ : ,J..~-. ""' .. ... ~., . ,. '"""' i . j 1. : . : ' ,..,. ""\ ... ... ,, (,.,,. ... ; .,; _,.. .. .. .. C '\ = ... ~ !. ... -.. :v ... .-!>1~, .g;;_ 72 Figure 13 Morpho l ogica l changes in the salivary g l ands of NOD-scid mice Hematoxylin/eosin stained tissue sections of submandib ul ar glands from 10 wk o l d NOD scid (A: 100:X, B : 200X) 30 wk o l d NOD scid (C : IOOX D : 200X) and 20 wk o l d NOD 1 nice (E : 1 OOX, F : 200X) .

PAGE 83

7 3 (la11e 6) Again, lysates of BALB / c parotid glands contained only the 32 kDa protein (lane 4), cornparable witl1 tl1e profile of young NOD. ~cid n1ice Ir1terestingly, the anti-PSP antibody bound to a protein band of approximately 65 kDa present in the parotid gland 1ysates of both BALB/c and 10 wk old NOD-scid 111.ice~ l1owever, this protei11 band has not been studied These results sho" the t e 1nporal nature of tl1e appearance of the 27 kDa isoform of PSP in the salivary glands and the abnormal expression of PSP by submandibular glands of NOD-scid nlice Dete c tion of Proline-Rich Protein (PRP) in the Saliva and Salivary Glands of NOD and NOD-s c id mice The PRPs are a set of proteins whose synthesis are induced in the salivary glands of mice following chronic P-adrenergic agonist treatn1ent or by the introduction of deleterious dietary char1ges (104) Saliva from NOD-scid diabetic or prediabetic NOD mice, a s well as BALB / c, CBA, and C3H strains were evaluated by Western blot for the presence of PRP As i11dicated in Fig 12A, antibody to rat PRP was able to cross-react with a protein of approximately 33 kDa in saliva of diabetic NOD, 10 wk and 25 wk NOD-scid mice 1n contrast, very little of this protein was detectable in the saliva from control BALB / c and prediabetic NOD olice No reactivity was detected in the saliva of C3H or CBA mice N-terminal amino acid seque11ce analysis confirmed the identity of this protein as PRP ( data not shown) An exanlination of parotid and submandibular gland lysates revealed that PRP expression was occurring in both glands of the NOD-s c id, prediabetic, and diabetic NOD mice, but not in control BALB/c animals (Fig 12B).

PAGE 84

74 1-listological Cl1a11ges in tl1e Sub111andibular Gla11ds of NOD-scid 111ice Histological sections of submandibular glands from 15 individual NOD-scid mice rangi11g in age fro1n 3 to 30 wks were evaluated for structural cl1a11ges Figure 13 shows hematoxylin/eosin stained sections of subn1andibular glands fro1n 10 wk (Fig 13A & 13B) and JO wk (Fig l 3C & 130) old female NOD-scid n1ice A comparison of tl1ese two time points reveal a marked, progressive loss of acinar tissue and a decline in the acinar to ductal cell ratio The histology of the parotid gland was also exanuned. However, there were no observable cl1anges in the celJular or glandular n1orpl1ology between IO wk and 30 wk NOD-scid mice ( data not shown) as was seen i.J1 the submandibular gland As expected, the NOD-scid tissue displayed no signs of immune cell infiltration ln contrast, subrnandibular gland tissue from diabetic NOD mice, which appear to have 011ly slight loss of acinar tissue, is highly infiltrated with 1nononuclear cells (Fig l 3E & 13F) Discussion The clirucal syn1pton1 of xerostomia, or oral dryness, associated with human Sjogren's Syndrome is commoruy attributed to two problems : tl1e loss of the fluid pt1ase of saliva and changes in salivary protein composition (I) Both problems are considered a result of a progressive autoimmune response against the salivary glands. This hypothesis is supported by studies using the NOD n1ouse model in which cl1anges in both salivary flow rates and protem composition are associated with the appearance of immune cells in the salivary glands (67) Unfortunately, these studies in man and the NOD mouse fail to

PAGE 85

75 detennine if the loss of salivary function is a direct re s ult of an autoin1111une attack or if the autoimmunity is in response to specific cl1a11ges i11 the exocrine glands In the present study, I l1ave attempted to disso c iate i111mune a11d non-imn1une factors which may contribute to the loss of salivary gland function by using the NOD-scid mouse As expected l1istological analyses of salivary gla11ds from NOD-scid mice did not reveal the pre se 11ce of lympl1ocytic infiltrates Furthermore, total salivary flow and protein concentration measured in NOD-scid mice appeared similar to the published values of BALB /c mice a11d prediabetic NOD 1nice (67) The composition of saliva proteins, as shown by EGF and amylase concentration in NOD-scid saliva, l1owever, showed significant cha11ges with age, wl1ereas tl1e levels detected in BALB/c saliva remained relatively constant over the same ti1ne period (67 71) Tlus was further reflected by greatly reduced amylase activity (90/o r e duction) in parotid gland lysates of NOD-scid mice as compared to BALB / c In addition, amylase activity detected in NOD-s c id parotid ly sat es declined nearly 50% between IO and 25 wks of age Temporal analyses of NOD-s c id saliva by SDS-PAGE again reveal e d tl1e age related decline of amylase as well as several additional proteins Age-related changes in n1urine saliva volume and composition in non -N OD strains does not typically occur until after 12 months of age (54) Tl1e most striking findings however, were the di scov ery of a novel expression of PRP and an internally cleaved PSP isoform (27 kDa) wllich was prominent in 15 wk NOD-scid saliva but was not detected in s aliva from BALB / c mice or younger NOD-scid. The abundant appearance of these proteins may explain why total saliva protein concentration remained constant despite the decline in other major proteins Praline-rich

PAGE 86

76 protein is a latent constituent of' rnuri11e saliva wl1ich is induced in response to glandular trau111a ( 104 ) 111 normal mice PSP is a 20 k.Da leucine ricl1 glycoprotein of unk11own function tl1at is secreted predon1inately by amylase produci11g acinar cells of the parotid gla11d (105) Studies have shown a developme11tal coordination of murine PSP and salivary amylase expression in tl1e parotid gland of adult rnice wl1ere tl1e two proteins appear in constant ratios (107,109) With the onset of diabetes in NOD mice ( > 14 wks) and the aging ofNOD-scid mice( > 15 wks), both tl1e 32 k.Da and 20 kDa isoforms of PSP are repla c ed with a 27 k.Da protein band detected with the anti-PSP antibody Sequence a11alysis of tl1e N-terminal anu110 acid residues of tl1e 27 kDa i s oform re v ealed tl1at the protein started at an internal region ( +27 aa) of the PSP protein Interestingly, all tluee isoforms of PSP appeared in the lysates of the submandibular glands of older NOD-s cid mice but not of no1111al BALB / c mice Tl1ese findings suggest that, first, the glandular specificity of PSP gene expression is lost over time in NOD and NOD-s c id mice, and second, the transition t o a new isof o rm of PSP might involve differential spli c ing and/or alteration of post-translational n1odifications Taken together, these fi11dings s uggest tl1at both submandibular and parotid gland function, as shown by alterations in gland specific PSP a11d PRP gene regulation and salivary EGF and amylase concentrations are altered in NOD-scid animals between 10 and 25 wks of age Therefore, changes in saliva protein content in the NOD-scid mice are probably not due to the insufficiencies of an individual gland but are 1nultiglandular and potentially affecting other exocrine gland function The low level of detection of amylase activity in parotid gland lysates, ectopic exp r ession of PRP, and the appearance of a novel

PAGE 87

77 isotorm of PSP in botl1 tl1e parotid and st1bma11dibular glands furtl1er suggest that these cl1a11ges take place at tl1e intracellular level Histological examination of the subma n dibu lar gland, but not the parotid gland of aging NOD-.s c id mice revealed a ren1ark ab le decline in the acinar to ductal cell ratio This may be due to acinar cell loss, the hyperprolif e rati o n of ductal cells, or a combination of botl1 Acinar ce ll loss in the submandibular gland may be triggered by the observed defe c ts in protei11 syntl1esis or processing which co uld pote11tially lead to celJ death Since whole salivary flow rates in the older NOD-scid animals do not decline de s pite this appare nt lo ss of s ub1n and ibu)ar acini, it is po ss ible that the parotid sub lin gual, and minor sa livary g lands in the oral mu cosa ha ve i11 crease d sa livary output in order to compensate for this loss of acinar ce lls (143,144) An inter es ting alternate hypothesis to acinar cell loss is the hyperproliferation of the s ubn1 andib ular du c tal cells, wl1ich are known to have se lf-ren ewing ca pacity Hyperplas ia and metapla s ia of tl1e sa livary ductal epithelium i s a hallmark feature see n in labial sa livary biop s i es of l1uman Sjogren's patients ( 15) It shou ld be noted too tl1 a t a b r1orn1al proliferation of differentiated tissue is often accompanied by tl1e loss of differentiated cell protein sy nthesis and fun c tion (145) This hypothesis is especially attractive in li g ht of studies sl1owing that the ductal cells of the NOD exocrine pancreas can under go hyperproliferation ( 146,147) Therefore, it is altogether pos si ble tl1at simi lar de ve lopmental abnor1nalities are occurring in the submandibular gland and pancre as of NOD mice that may precede the d eve lopment of sia loadenitis and insuljtis respe ct ively, and be inherently involved in the pathogenesis of these autoimn1une lesions The de 1101 1 0 production of the 27 kDa PSP isofor111 in aging NODsc id mice suggests that aberrant proteolytic proce ss ing n1ay play a role in the generation of

PAGE 88

78 otl1erwise hidden cryptic antige11s, prin1ing tt1e i111111une system for a11 autoimn1ur1e response The tin1e at wl1icl1 tl1is new isofor111 of PSP appears in tl1e saliva and sub1nandibular glands of NOD-scid mice coincides with the tin1e of appearance of lyn1phocytic infiltrates in the salivary gla11ds of NOD mice In addition, morphological cl1anges in tl1e saJivary glands of NOD scid mice involving loss of acinar tissue are detectable by histology in the absence of lymphocytic infiltrates. These observations suggest that cl1anges in the salivary glands of NOD ,nice occur independently of lyrnphocytic i11filtration and that deve)opn1ent of tl1e associated autoimmune activity may actually occur in response to tl1ese cl1anges. This is consistent with the proposed model for the induction of human Sjogren's Syndrome ( l ), except that tl1e irutiaJ "injury" to the gland is intrinsic i e genetically programn1ed in the NOD mouse ratl1er than extrinsic i e. a consequence of viral infection or other insult A newly emerging model of pathogenesis of autoimmune sialoadenitis in the NOD mouse suggests that tl1e initial trigger may re s ide in a defect in salivary gland homeostasis, leading to the production of new or altered proteins As antigenic forms of cellular and secretory proteins are released, the in1n1une s ystem responds by initiating the honling of i11unune cells to the exocrine tissue. Production of cytotoxic cytokines and direct cell killing by activated T cells may play a role in furthering glandular damage. However, the presence of a subclass of autoantibodies recognizing the f3-adrenergic and muscarinic cl1 o linergic receptors alludes to a potential 1nechanis1n for the decline in saliva production and protein output which involves antibody-mediated impairment of neuro-glandular stimulation Tl1us, the presence of autoantibodies may be responsible for secondary effects on gland function following a primary immunological disturbance generated by a

PAGE 89

79 genetic defect in the salivary glands Tl1is n1odel is useful in explaining why first, the pre s e1tce of activated l y 111phocytes is necessary to develop the loss of tl1e fluid phase of saliva in the NOD 1nouse while NOD-.s cid salivary flow rates ren1ain normal, and second, how an autoin1mune response against relatively sn1all portions of the total area of the subn1andibular gland may dran1atically affect the total salivary output of the parotid sublingual and submandibular glands con1bined In co11clusion by usi11g tl1e NOD-scid mouse model, I 11ave begun to dissociate elements of salivary dysfunction in tl1e NOD mouse attributable to tl1e progression of the autoimmune di s ease from nom1al/abnor1nal events dictated by the genetic background of the ani111al These findings are striking in that they suggest an underlying defect in salivary gland homeo s tasis in NOD n1ice which results in a }1istopatl1ological and functional phenotype similar to human Sjogren's syndrome

PAGE 90

CHAPTER4 EXPRESSION OF PAROTID SECRETORY PROTEIN (PSP) rN LACRIMAL GLANDS AND ITS POSSIBLE FUNCTJON AS A BACTERIAL BINDING PROTEIN [N EXOCRlNE SECRETIONS lntroduction The protein and 1nucin-rich secretions derived from the salivary, lacrimal, a nd other minor exocriI1e tissues, i e labial and l1 ardarian glands, are es se ntial for maintaining the healtl1 and integrity of the oral and ocular s urfaces (9) For the most part, both tear and saliva secretions serve similar functions and contain many of the same protein con s tituents, e g EGF, NGF, TGF-a, la ctoferrin, lysozyme, a11d immunoglobulins (9, 10) At the san1e tin1e, however, saliva a11d/or tear specific secretory proteins, as evidenced by sa livary amylase and digestive enzymes, provide for specialized physiological functions of the individual secretory fluids (9) Cl1apter 3 of this dis se rtation documented unique changes in the synthesis of several proteins noted specifically in saliva, including de 11ovo synthesis of PSP as welJ as decreased concentrations of amylase and EGF in NOD mice (121) Similarly, protein expression ab11ormalities were detected in NOD-scid mice These findings suggest that changes in saliva protein constituents in the NOD strain are independent of tl1e autoimmune destruction of the glands The aberrant synthe s is of PSP in the submandibular glands of NOD mice led us to examine the question of whether PSP may 80

PAGE 91

81 be abnonnally syntl1esized in other exocrine tissue of NOD mice, and whether trar1scriptional splicing is likely to accou11t for the 11ovel PSP isofor111 described in Aim 3 l11 tl1is specific aim, I sl1ow tl1at wl1ile PSP is synthesized in the lacrimal glands of NOD mice, it is detected in the lac1in1al glands in several other laboratory mouse strai11s as well As a constitue11t in saliva a11d tears, I provide evidence of a potential anti-microbial function for PSP which may explain its normal sy11thesis by both the salivary and lacrimal glands In addition, NOD mice were found to have lost the normal regulation of PSP gene transcription in a number of orga11s/tissues No alten1atively spliced mRNA was detected, l1owever, the NOD PSP gene contains numerous base pair changes indicative of strain specific differences between mice Materials and Methods A.t1in1al s BALB/c, CBA/J, and NOD/Uf mice were bred and maintained under SPF co11ditions in tl1e mouse colo11y of the Department of Pathology and Laboratory Medicine at the University of Florida, Gainesville, FL. C3H/HeJ and NOD-scid mice were purchased from The Jackson Laboratories (Bar Harbor, ME). Both male and female mice ranging in age from 8 to 25 weeks were used NOD mice were routinely tested for blood glucose levels using Chen1strip bG reagent strips (Boel1ringer Mannheim, Indianapolis, IN) Consecutive e l evated fasting blood glucose levels >240 mb/dl were considered onset of diabetes, after which time the mice were mai11tained on daily insulin injections (67)

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82 NOD mice of 15 weeks of age we r e separated into diabetic and prediabetic san1ple groups for protein studies Bacteria Lys te, ia 111011oc ~ 1 -' IOg e ,1es and St1 e ploc c >c11s 1111,ta,1s were gifts from Dr F Southwick, Departmer1t of Infectious Diseases, University of Florida, and Dr. A Bleiweis, Departn1ent of Oral Biology, University of Florida Acti11obaci//11s acti110111ycete111con1ita11s was generously provided by Dr P Fives-Taylor, Departn1ent of Microbiology, University of Vermont Isolation of Tissues for Protein Studies Ti ss ues were excised from mice killed by cervical dislocation Glands were pooled from a minimum of two ageand sexmatched mice of each strain, freed of connective tissue fat, and any lymph nodes, then homog e ru z ed in 10 n1M Tris bu ff er (pH 7 4) The slurry was ce11trifuged at 500 g for 15 min to pellet cellular debris and protein co11ce11trati o ns of the re s ulting s upernatant were detemuned by the metl1od of Bradford (132) using bovir1e s erum albumin as the s tandard Supernatants w e re frozen at -70 C for Western blot analysis Isolation of total RNA Excised tissues were immediately homogeruzed in 2 ml of 4M Guarudinium lsothiocy11ate (GITC) as described by Chirgwin et al (148) Briefly, 100 l of 10% Sarkosyl and 14 2 I of 2-Mercaptoethanol were added to homogenates and total RNA

PAGE 93

83 was isolated by centrifugation at 35,000 g over a 2 M CsCI gradie11t RNA pellets were washed in 200 I of 80 / 20o/o etl1anol / Diethyl Pyrocarbonate (DEPC) treated water and then resuspended in 200 I of DEPC-treated water RNA was precipitated tl1rough tl1e addition of 500 l of 100/o ethanol and 8 l of 5M NaCl Quantitation of total RNA was deter1ni11ed by spectrophoto111etric analysis at 260 n111 wavelength RT-PCR and Southern Blot Detection of PSP PCR Products Total tissue RNA (2 g) was pelleted by centrifugation and reverse transcribed using Superscript II Reverse Transcriptase (Gibco BRL) ( 122) The r esulting cDNA was arnplified by PCR for 40 cycles t1sing a 94 C denaturation (1 min), 60 C primer annealing (1 min) and 72 C elongation (2 mi11) using S GCAGAGAAACAAGGATCTCG and 3' CACTGGAGAGTAGCCAGCAGG PSP-specific prirners These primers spanned a region preceding tl1e start codon and extending beyond tl1e translation ter11unation site ( 149) PCR products were separated on 1 2% agarose gels and transferred to 11ylon membrane by Southern blotting To confirm the identity of specific PCR products, hybridization of digoxigenin-labeled oligo11ucleotide internal probe ( 5' AATGCGACCGTTCTTGCC) specific for PSP cDNA was carried out using the Genius system of Boehringer-Mannheim (Indianapolis, IN) ( 150) Briefly, oligonucleotide probes were labeled with digox.igenin using terminal transferase Southern blot membranes were baked at 80 C for l hour, blocked with pre-hybridization buffer (Genius Kit), a11d hybridized with labeled probes overnight at 65C Colorimetric detection of PSP product was assayed using an anti-digoxigenin alkaline pbospl1atase conjugated antibody accordit1g to manufacturer's instructions Primers and probes specific for G3PDH were used as

PAGE 94

84 positive contr o ls for all PCR reactio11s All 11ucle o tide primers a11d probes were sy11tl1e s ized i11 the Univer s ity of Florida's ICBR DNA Syntl1esis Laboratory (Gaine s ville FL) Polyacryl a n1ide gel electropl1ore s is and We s tern blot analy s is Gla11d lysates (30 g of total protei11 per lane) were subjected to electrophoretic s eparation on a l 2 o/ o SDS-polyacrylanlide gel usi11g a n1odi.fied Tris-Glycine system of Pug s ley and S c lma i tman ( 136) Gels were stained by u s ing Coo111assie Brilliant Blue R250 ( 13 7) or tran s ferred to lmmobilon-P 1nen1bra11es (Millipore, Boston, MA) for 2 hr at 70v for Western blotting (138) The blocking buffer consisted of 3% nonfat dry milk and 3o/o BSA in Tris-buffered saline. Polyclonal rabbit anti-mouse parotid secretory protein (mPSP) IgG a11tibody, kindly provided by Dr. William Ball (Dept of Anatomy, Howard Utliversity) (139) was incubated with each membrane for 2 l1r at 25 C Following three 10 1ru11 wa s hes the n1en1bran e s were incubated i11 alkali11e pl1ospl1atase conjugated goat anti-rabbit inm1un o globulin (Si g ma Chemical Co ) and e x posed to substrate as previously described (95). Purification and Radio labeling of PSP Parotid secretory protein was purified using a one-step procedure by electrophoretic separation of saliva proteins on a 3 mm l Oo/o polyacrylan1ide prep gel Four l1undred I of wl1ole saliva from C3H/HeJ or BALB / c mice was separated using a single large well in tl1e stacking gel Molecular weight standards (Bio-Rad) and 0.5 cm of the sample well were cut and transferred to PVDF membrane for Western blotting

PAGE 95

85 f'ollowing detection of PSP migration, the similar region on the remai11i11g unfixed gel was removed, macerated by an electric hon1oge11izer in J n1l PBS co11taining 0 02o/o NaN 3 a11d 0 2o/o SOS, a11d placed on a11 orbital sl1ake r over11ight at 4 C Tl1e extracted protein was dialyzed against ddH20 and lyophilized to concentrate tl1e purified protein Typically, 1 52 g of pure PSP was recovered for 400 l of saliva protein Purity of PSP was determined by electrophoresis and staining of pol y acryla1nide gels with Coon1asie Brilliant Blue R-250, autoradiography and Western blot Pure PSP Mr ~ 25,000 was radiolabeled u s ing chJoramine-T a11d Na 1 2 5 1 obtai11ed througl1 Arner s ham (Arlington Heights, IL) Radiolabeled PSP was purified from free ( 1 2 5 1], potentially otl1er contaminating proteins and radiolabeled BSA in tl1e incubation buffer by molecular sieve chromatography on Sephadex G75 obtained from Pl1arma c ia (Uppsala Sweden) PSP Binding to Bacteria Four strains of bacteria, L 1 1 1 0 11o c yt o g e 11es, E coli S. 11111ta11s, and A a c li110111ycete111co1nila11s were grown i11 overnight cultures at 3 7 C The bacteria were pelleted by centrifugation at 4 C for 5 min at 15 000 x g and washed in PBS buffer containing 0 5% BSA, 0 02o/o NaN3, 1 mM CaCl 2 1 mM MgCI2 and I mM ZnCl2 The bacteria were resuspended in 1 0 ml of the above buffer at 10 8 cells/ml ( 1 2 5 1]Labeled PSP (10 4 cpm) was a dded to tl1e cells a11d incubated at 37 C or 23 C for 2 hr on an orbital sl1aker The cells were pelleted by centrifugation, washed twice, and the radiolabel bound to t he bacteria quantitated by a Beckman gamma counter Specificity of PSP binding was determined by pre-incubation of the cells with IO g/ml of unlabeled PSP Cation

PAGE 96

86 dependent bi11ding was determined by i11cubation wl1ere one or all of tJ1e salts were altered in the i11cubation buffer Liga11d blot assays were conducted as described previously ( 15 l ) In brief, bacteria were lysed by sonication ( 4 C in PBS), and the membrane fraction co llected following ce11trifugation Tl1e membranes were sonicated a second tin1e (in buffer containing 0 5% SOS), followed by centrifugation a11d the soluble material heated to 100 C for S min. Sample (tl1e optical density adjusted to A 2 s o= 2 6 units / ml : 1 Ol ( ~ 0 1 units) was n1i xe d with SOS contai1ung sample buffer, s e parated on I 0% polya cry lamide gel and transferred to PVDF The membrane was blocked using a modification of the method of Hossenlopp et al ( 1 S 1 ) Briefly, the blot was i11cubated for 30 min in IO mM Tris-HCI, pH 7 4 co11taining 150 mM NaCl, 0 02% NaN 3, and 3% NP-40, 30 min i11 Tris buffer containing l % BSA in place of detergent, and 30 mjn in Tris buffer containing 0 1 % Tween-2 0 The men1brane was incubated overnigl1t in TBS co11taining I% BSA, 0 1 % Tween-20, and I 0 5 cpm [ 1 25 1]-PSP The blot was washed 3 times in TBS and exposed to X-ray film for 12 hr at -80 C u s ing Kodak XAR-5 film Specificity of PSP binding to bacterial proteins was determined by pre-incubation of the filter witl1 unlabeled protein Ligand binding blots were run on 3 separate occasions for reproducibility using 2 separate preparations of [ 125 1]-PSP fron1 C3H/HeJ and BALB / c saliva Amylase Assay The activity of human sal ivary amylase (SIGMA Chemical) was determined in the presence of varying concentrations of pure PSP, proline-rich protein (PRP), and BSA Amylase activity was determined by the method of Bernfeld using starch as a standard

PAGE 97

87 substrate ( 133 ) Hur11an a111ylase ( 1 g/ml) was resuspended in PBS. The incubation solution was con1prised of 0 4 g soluble starcl1 in 60 nlM Tris-HCI contai11ing 0 15M NaCl a11d 3 mM CaCl 2 and ZnCl 2 Tl1e stop solution consisted of 0 45% 12 0 045% Kl a11d 0 03 N HCI After tennination of the reaction at 5, 10, 15, and 20 1nin, the enzyme activity was d e fi11ed as the amount tl1at hydrolyzed l mg s tarclvn1in/mg of protein All values are expressed as mean S E for 3 separate determinations Results Detection of PSP RNA Transcripts in Murine Lacrimal Gland Total RNA derived from lacrimal tissue isolated fron1 NOD BALB/c a11d C3H/HeJ nuce was assayed for tl1e presence of PSP RNA transcripts by RT-PCR a11d Soutl1ern-blotting As shown in Fig 14~ a stro ng PCR b an d was observed at 785 bp in lacrin1al RNA of NOD but not C3H/HeJ or BALB / c nuce When hybridized with a PSP specific internal oLigonucleotide-probe both NOD and BALB / c developed a band at the expected base pair size (Fig 14B) while the C3H/HeJ remained negative 1J1terestingly, the lack of PSP expression in C3H/HeJ mice is in concordance with previous reports by Hjorth et al ( 105) describing the parotid specificity of PSP gene expression using C3WHeJ nuce Detection of the housekeeping gene, G3PDH, was used as a positive control to indicate that sinu1ar amounts of RNA was utiljzed in ea ch PCR reaction (Fig 14C) G3PDH bands of 983 base pairs were prese11t in all samples.

PAGE 98

A 800 bp 700 bp B 800 bp 700 bp C 1,000 bp 900 bp C en ::c M (.) C 0 z ..,CJ .., aJ ..J <( aJ 88 ~PSP ~PSP ~G3PDH Figure 14 RT-PCR and Southern blot detection of PSP mRNA isolated from murine lacrimal glands Total tissue RNA (2g) was reverse-transcribed and resulting cDNA amplified by PCR using PSP-specific primers spanning the entire of length of the PSP transcript (785 bp ) Panel A ; Ethidium bromide-stained agarose gel containing PSP amplified PCR product (10 /lane) from 8 week C3H/HeJ (Lane 1), NOD (Lane 2) and BALB / c (Lane 3) lacrimal tissues Panel B ; Agarose gel (from Panel A) was transferred to a nylon membrane by Southern blotting and hybridized with a digoxigenin-labeled oligonucleotide probe specific for an internal PSP sequence Blots were developed using ar1 alkaline phosphatase labeled anti-digoxigenin antibody Panel C ; Positive control of RT-PCR and Southern Blot procedure using G3PDH-specific primers (983 bp product) and probe for CJH/HeJ (Lane I), NOD (Lane 2), and BALB / c (Lane 3) RNA samples

PAGE 99

84 0 53 2 34.9 28.7 kDa C .... Cl) o+... Q. :c ('t) 0 \, 0 o+otC\I 0 0 C\I C\I al :c < ...J < ('t) al 0 0 al 89 Lacrimal o+\, Of0 C\I \, otu, u, u al co co "' C C C C C ...J < 0 0 0 0 0 al z z z z z Figure 15 Western blot detectjon of parotid secretory protein in murine lacrimal gla11ds. Tota l protein in pooled tissue lysates (11 = 3 mice/sample) from C3H/HeJ parotid gland (Lane 2~ 1 g/lane) was compared with pooled lacrimal gland lysates (30 g/ l ane) from C3H/HeJ (Lane 3), CBA/J (La11e 4), BALB/c (Lanes 5 and 6) N OD (Lanes 7-10) and NOD-.s c i d (Lane 11) mouse strains Age ( i n weeks) and sex of sample groups are labeled above with 15 week NOD mice being diabetic Briefly proteins were separated on 12% SDS-P AGE gels under reducing conditions, transferred to lmmobi l on P membranes, and i ncubated witl1 rabbit a 1 1ti-PSP ant i bod y B l ots were deve l oped witl1 alkaline phosphatase conjugated goat anti rabbit antibody and substrate as per methods section Prestained mo l ecular weight markers are as fo l lows ; Bovine serum albumin, 84. 0 kDa; Ovalbumin, 53 2 k.Da; Carbonic Anhydrase 34 9 kDa ; ar1d Soybean trypsi11 inhibitor 28 7 kDa

PAGE 100

116 84 53 35 28 kDa C ... 1 Cl) [j 116 84 53 35 28 kDa C ... 1 Cl) [Q] 11684 533528 kDa 9 0 C 1 ... 2 Cl) I Figure 16 P u rification of Parotid Secretory Prote i n Four hundred I of total sa l iva was separated in a prep ge l well (Pane l A) Parotid secretory protein was identified by Western blot using antibody to PSP (Pane l B) and cut from the ge l The p u rified protein was dialyzed radio l abeled and reanalyzed for pur i ty by autora d iography a11d Western blot reactivity (Pane l C lanes 2 and 3, respective l y) Mo l ecu l ar we i ght standards (Bio Rad) are Pl 1 osphorylase B 116 k.Da ; Bovine Serum Albumin 84 kDa ; Ova l bumi n 53 kDa ; Carbonic Anhydrase, 3 5 kDa ~ and Soybean Trypsin I n h ibitor 28 kDa

PAGE 101

91 I 15 000 15 000 E T T E -c:: > c:: > E 10 000 E 10 000 u u < -:: < t:: Q) 0. 0. en Q) (J) en .c (J) CV (J 5 000 C\S 5,000 .. > .. n, > E n, en E 0 < a, < 0, E E -0 .._.. 0 0 0 1 1 0 5 10 25 0 0 1 1 0 5 10 25 PAP Concentration PSP Concentration ( M ) ( M ) [g 15 000 > E -c:: > E 10 000 u -< Q) 0. 0 (J) .c CV (J 5 000 ... > n, E 0 < 0, E 0 0 1 1. 0 5 10 2 5 BSA Concentration ( M ) Figure 17 Amylase enzyme activity in the presence of saliva proteins One g of human saliva amylase was incubated i11 i11 1 it, ~ o enzyme assays using starcl1 as substrate Amylase activity was assayed in tl1e presence of increasing proline-ricl1 protein (PRP ~ Panel A) PSP (Panel B) or BSA (Panel C) concentrations All values represent the mean + S E for 3 separate determinations performed in duplicate

PAGE 102

92 Detection of PSP in Lacri111al Gland Lysates The transcribed and secreted PSP protei11 of n1i ce l1as a molecular weight of 25 7 kDa wl1en subjected to electropl1ore s is on 12 % SOS-PAGE gels We s tern blot analyses using an anti-PSP polyclonal antibody were used to investigate the presence of PSP pr ote in in la cri n1al tissues As depicted in Fig 15 PSP bands were detected ir1 tl1 e l acrin1a l glands of CBNJ, BALB / c, NOD, and NOD-scid, but not C3H/HeJ mice There appeared to be ir1ter -s trai11 variability in the levels of PSP production with CBNJ > BALB/c > NOD > C3H/HeJ Rel ative expres s ion of PSP proved to be independe11t of age, how ever fen1aJe mice di s played lower concentrations of PSP when compa red witl1 synge11eic ma les B eca u se C3H/HeJ mice did express PSP in tl1eir parotid g l a nds, it is unknown why they fail to express PSP in tl1eir lacrimal glands Amyla s e Activity in the Pr ese n ce of PSP The coordinate synthesis and secretion of an1y la se and PSP into sa li va implied a potenti al re gu lat ory or chaperone function for PSP Therefore, I examined the p o tential of seve ral prot e in s in c ludin g PSP, PRP and BSA (as a co11tro l) to n1odify amylase enzy me activity i11 1 itr o. PSP and PRP were i so lated by prep ara tive gel electrophoresis and checked for purity by staining with Coomasie Brilliant Blue and autoradiography of isolated PSP (Fig 16) As shown in Fig 17, PSP, PRP, and BSA had little, if any, influe11ce on amylase activity

PAGE 103

a. en a. I j I Lt) N ,... I I 9 3 E ~o il 3000 S. ruu1un T L OI O DQCJ r o geno T p,. \ 1 11 1 lnom) ekru com lt"n, C/l T p,. I 2 000 T ,-, ., 4 T H [ E Lfl 0 C. N .-i (.) ....... ,,, a 1 1100 H (.) E E :: & i a:s zrf + w C o ncentrati o n o f en
PAGE 104

94 A B E E (.) ca E E (.) ca ,,. w <( Cl) -I w <( ca Cl) ..J C 116 116..., 84 84 ... .r:. C) 53 53 (1) 3: ... 35 35 ca 28 28:::, (.) (1) 0 20 20 Figure 19 Autoradiograpl1 of PSP binding t o bacterial 1 nembrane proteins Fifty g of membrane protein was separated on a 10% polyacry l arnide gel and transferred to PVDF membrane T l 1e ftlter was incubated with 5 x 10 5 cpm [ 1 25 1]-PSP washed and exposed to X-ray film Pane l A represents binding of C3H/HeJ PSP protein however similar results were obtained using radiolabe l ed BALB / c-derived PSP Panel B represents a duplicate blot pre-incubated with 10 g/rnl unlabe l ed PSP for 2 l1r prior to the addit i on of [ 1 25 1] PSP T l 1e bit1ding assay was perforn1ed on 3 separate occasions for reproducibility

PAGE 105

95 Bacteria Bi11ding of PSP As 011e of the 111ain functions of botl1 saliva and tear s is to s erve as a no11-imn1une barrier to bacterial infe c tion from the e x t e rnal environme11t PSP was examined for its ability to bind to oral or gastrointesti11al tract patl1ogens When radiolabeled PSP was incubated with intact bacteria (10 8 cells) in tl1e presence of NaN ~ binding ranged frorn 1828% of the radiolabeled protein added (Fig 18, Panel A) Pre-u1cubation of bacteria with unlabeled PSP reduced the level of binding to 3-5% of the total counts added The additio11 of NaN 3 to the incubation buffer m e taboli c ally kills bacterial cells tl1us eliminating the potential for active uptake of PSP rather tl1 a n strictly bu1ding of PSP to the cell surface Since PSP is a leu c ine rich protein, tl1e potential exists for protein-protein intera c tions that are dependent on the pre se nce of Zn 2 +. To e x anune this p o tential, PSP binding to E co li S M11ta11s A a c ti11 0 111yc e l e 111 co 111ita11s, and L ll1011o cy tog e 11es was evaluated with each of three cations (Ca 2 +, Mg 2 + and Zn 2 + ) add e d separately t o the binding buffer As shown in Fig 18 Panel B, binding to intact bacteria was dependent on the pre s ence of Zn 2 +. Maximal binding occurred at 100 M ZnCl 2 for all the bacteria except A. acti1101nycete,11co111ita11 s which showed maximal binding at I mM of Zn 2 + cation No binding above background was detected in buffer containi11g CaCl 2 or MgCl 2 ( data not shown) Specific Interaction of PSP with Bacterial Membrane Proteins Binding of radiolabeled PSP to specific bacteria I proteins was detertnined utilizing a ligand binding a s say witl1 solublized membra11e proteins. As shown in Fig 19,

PAGE 106

A 800 bp 600 bp B 7 00 bp 500 bp C C .... "' C: Q. c I I C 0 0 z (.) z E .. ca "' Q. ...J I C I 'C :c :c 0 0 M M (.) z (.) z (.) 96 .. > '0 ..., al ...J :c I c I c I 'c I I C C :c :c :c 0 0 0 0 .... M M M "' z (.) z (.) z (.) z (.) I" t Fig u re 20 RT-PCR and Southern blot detection of PSP transcripts in NOD and C3H/HeJ tissues Total tissue RNA from 8 wk NOD and C3H/HeJ mice was co l lected and amplified using PSP specific primers as previous l y described (see Fig 1) Panel A, Ethldium bromide stained agarose gels containing PSP pri1ner-amp li fied PCR product ( I 0 I/lane) from NOD (odd numbered l anes) or C3H/HeJ (even 11un1bered l anes) tissues Tissues are labe l ed as fo l lows : Pn, Pancreas ; Sm, Submandibular ; Pr, Parotid; Le Lacrimal ; Br Brain ; Lv Liver ; Kd, Kidney; and Ht Heart Panel B ; The ge l from Pane l A was transferred to ny l on membrane by Southern b l otting hybridized with a PSP specific, digoxigenin l abe l ed o l jgon u c l eotide probe and deve l oped with a l kaline phospl1atase con j ugated anti-digoxigenin antibody and substrate Panel C. Positive control of RT-PCR and Southern blot procedure usi 1 1g G3PDH-specific prin1ers (983 bp product) and probes for eacl1 RNA sa1np l e I

PAGE 107

50.6 35.5 29 1 20.9 kDa I C tCl) s ..J I I IC C 0 0 z 0 z C .., a. :I: I I I IC I :I: 0 CW) 0 z 0 97 -c > '..J m J I I ''c I I C I I C I a. :I: :I: :I: e o 0 CW) 0 CW) CW) Cl) z 0 zo z 0 0 Figure 21 Western b l ot detectio11 of parotid secretory protein i11 8 week NOD a11d C3H/HeJ tissues lysates Tissue proteit1 (30 g/lane ; parotid lane contained 1 g/la11e) i n pooled tissue lysates (n = 3 mice / samp l e) from NOD (odd numbered lanes) and C3H/HeJ ( eve11 numbered lanes) mice we1e separated on 12% SDS-PAGE gels under reducing conditions transferred to lmn1obilon-P membra11es a 1 1 d incubated with rabb i t anti PSP ant i body Blots were deve l oped witl1 a l kal i ne phosphatase conj u gated goat anti-rabbit a11tibody and substrate Similar resu l t were obtained u sing diabetic NOD mice (15 weeks) Tissues are l abeled as fo ll ows : Le Lac r i1na l ; Pn Pancreas ~ Ht Heart ; Kd Ki d ney ; Lv Liver ; and Br Brain Prestained molecu l ar weight 1narkers are as indicate d in Fig 7.

PAGE 108

N OD PSP mPSP ( ,n1 scr c < I l? e,r ~i, 11 I / I 'i I / / / ( ,'/11 I / 3 () I /33 I / / / /11et I of I o r 1 1h/c llc ,Q 1<>11 I / 77 I/ ,' ~(} \('r 7 r l 1 ul l t t! . I / '( ) I t i I . I / <) I( l ., e ll I 1 / (,II' I _~} 7 I ~36 1 s 11 5 I<>/> /\ l' G G( iG . .. ......... .. ... ( i/\( j .... A r ci .. ( ; .. r . .. .' I c r ........ l'( A l' ACc; r c ( r e .. (i' l I ....... ( I' ( ....... GG( ....... AA I .' l'(iA t t t t t t tt t t t t t t A r G .. G(i A .. ....... . ........ c; A 1 ... A c c ; .. A .. r ... _. c c .. .. _. r A c c c A r e A 1 c .. A r 1 ...... c ; r e ....... AG c ....... AG r _. r e, A A 1 e t (ilr . l.'>1/> T/1 r lie \ 'e r Le,, l 'r,, lie lie li e I u/ 5ier 5ie r Figure 22 Sequence of parotid secretory protein cDNA derived from the parotid gland mRNA from the NOD mouse strain PCR product obtained from PSP primer amplification of NOD parotid and lacrimal tissue RNA ( see Fig 3) was eluted from agarose gels purified and sequenced The sequences spanned from the 5 leader sequence and structural gene into the 3 untranslated region preceding the polyadenylation initiation site The sequence comparisons are based on the published murine PSP cDNA sequence from Madsen et al ( 141 ) Onl y areas of nucleotide base pair differences between NOD PSP and the published sequence are shown, as well as any expected amino acid coding changes indicated by arrows 00

PAGE 109

99 111en1bra11e protei11s from all four bacteria te s ted bound PSP Bindi11g of PSP to three di s tin c t proteir1s was detected f o r all ba c teria except S. 11111ta11s which contained a sing le PSP-bindi11g protein at M., = 53k0a Tl1e other proteins ra11ged in size between 40-55 kDa When the blots were pre-incubated with unlabeled PSP, tl1e binding of radiolabeled PSP was ei tl1er completely co mpeted away (S 11111ta11 s and L 111 0 11 ocy t oge 11 es) or substa 11tially reduced (E. coli and A acti11on1ycete111co1nita11s) The sa1ne bacteria binding pattern was obtained whether the so urce of PSP was from C3 H/HeJ o r BALB / c n11ce PSP Gene Dy sregula ti o n of the NOD mice In addition to tl1e lacrimal glands, a number of other organs and tissues were exan1ined for tl1e expression of PSP tran scr ipts in c luding heart (atrial tis s ue) pan crea s su bmandibular g lands kidney, liver and brain Messenger RNA from each ti ss ue was re verse -tran s cribed and the PSP and G3PDH ge nes amplified by PCR u s ing specific primers As s l1own in Fig 20, ethidium bromide s tainin g r evea led PCR ba11ds in la cri mal, s ubmandibular par ot id pancreas, and he art, but not in brain, kidney or li ve r ti ss ues of N OD mice In contrast, only the par o tid glan d of C3H/HeJ mi ce yielded a visible PSP PCR band S ou thern blot analy s is (Fig 20B) confirn1ed the identity of the 785 bp bands as PSP Note that weaker-staining bands of incorre c t base pair size detected by ethidium bromide were not recognized by the PSP-specific probe BALB / c nuce displayed similar profiles to C3H/HeJ except that a band was d e t ec ted in the lacrimal samples ( data not s h ow n). Positive co ntrol G3PDH bands were d etec ted in all of the tissue samples (Fig 20C)

PAGE 110

100 Westerr1 Blot Ai,alysis of NOD and C3H/HeJ Tisst1es for the Presence of PSP The above data indicated tl1at PSP tran s cripts are expressed in tl1e heart, pancreas submandibular, parotid and lacrin1al glar1ds of NOD n1ice For tl,is reason, I analyzed by Western blotting a pa11el of tissue lysates derived from NOD and C3H/HeJ rnjce for the presence of PSP protein (Fig 21 ) In NOD n1ice PSP protein was detected onJy in the parotid a11d lacrimal glands In contrast, PSP was fou11d only in the lysate of parotid glands from C3 H/HeJ 1nice Despite the presence of detectable nl.RNA transcripts in l1eart and pa11creas of NOD mice, I was unable to detect the presence of PSP at tl1e protein level Sequence Analysis of tl1e NOD PSP Gene The open reading frame of tl1e PSP gene of NOD mice was sequenced using cDNA amplified fron1 mRNA isolated from both the lacrimal and parotid glands The PCR products were isolated from agarose gels and the full length DNA products sequenced by primer extension (Fig 22) As expected, tl1e sequences of the lacrin1al and parotid gland PCR products were identical, and revealed a 98% hon1ology with the publisl1ed n1urine PSP DNA sequence (14 base pair differences out of 705) Two regions corresponding to amino acid residues 111-144 and amino acid residues 198-227, contain a few random mutations ; however two additional regions are of particular interest The first, containing the first 110 amino acid residues, represents a highly conserved region where no amino acid cl1anges were detected between the NOD and the published mouse PSP sequence The second, containing 7 amino acid residues at position 177-183, is a highJy substituted region in the NOD PSP gene sequence These amino acid changes,

PAGE 111

IO I i11cluding a proline substitutio11 at anlino acid 178 could p o tentially cause n1aj o r structuraJ differen ces between tl1e two PSP protei11s Discussion Th es e re s ults show that PSP is produced in the la c rin1al glands of all normal mice t es ted with the excepti o n of C3H/HeJ, s uggesting that this secretory protein is not re s tricted to the parotid and sub lin gua l g la nd as first r epo rted ( I OS) T11e lack of expression in the la cri1na l gland of C3 H/HeJ mice supports the findings o f Hjorth et al ( I 05), but raise s furtl1er questions about tl1e in1portance a nd/or functions of PSP as a secretory protein These d a ta s ug ges t that although coordinately expressed with am y lase the protein doe s not influe11ce enzyme activity ( I 06). Further, d e tection of PSP in the la c rim a l g1at1ds would i ndica te that the pr ote in i s not inv o lved in sa liva specifi c functions s uch as di ges ti o t1 By far the n1 os t widely recogni ze d fun c tion in sa liva is tl1at of growth co ntrol of micr oorganis ms The ability of PSP to bind to bacterial surface proteins in a Zn 2 + dependent manner s u gges t that tl1e protein may function as anotl1er host protein capable of modulating bacterial growth or colonization rates ( 152) The sequence analysis of PSP mRNA of NOD mice revealed a number of amino acid differences in the ca rboxy-terminal region of the protein suggesting the presence of s trains pecific i soform s (141 ) Whether or not th ese changes influence the functional capacity is unknown; however comp a ri so n of PSP with bacterial surface proteins f or homologous leucine-rich regions revealed that potential leucine-rich interactions are pre se nt at the highly conserved amino terminal end of PSP The observation of Zn 2 +

PAGE 112

102 dependen ce for bacterial interactions sugge s ts tl1at leucine-rich domains may be respon si ble for tl1i s property of PSP ( l 53 ) This 111ecl1ani s n1 has bee11 recently detailed for L 111011 oc; 1 t o g e 11 es interaction with epithelial cell E-cadl1erin receptors leading to bacterial internalization ( l 54) Though tl1e se data clearly show that PSP is both transcribed and translated in the lacrimal glands of most normal mice, the levels of PSP produced varied between strains This inter-strain variability is con s istent with earlier published data ( 105) which revealed that total levels of PSP a11d amylase were highly variable a1no11g 111ou se strains but that these two proteins maintained similar ratios in saliva ( l 06) While studies by Shaw et al. (107,109) sl1owed a concomitant appearance of PSP and amylase in the saliva of neonatal 1nice, this coincidental co-appeara11ce of secreted protein was not controlled at the level of gene transcription since PSP but no t amylase, transcripts were detected in neonates at birth Thus, ger1e expression of PSP and amylase in sa li vary tis s ue may be co 11tr o ll e d by different and di s tinct transcriptional re g ulators Thjs ma y explain how PSP transcription occurs in the lacrimal tissues in the absence of an1ylase gene activation Tl1e presence of PSP tra11scripts in the s ubmandibular gland, pancreas and heart of NOD rruce is unique among the strains examined However, PSP s pecific-primers spanning the cap to the untranslated region re vea led o nl y one PCR product, indicating that the novel PSP isoform wruch predon1inates aging NOD and NOD-scid mice saliva is most likely not attributed to alternate splicing events. Despite tl1e presence of aberrant PSP transcription in several tissues of NOD mice posse ss ing exocrine function, protein production was detected only i11 tl1e submandibular, lacrimal and parotid glands, and not in the pancreas or heart F urther1nore non-exocrine tissue (e.g brain, liver, and kidney) did

PAGE 113

103 not express PSP 111RNA transcripts One explanation is tl1at only the salivary and lacrimal tissues possess both PSP transcripts and the necessary cytoplasmic fa c tors for proper tran sc ript processing and post-translatio11al m o dification Evidence for this was first described by Shaw and Schibi er ( I 07) who found abunda11t PSP transcription in the developing muri11e parotid gland at birth, yet tran s lated protein was not detect e d ur1til 12 days of age ( 155). Tlus apparent lag between PSP gene expression and protein translation was attributed to rapid turnover of PSP transcripts i11 tl1e cytoplasm Submandibular glands of neonatal 11uce produce PSP until about day 5 t l1en gene expression and secretion subsides (108). Under normal conditions, cellular activity necessary for the production of PSP protein might still be present in adult subrnandibular glar1ds, but PSP transcription would have ceased. Reappearance of PSP mRNA transcripts in the subr11andibular glands of NOD nuce ( 121) in addition to the presence of appropriate cytopla s mic factors could explain wh y abnor111al protein production of PSP is present only in the submandjbular gland, but n o t tl1e heart or pancreas Whetl1er trus dysregulation observed in NOD nuce is directly i11volved in tl1e pathogenesis of tl1e autoimmune activity and loss of immunological tolerance to the subtnandibular and lacrimal tissues or is merely a biological marker of ge11eralized transcriptional alterations remains unknown and requires additional investjgation In conclusion, these findings indicate that : first, PSP is nortnally expressed in the lacrimal glands of rodents, including NOD ,nice where it potentially functions to control n1icrobial growth or colonization~ second, in addition to expression of PSP in lacrimal glands, NOD rruce aberrantly express PSP transcripts (but not protein) in the pancreas and heart; and third, based on the cDNA sequence of PSP from NOD subma11dibular and

PAGE 114

1 0 4 lacri111al glands, tl1e PSP protei1 1 appears to co11tai11 a higl1ly altered region \~hich could re s ult in strain-specific PSP isof o rms

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CHAPTER 5 EVALUATION OF NOVEL PROTEOLYTIC ACTIVJTY DETECTED IN THE SALlV A OF AGING NOD MICE Introduction Results in this dissertation suggest tl1at PSP is enzymatically cleaved in older NOD saliva The cleavage occurs between leucine a11d asparagine at the 26 th and 27 th an1ino acids of the protein. Database searches did not reveal any known enzymes which may be responsible for this cleavage. However, since PSP cleavage corresponds to the time of dramatic aci11ar cell loss in the NOD mouse, I i11vestigated the potential of both matrix metalloproteinases (MMPs) and apoptotic cysteine proteases to cleave PSP through bysta11der activity These enzy111es, involved in glandular restructuring and apoptotic cell death, respectively, may play a k e y role in the loss of submandibular acini in NOD and NOD-scid mice Therefore, a novel proteolytic activity detected in aging NOD and NOD scid saliva n1ay be indicative of glandular traun1a as well as further establishing novel protein expression in the exocrine glands. In this specific ailn, proteolytic activity in NOD saliva was evaluated for the ability to cleave purified PSP or PSP from control mice In addition, since programmed cell deatl1 appears i11deper1dently of lymphocyte infiltration of the submandibular gland, I investigated the potential activation of apoptotic proteases in the saliva and salivary gland lysates of NOD nlice The best characterized of the apoptotic cysteine proteases is the 105

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106 interleukin-] p converting enzyn1e (ICE) which is required for inductio,1 of apoptosis through tl1e CD95 (FAS) sigr1aling patl1way ( 119) 111 tl1is specific ai1r1 it1crea ses in cysteine protea se activity were detected by both a11 i11 vilt O a c tivity assay and W e s tern blot analyses of saliva and sub111andibular, but not parotid glands, from NOD and NOD-scid, as compared to BALB / c and young NOD mice However, tl1e ability of sa liva and both submandibular and parotid gland lysates to generate tl1e aberrant NOD PSP cleavage product s ug g est that additio11al c e llular protease s are activated i11 these tis s ues as well Zymogram analysis was used to demon s trate that unique MMP activity is pre s ent in tl1e s aliva and gland lysates from NOD mice These proteases are responsible for the breakdown of extracellular 1natrix components and 1nay contribute to glandular re s tructuring in NOD mice (111 ) In addition, the ability of NOD saliva to cleave BALB / c PSP was inl1ibited by the presence of EDT A EDT A is a metal iron chelator u s ed as a ubiquitous inhibitor of MMP activity thr o u g h its ability to se que s ter zinc ions nece ss ary for l\1MP prot eo lytic activity ( 111 ) EGT a calcium chelator, was unable to inhibit PSP cleavage Together these findings confirm that the aberrant cleavage of PSP is due to unique protease activity in NOD saliva While the protease ren1ains ur1known the detection of both a unique MMP activity in NOD saliva and EDT A inhibition suggest that MMP activity may be involved in the cleavage of PSP

PAGE 117

I <)7 Materials and Metl1ods Materials BALB / c and NOD / Uf mice were bred and 1naintained under SPF conditions in the mouse facility of the Department of Patl1ology and Laboratory Medicine at tl1e University of Florida, Gainesville, FL NOD-scid mice were purchased from the Jackson Laboratories (Bar Harbor, ME) Both n1ale and female n1ice at ages 8 weeks and 20 weeks of age were u s ed. NOD mice were routi11ely tested for blood glucose levels u s ing Chem s trip bG reagent strips (Boehringer Mannl1eim, Indianapolis, IN) Consecutive elevated fasting blood glucose levels > 240 mg/di were considered onset of diabetes, after which the mice were maintained on daily i11 s ulin injections NOD mice at 20 weeks of age were s eparated into diabetic and pre-diabetic groups for protease assays Antibody for rat cystatin ( 156) was a generous gift from Dr Gurrinder Bedi Magainin Pharma ce uticals Plymouth Meeting PA PiJocarpine d 1-Isoproteren o l phenyl methylsulfonyl fluoride (PMSF) ditluotl1reitol (DTT), dimethyl sulf oxide, papain, alkaline phosphatase-conjugated goat anti-rabbit i1nmunoglobulin and sodium benzoyl-dl-arginine p-nitroanilide (BAPNA) were obtained from SIGMA Chemical Co. (St Louis, MO) Reagents for polyacrylarnide gel electrophoresis were purchased from Bio-Rad (Richmond, CA) Saliva Collection and Preparation of Gland Lysates Saliva was collected from control and experimental groups of mice following stirnulation of secretion using isoproterenol (0 20 mg/100 g body weight) and pilocarpine

PAGE 118

lt>8 (0.05 mg/ I OOg body weigl1t) dissolved i11 sali11e Tl1e secretagogue cocktail was injected (0 1 rnl volu111e) intraperito11eally, ,~itl1 saliva subseque11tly collected from tl1e oral cavity by mi c ropipet and placed i11to chilled 1 5 ml micr o fuge tubes (67) Saliva samples were collected from groups of 6 mice and then froze11 at -80 C until analyzed for ten1poral changes by enzyme a ssa y, SDS-polyacrylamide gels, and Western bl o tti11g Parotid and submandibular glands were excised from n1ice killed by cervical dislocation Eacl1 gland was separated from connective tissue, fat and lymph nodes, tl1en l1omogenized in 10 mM Tris buffer (pH 7 4) and i1nmediately frozen at -80C. Protein assays of both sa liva and gland lysates were performed u s ing the method of Bradford (132) with bovine serum albumin as the standard. Cysteine Protea s e Assays Protea s e activity in saliva and glar1d ly sa tes were detern1ined usi11g a standard protease assay as described elsewhere (157 158) The as sa y relies on the cleavage oftl1e chron1agenic reagent, BAPNA The in c ubation buffer consisted of 25 I I 00 mM BAPNA in DMSO, 10 I unknown sample 190 I PMSF buffer co11sisting of 0 2 mg/ml DTT, 0 5 mg/ml Na 2 E DTA and 1 0 mM PMSF in 100 mM phosphate buffer (pH 6 0) Experimental samples as well as a dilution profile of papai11 were incubated at 3 7C for one hr The reactions were ternunated by the addition of 25 I of glacial acetic acid adjusted to 1 0 ml with ddH 2 0, and the optical density detem1ined at OD40s 1un to dete11nine the an1ount of p-nitroaniline released A standard curve was generated from the papain. All values were expressed for the mean standard error for all samples performed in duplicate on three separate occasions

PAGE 119

109 Polyacryla1nide Gel Electropl1oresis. Zymogram. a11d Western Blot Analy s is Total salivary proteins (IS g/well) or gland lysates (50 g/well) were subjected to electropl1oretic separation on a 1 5-mm-t11ick 1 Oo/o or 12% SDS-polyacrylamide gel using the 111odified Trisg ly c i11e syste111 of Pugsley a11d Schnaitrnan ( 136) For zyn1ogram gels, 3% gelatin wa s added to the gel mix prior to casting Following electrophoresis, tl1e proteins were transferred for 2 hr at 70V to lmmobilon-P membranes (Millipore, Bedford, MA) for Western Blotting or washed and incubated for 24 hr in Tris buffer with zinc for zymogram analysis ( 138) Polyclonal antibodies specific for murine ICE and Nedd-2 (Santa Cruz Biotechnology, Santa Cruz, CA), and Apopain/CPP 32 (UBI, Lake Placid, NY) were used to detect specific apoptotic proteases at dilutions specified by the mai1ufacturer Polyclonal rabbit anti-rat cy s tatin ( l : 500 dilution) was incubated witl1 each 111e1nbra11e for 12 hr at 25 C The blocki11 g buffer con s isted of 3% nonfat dry milk and 3% BSA in Tris-buffered saline Saliva from chronically i s oproterenol-treated rats was used as a po s itive control for a s sessing the performance of the anti-cystatin antibody, while pre incubation of the anti-cysteine prote a se antibodies with the specific peptide antigen served to deter1nine the specificity of the antibody-antigen reactions Following three IO-min washes, the membranes were incubated with alkaline phosphatase-conjugated goat anti rabbit immunoglobulin and exposed to substrate as previously described (121)

PAGE 120

110 Proteolytic Dige stio n of PSP For assays using radiolabeled purified PSP, IO ~ti sa liva or 30 ~1g sa livary gland lysates (total protein) was incubated witl1 l 0 4 cpm of [ 1 2 5 1]-PSP in a 50 l total volume for 4-6 hr at 37C The digestion was stopped by the addition of SOS-PAGE s a n 1p)e buffer and heating to l 00 C for 5 111in prior to electrophoretic separation 011 a 1 2% SDS poly ac rylamide gel as described above The gels were dried and exposed to Kodak XAR5 X-ray film for 48 hr. For saliva mixing assays, equal volumes of NOD or BALB / c saliva (2 5 ~ti) were diluted up to a total volume of 20 I in PBS and incubated at 37 C for 2-6 hr The additio11 of EDT A or EGTA to a final concentration of 1 nlM to 1001nM was used to assess p roteo lytic di gestio n upon ren1oval of ca tions The reaction was s topped by the addition of S l of SOS-PAGE sample buffer and l1eated to 95 C for 5 minutes Electrophoresis and PSP Western blot dete ctio n were conducted as d esc ribed above Stati s tical Analysis All mea s ure s of variance are given as standard deviations of the mean Tests of significance for differences between independe11t means were performed witl1 the unpaired Student t test Results in which P < 0 05 were considered s ignificant

PAGE 121

20 5 1 7 0 k Da 0.. (/) 0.. cc ...J < cc > > (/) :E (/) 0 0.. cc ...J 0 < 0 cc z 111 X :E > X X (/) a: :E < (/) :E :E (/) 0.. (/) 0 :E :E :E 0 0.. 0 0 cc 0 ...J 0 0 0 0 < 0 0 0 z cc z z z Figure 23 Autoradiogram of differential PSP n1i g ratio11 following incubation with saliva or salivary gland ly s ates 10 4 cpm of [ 1 25 1] PSP purified fro1n BALB / c saliva wa s i11cubated for 4-6 hours at 37 C with whole saliva submandibular (SMX) lysates or parotid (PAR) lysates from BALB / c or NOD mice NOD P-DM prediabetic NOD ; NOD DM diabetic N OD Radiolabeled BALB / c PSP (Lane 1) incubated in PBS served as control migration

PAGE 122

50 0 35 0 29 0 20.9 kDa .. s::::. 0 u ...... al ..J < al .. s::::. .. "2" s::::. u 0 ...... C al ..J 0 < z al 11 2 .. .. .. .. s::::. s::::. s::::. s::::. C\I "2" (0 C0 >< >< >< >< :E :E :E :E Figure 24 Western b l ot of differential PSP migration fol l owing incubat i on of NOD and BALB / c saliva Saliva (2 5 I ) from BALB / c (0 hr inc u bation ; Lane 2) NOD (0 hr i11cubation ; Lane 3) BALB / c ( 4 hr incubation~ Lane 4) was separated on a 1 Oo/o SDS PAGE gel and immunoreacted with anti-PSP Lanes 5 8 depict equa l volumes ofBALB / c and NOD saliva (2 5 I ) mixed ru 1 d incubated in 15 I PBS for 2hr 4 hr 6 hr and 8 hr, respectively. Molecular weight standards are as fo ll ows : bovine serum albumin 84000 Da ; ovalb u min 54000 Da ; carbonic anhydrase 3 5000 Da ; and soybean trypsin inhibitor 28000 Da.

PAGE 123

50 6 29 1 20.1 ... s::. 0 (.) ...... CD ...I < CD ... s::. ... .J::. (.) 0 ...... C CD ...I 0 < >< >< >< >< :E :E :E :E :E Figure 25 Wester11 blot depicting EDT A in l libitio n of the proteo l ytic cleavage of PSP Saliva (2.5 I ) from BALB / c (0 l 1r i ncubat i o n ; La 11 e 1 ), NOD (0 h r incubat i on ; Lane 2) B ALB / c (4 hr incubation ; Lane 3) was separated on a 10 % SDS PAGE gel and immunoreacted with a11t iPSP Lanes 4-8 depict equal volumes of BALB / c and NOD sal i va (2 5 I ) 1nixed and in cubated i n 15 I PBS with 0 100 50 10 and 1 1nM concentrations of EDT A, respect i ve l y Mo l ecular weight sta 1 1dards are as described in Fig 24

PAGE 124

50 6 29.1 20 1 0 (.) ...... al ..J < >< < >< >< :E :E :E Figure 26 Western blot depicting proteolytic c l eavage of PSP in tl1e presence of EGT A Sal i va (2 5 1 ) from BALB / c (0 hr inc u bation ; Lane 1) NOD (0 hr inc u bation; Lane 2) BALB / c (4 hr incubation; Lane 3) was separated on a 10% SDS PAGE gel and immunoreacted with ant i -PSP Lanes 4 8 depict equal volumes of BALB / c and NOD saliva (2 5 l) mixed at1d incubated i n 15 I PBS w i t h 0 100 50 10 and 1 mM concentrations ofEGT A, respective l y Mo l ecu l ar weight standards are as described in Fig 24

PAGE 125

Table 5. Cy s teine Protease Activity in Saliva ACTIVITY g/ml g/mg protein 8 week 20 week 8 week 20 week BALB/c 15.2 2 6 16 7 2.7 15.9 2. 7 12 8 2.1 NOD P-DM 14.8 1.9 45 8 5.3 10.3.9 39.2 4 8 NODDM N.A. T 40.3 6.8 N.A I 39 5 3 7 NOD-scid 12.1 3.2 67.5 12.3 13.6 2.2 + 67.0 + 4.7 All values expre s sed as the mean standard error for n=6 animals/group The a ss ay s were perf orrned in duplicate on three separate occa s ion s. ~ A = not available due to the later age of diabete s onset in the Univer s ity of Florida NOD c olony n=2 U'I

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116 Re s ult s Gen er ation of the Aberr a ntly Pr o cessed l 7kDa PSP Isoform As presented in Chapters 2, 3 and 4, NOD and NOD-scid mice abnormally expre s s PSP in tl1eir submandibular glands and tl1at with increasi11g age thi s PSP is aberrantly processed through enzymatic digestion in their submandibular glar1ds ( 121 ) Using radiolabeled PSP purified fro1n BALB / c sali v a I assessed tl1e ability of saliva and g land lysates to enzyn1atically digest PSP to the aberrant i s oform. Incubation of BALB / derived 1 25 1-labeled PSP with NOD saliva resulted in tl1e di g estion of the 20 5 kDa PSP isoform to the 17 0 kDa PSP within 4 to 8 hr in c ubation at 37 C Incubation for more than 8 hr caused a complete degradation of the radio labeled PSP ( data not s hown) Incubation of PSP witl1 BALB / c sali v a or subma11dibular gland ly s ates failed to generate the 17 0 kDa PSP isoform (Fig 23) and th e re was no evidence for partial d eg r a dation of PSP by incubation of the prot e in at 3 7 C in PBS Al s o s l1 o wn in Fi g 23 s ub ma ndibular gland ly s ates from both 20 week old pre diabetic and diabetic NOD mice were able to cleave tl1e BALB / c PSP to the aberrant 17 0 k.Da i so forn1 In addition to assays using radio labeled PSP, PSP cleavage was demonstrated through the mixture of pooled l 8wk NOD and BALB / c saliva Since NOD saliva contained only tl1e cl e aved PSP isofortn while BALB / c contai11ed only native PSP, cleavage of BALB / c PSP could be differentiated by shifts in the m o lecular weight as the protein was digested As shown in Fig 24 complete dig e stion of PSP in BALB / c saliva was achieved following a 2 hr incub a tion BALB / c saliva in the ab s ence of NOD saliva failed to generate tl1e cleaved PSP isoform after 4 hr of incubation

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117 l11llibition of PSP Dige s tion with EDT A To t es t t l1 e ability of divale11t c atio11 ch e lat o r s t o inhibit the pr o teolytic dige s tion of PSP NOD a11d BALB / c s a liva were incubated at 3 7 C for 2 hr in the pre se n c e of E DT A or EGT A A s s hown in Fig 25 dig es tion of BALB / c PSP was inhibited by I 00 50 and I O mM final c oncentratio11s of EDT A Inh i bition was greate s t at hi g her EDT A concentrations and was dose-dependent At I mM EDTA concentration, the n1ajority of BALB / c PSP was cleaved No inhibition was det ec t e d upon the addition of I 00, 50 I 0, or 1 mM EGT A to the s aliva mixture (Fi g 26) Th i s indi c ates that the pr e s e r1 c e of div a lent n1etal ions, but not ca l c ium ion s, is 11ec ess ary for p ro teolytic c leavage of PSP Elevated Cy s tei11e Prote a se Activity in NOD Mice Change s in c ys t e ine protease activity in the saliva of N OD and BALB / c c o ntrol mice was as s e sse d to correlate c hange s in the hi sto logy witl1 p o tential apopto s i s of acinar cells as obse rv ed previously ( 121) As pre se nt e d in Table 5, there wa s an a p p roximate 3fold in c rease in cy s t e ine prote ase a c tivity in the sa liva of 20 week old pre diabetic ai1d diabetic NOD mi c e wl1en compared to age-mat c h e d BALB / c c o ntrols or 8 w e ek old NOD animals (P < O 01 ) In N OD-~ c id mice tl1is incre as e in prote o lytic activity was eve11 greater (67 5 , s 16 7 ~ P < 0 005) Saliva concentrations of cysteine protease ac tivity ranging between 14-17 g/ml saliva were measured in BALB / c pre-diabetic N OD and NOD-s c id mice 8 weeks of age The s a me trends in r e l a ti v e protease activity in saliva did not vary greatly if prot eo lysis was calculated relative to con s tant protein rather than to constant volume (Table 5). There were no differences in tl1e levels of cysteine protease activity

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118 between 8 and 20 wks of age i11 BALB / c anin1als or between 111ale and female mice (P > 0 05) To further define the s ource of tl1e increased proteolytic activity in saliva both parotid and submandibular gland lysates were prepared from 20 week old mice Parotid gland lysates fro1n BALB / c, 8 wk old prediabetic (NOD P-DM) and diabetic NOD (NOD DM) as well as NOD-scid mice showed si1n.ilar levels of proteolytic activity, approximately 15 g protease / mg total gland lysate protein (Fig 27~ P > 0 05) In contrast, gland lysates prepared from the submandibular gla11ds of older mice revealed an increased cysteine protease activity in pre-diabetic NOD n1ice tl1at was even greater with on s et of diabetes (37 3 , s 78 8 g protease/mg gland protein respectively; P < 0 01) In submandibular gland 1ysates of NOD-scid mice, proteolytic activity proved to be the greatest correlating with previous histological analyses and suggesting loss of acinar cells tl1rough cell de.atl1 (121 ) Detection of Elevated ICE, Nedd-2, and Apopain/CPP 32 in NOD Saliva and Gland Lysates Saliva and gland lysates were evaluated by Western blot to confirm the increased activity of apoptotic cysteine proteases in NOD mice As presented in Fig 28, whole saliva possessed an increase in inm1unoreactive material for ICE, Nedd-2 and Apopain/cpp32 in older NOD and NOD-scid mice Potential pro-enzyme forms (Mr = 3 550 kDa) were detected in saliva from 8 and 20 wk NOD as well as the NOD-scid animals, but not in BALB/c mice The active subunits detected by the antibodi e s were : ICE (pl 0), Nedd-2 (pl2) and Apopain (pl2) (25-27) Thus, the different 111obilities of the proteins

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75 > C: ..., Q) (.) 0 < Q. 50 Cl) 0) "' E ca Cl) 0) ..., 0 :::::t 25 lo. a. 0 >< cc < Cl') 0. BALB / c GLAND LYSATES >< cc < Cl') 0. NOD P re D M >< cc < Cl') 0. N OD -OM >< cc < Cl') 0. NOD-scid 119 Figure 27 Histogram of cysteine protease activity in salivary gland lysates Parotid (PAR) and submandibular (SMX) gland lysates were incubated with chromage11ic substrate, BAPNA, for 60 min at 37 C Sta11dard curve was ge nerated by linear regression analysis of papain digestion of BAPNA All values represent the mean S E performed in dup l icate on three separate occasio11s

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C ICl) so s-s 35 529 1. ... 20 9kDa 3 0 C\I (,) al ..J <( al 3 0 C\I I 3 0 (.) co C\I (I) I C C C 0 0 0 z z z to --.., \_ -,.-, lE d '1 I ICE 3 0 3 C\I 3 0 as co C\I C ..J C 0 I<( 0 0 Cl) al z z u ,. F ; r ' a ; .._ Nedd-2 3 3 0 0 3 C\I C\I 0 3 C\I 3 0 (.) (,) (.) (I) co C\I (I) al I I C C ..J 0 0 0 0 I<( 0 0 0 z CJ) al z z z '. .. 1 ; IC,.~-1 .. .. Apopain Figure 28 W estem blot analysis of saliva for the presence of apoptotic proteases Whole saliva ( 15 g) was separated on a 12% SDS-PAGE and evaluated for the presence of murine ICE Nedd-2 and Apopain/CPP 32 using rabbit polyclonal antibodies The fidelity of the antibody reacti on was established by pre-incubation of the antibodies with the peptide antigen (1 g/ml) prior to reaction with the nitrocellulose membrane Cleavage of the ICE p45 pro-enzyme generates p20 and p 10 active subunits Proteolytic cleavage of Nedd-2 the p50 pro-enzyme generates the p20 and p 12 active subunits cleavage of the Apopain/CPP 32 p35 pro-enzyme produces the p 17 and p 12 subunits of the mature protease Prestained molecular weight s tandards (Bio-Rad) are as in Fig 24 N 0

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84 533528 kDa 0 ICl) 0 Cl) + .... ca a: 0 Cl) + "' "' "' == == == 0 0 C0 N N 0 0 0 " m m m _,J _,J _,J
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l23 recognized i11 tl1e gel could be non-djssociated complexes of the active apoptotic protease su bunit s or dige s tion pr od u cts fro111 intera c tion witl1 protea s es fron1 sa liva The antibodies failed to react with material in gland ly sa tes potentially du e to t}1eir low concentration in total protein preparations As a control f or tl1e proper identifi c ation of cystei11e proteas es in saliva, the same prepa ra tions were reacted with primary antibody pre-incubated with the p e ptide antigen s upplied by the n1anufacturers Under these conditions, only the higl1est 111olecular weight band (Mr :::: 55 kDa) was detected wit h the ad dition of seco ndary antibody and chromagenic substrate Deter 1nination of Cystatin Levels by We ste rn blot Cystatins are a se ries of cellular proteins produ ce d by various tissues to linut proteolytic activity in re s ponse to injury ( 158) Synthesis of these proteins can be induced in salivary glands by chronic treatment with tl1e P-adrenergic agonis t i soprotereno l ( 15 6) Sali v a was analyzed for the l eve ls of cysta tin by We stern blotting usin g a polyclonal antibody to rat cys tatin As pre se nted in Fig 29, a protein of approximately 15 k.Da tl1at n1igrated si1nilarly to tl1e cystati11 protein present in whole rat s aliva isolated following chronic isoproterenol treatment, was observed i11 each saliva te s ted There were no differences in the levels of cystati11 det ec ted ii1 saliva from BALB / c or NOD mice irre s p ec tive of age Inter es tingly chronic treatment of mice with isoproterenol appeared to d ec rease the levels of cystatin pr es ent (Fig 29). This was reflected in a BAPNA digestion assay where proteolytic activity increased fr om 14 to 31 g protease/ml and 43 to 51 g protease/nu in BALB /c and pre-di a betic NOD mice, respe c tively(P < 0.05)

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124 D e tection of Novel MM.P Activity i11 NOD Saliva and Gland Ly sa tes To evaluate the presen ce of matrix met a lloprote inase activity in NOD exocrine ti ss ues, gland lysates or saliva from NOD, NOD-scid, or BALB /c mice were analyzed by zy1nogran1 gel As s hown in Fi g 30, unique zones of gela t in clearac1ce were detected at approxi111ately 98 kDa and 60 k.Da in NOD and NOD-scid g land lysates High levels of MMP activity were detected at 55 kDa i11 BALB / c nlice wllile lesser activity at this mole cu lar weigl1t was pre se nt in N OD strai11s. Gelatin clearance wa s detected at ~2 0 kDa in all sa 1nples~ however this activity was weakest in 20 \\ ~ k NOD mice Total MMP activity in NOD a11d BALB /c tis s ue ly sa tes was f o und to be similar by azocoll clrromagenic as s ay (data not s h ow n) These findings confirm tl1at a c tive MMPs are present in both NOD and control BALB /c mice, althougQ unique MMP activities are detected only in NOD mice Di sc u ss i o n The re s ults pre se nted in this aim confirm that the appearance of tl1e cleaved PSP isofonn in the saliva of aging N OD and NOD-scid nlice is due to unique proteolytic proce ss ing While the protease re s pon s ible for this enzymatic activity is unknown at this tin1e, the inliibition of PSP cl eavag e by tl1e additio11 of E DT A, but not EGT A, suggests that the presence of metal ion, but not calc i u~ is 11ecessary for this activity The matrix metalloprot e inases (MMPs) are a class of z inc-dep e ndent enzymes which, in conjunction with their specific inhibitors (TIMPS), are responsible for restructuring and maintenance

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125 of the extracellular matrix ( 110) Over 12 MMPs have been d esc ribed and include tlte c0 Jla ge 11a ses, ge latin ases ( collagen type IV) s tr o 111al ysi n s ( lamin ases ), a11d e la s tin s which are able to cleave v irtually all con1ponents of the extracellular matrix ( 111 ) Expressio11 of specific MMPs during the de ve lopmental process is r espo nsible fo r resculpturing tl1e ext r ace llular environn1ent and tl1 e reby dictating ce llular turn ove r and differe11tiation Si11ce MMP activity is lli gh ly dependent on the presence of Zn 2 -+ EDT A is com.Jnonly u se d as a pot e nt inhibitor o f MMP activity ( 110) Inhibition of MMP activity by E DTA is typically d etecte d at 4 mM co11centrations. In tl1e pre se nt studies, a dose d epende nt inhibition of PSP digestion was d e te c ted between 100 and 1 mM EDT A co n ce ntrations Intere st ingly, maximal i1lhibition by EDT A was dete c ted at IO m!\'L but not 1 mM, E DT A concentrations Tlus indicates th a t both MMPs and the enzyn1e responsible for PSP cleavage are inlubit e d by similar co ncentrations of EDT A Furtl1ern1ore, unique MMP activity was dete cted in the sa liva and gland ly sa tes of NO D mice that was not d e te c ted in BALB / c mice. Tl1ese re s ult s sugges t tl1at no ve l MMJ> activi ty in NO D exocrine tissues remains a s trong candidate for PSP c leava ge Cellular h o m eos ta sis depends on r eg ulated ce ll pr o liferati o n coupled to ce ll death Extracellular signali11g molecules are capable of regulating glandular cell populations through a se ries of intracellular events tern1ed prograrru11ed cell death or apoptosis Tl1e cytokine tumor ne cros is factor (TNF) and the related protein CD95 (FAS) are immune syste m mole c ules ca pable of triggering apoptosis Programmed cell death is mediated intracellularly by a proteolytic cascade involving m e mbers of the cystei ne protease fanuly wl1ich cleave important c e llular proteins including pro-enzymes of other members of this class of proteases ( 115) A prototype protein of tl1e cysteine proteases is interleukinIP

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126 convcrti11g e11zyme (ICE) whicl1 has been characterized as the activat o r of the cytokine interleukinI~ throu gh cleavage of its precursor at Asp 116 / Ala 117 ( l 20) All members of the ICE-related cy s teine protease fan1ily cleave tl1eir substrates after ru1 a spartate re s idue followed by a small runino acid residue which is important for substrate consensus recognition (120) Therefore l1igl1 levels of cystei11e protea s e activity may be indicative of both tl1e activation of apoptotic mechanisms as well as the processing of proinflammatory cytokine precursors In the present study I have found that NOD and NOD-scid mice had high co11 c er1trations of cysteine protease a11d MMP activity in their saliva and submandibular gla11d lysates consi s tent with tl1e histological observations suggestive of submandibular acinar cell death However the parotid glands fron1 NOD mice, which do not show evidence of a strong autoimmune attack possessed similar levels of cysteine protease a ctivity as tl1at seen in tissue from BALB / c control mice The increased cysteine protease activity in the submandibular glands correlated with the time at whicl1 autoimmune leukocytes generally appear in tl1e glands and subsequently display secretory dysfunction Tl1e greatest l e vel of protease activity was present in the NOD-scid mice despite the absence of functional Band T-lymphocytes Autoimmune lymphocytes are not therefore, tl1e main underlying factor for increased acinar cell turnover The increased cysteine protease activity in NOD lysates did not appear to be the result of a loss in the control of synthesis of the specific inhibitor cystatin, as evidenced by similar saliva protein levels in BALB/c and NOD mice Thus, the detection of cysteine protease activity in the submandibular gland is consistent with the concept that NOD mice possess a genetic

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12 7 predi s p os iti o t1 for glandular loss of function leadi11g to activation of tl1e apoptotic pathway and s ub seq u e 11t activation of tl1e irnmu11e sys te111 ( 121 ) While parotid g lands of aging NOD mice are able to ge11erate the aberrant PSP 17 0 kDa isoform, le ve ls of cysteine protea s e activity are similar to that of BALB / c a11imals Tl1e further observation that tl1e unique cleavage s ite of PSP (Asn26/Leu27) is not an amino acid consensus se quence for cysteine proteases, suggests tl1at aberrant proteolytic proces s ing of PSP in NOD mi ce is independe11t of the activation of tliis class of enzymes Considered as a whole, these s tudies are painting a con1plex picture as to tl1e phy s i o l og ical state of the s aliva ry glands of NOD mice Rep o rts (107, 108) indicate that PSP is synthesized in the neo11atal submandibular gland acinar cells of normal mice up to 5 days of age after which time syn the s is ceases, while synthesis i11 tl1e parotid g l an d continues In the NOD mou se the reapp eara nce of tran scri pt s and tran s l ate d PSP in the subn1andibular gland sugges t that there i s a breakdown in cell differentiation and re expression of developmental proteins This loss of devel o pmental c o n trol must exte11d to the parotid gland with the s ub se quent ab11ormal proces s ing of PSP in both glands and may be dependent on a berrant MMP activity The more extensive g landular disruption of the submandibular gland could pot e ntially activate programmed cell death in the acinar cells leading to the observed histopathology seen in tl1e NODscid backgr o und The re s ulting cell death may then contribute to the autoimn1une path ology throu g l1 tl1e activation of immune system activators and effectors such as cytokines and the generation of autoantibodies which exacerbate the glandular dysfunction leading to loss of secretory

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128 fut1ction Tl1e end re s ult of tl1is process on exocri11e ti ss ues would be cli11ical prese11tations of xeroslon1ia a nd xeropl1thal111ia

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CHAPTER6 CONCLUSION AND FUTURE DIRECTION It is generally believed tl1at the physiological and biochemical changes talcing place i11 tl1e exocrine glands of Sjogre11's Sy11drome patients and animal models of this disease are a direct result of the lymphocytic attack on tl1e exocrine ti s sues For this reason, most research endeavors have focused on tl1e role of T a11 d B lyn1phocytes, autoantibodies, and cytokines in tl1e pathogenesis of anti-exocrine autoin1mun.ity These studies l1ave overwhelmingly shown that strikingly similar lyn1phocyte pl1enotypes and cytok.ine expression are detected in the salivary a11d lacrimal glands In tl1e present study I have den1onstrated that, in tl1e scope of tl1e 111arkers tested these features of the anti-exocrine response in NOD mice resen1ble those detected in patient biopsies and other disease models Since normal salivary flow rates are detected in immunoco1npromised NOD-scid mice, tl1ere is little doubt that the lymphoc)rtic component is crucial for the loss of secretory function in NOD mice Therefore, tllis work confin11s tl1e NOD mouse as a valid model for the study of autoim1nune-induced sicca syndrome Of considerable interest is tl1e fact tl1at, while numerous anitnal strains (i e. MRL/lpr, NZB/NZW) develop lymphocytic foci in the exocrine tissues, only the NOD mouse exhibits a Joss of secretory fu11ction sin1ilar to that of }1uman patients This suggests that similar to tl1e observation of insu1itis in non-djabetic NOD mice, the mere presence of the lyinphocytes in the exocrine tissues is not sufficient for the loss of 129

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130 glandular fu11ctio11 Tl1erefore, tl1e in1111L111ological and ph ysio logical events occurring in tl1e exocri11e ti ssues of NOD 1nice mu s t be u11ique from tl1 ose taking place in other anin1al 111odels displayi11g sialade11itis Co1nparing tl1e sirnilarities and differec1ces betw ee n sialadenitis in tt1ese a11imaJ models should provide insight into the autoinunune effector n1e c hani s ms whi c h result in secretory dysfun c tion Focal le sio ns of the submandibular glands of NOD mice cover only a small portion of the total tis s ue area, and the parotid glands do not develop extensive lymphocytic ittliltration Furtl1er1nore, the parotid glands of normal rni ce are able to compen sa te for the surgical recnoval of the s ubmandibular glands by i11crea si ng secretory flow to volun1es comparable to tl1at seen prior to surgery. De s pite this fact, 11early 90% of secretory fu 1 1ction is lo s t in NOD mice between 8 and 20 weeks of age Tl1erefore, both submandibular and parotid gland function i s affected by the autoimmune processes These findings further sugges t that dire c t lymph ocyten1 ed iated cell killi11g is not the principle effector mechanism for the loss of sec retory function Cytokines such as IFNy and TNFa which are prevalent in the focal le s ions of human labial ljp biopsies and exocrine ti ss ue s of animal n1 o de)s have been studied for their p o tential cytotoxic properties towards exocrine cells. For example i11 vitro studies have shown that IFNy and TNFa are cytotoxic to human sali\ ary duct cell lines, hinting that similar events may be taking place i1z vivo In tl1e parotid glands of NOD mice, little or no mRNA transcripts for IFNy are detectable by RT-PCR. Furtl1 e rmore, the presence of large quantities of these cytokines in the exocrine tissues of MR.L / lpr and NZB/NZW mice which do not lose secretory function strongly indicates that bystander cytotoxic activity of these cytokines is not s ufficient to cause the loss of secretory function It should be noted

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131 tl1at tl1is does not exclude a potential role of cytokir1e cytotoxicity i11 111ediating secretory dysfu11ctio11 i11 NOD 111ice by other, as yet unidentified, contributing factors Tl1e finding of autoantibodjes directed against tl1e P-adrenergic a11d n1u s carinic receptors provides a plausible explanation for tl1e loss of both parotid and submandibular secre tory function, as antibodies in the se ra could influence functio11 of either tissues Tl1ese cell surface receptors are down-regulated in the salivary gla11ds of NOD n1ice, which may indicate tl1e n1echanism by wllich tl1ese autoa11tibodies could contribute to secre tory dy sfu nction To confirm tl1e pathogenic role of these antibodies is an area of intense interest Researchers have yet to evaluate tl1e presence of sinlilar autoantibodies in l1u1nan sera, a11d the role of autoantibodies in Sjogre11 s Syndrome is unclear The recently developed ~t-cl1ain knockout NOD truce can be used to test the pathogenicity of specific autoantibodies to the loss of secretory function through selective B cell or autoantibody transfer Since T cell help is thou gh t to be a necessary component dri v ing antibody ge 1 1er ation, this model is better suited for the study of B cell contributions to disease than is the NOD-scid mouse Wh e ther sialadenitis or sec retory dy sfu nction occurs in -chain knockout NOD mice is not y et known however, the study of thls strain sl1ould provide exciting new insights to the effector mechanisms of secretory dysfunction in the NOD mouse. Perhaps the most intriguing aspect of the work in trus dissertation is the finding of gla11dular changes in the abse nce of lymphocytic infiltration Tl1ese changes include morphological abnor111alities, changes in tissue specific gene expression, changes in secretory proteins, increased apoptotic protease activity, and novel matrix1netalloproteinase activity Moreover, these changes occur at the time in which

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1 32 ly111pl1 o c y te s ir1filtrale tl1e exocri11e ti ss u es a11d d es troy glandular f unctio11 It is generally believed tl1at l1i sto l ogica l a11d biochen1ical c l1 a r1g es taki11 g place in the sa liv ary and la cr i111al g lands of Sjogren's Sy11drome patie11t s a11d anit11al m o dels is a result o f autoimmu11e activity T l 1ese data s trongly suggest, h owe ver, that the dev e lopment of autoimn 1u nity in tl1e se tis s ue s 1na y actually be a re su lt of u11derlyin g physiological and biocl1 e mical changes within the target tissue itself Therefore the target ti ss ue organs of individuals predisposed to autoim1nunity tnay be actively re spo n s ible for tri gge ring or enhancing the a ut o i1nmune response While th ese ti ss ue abnormalities are intrin s ic in the NOD ge11etic background, sim ilar or exacerbating ti ssue changes precipitated by viral infection may explain how environmental factors contribute to autoimmune devel op ment in pr edis po se d individuaJs This the ory is intri g uing i11 tl1at while 110 specific vi ru s ha s been implicated in Sjogren' s Syndrome evide nc e of v iral infection or activity i s often detected in aut oi mmune sites. Tl1is suggests that no si n g le v iru s o r e nvironn1 e ntal agent will be found to pr ecipi tate autoimmune development Instead any number o f environmental agents which promote a s itnilar array o f glandular c hanges co uld lead to autoimmu11e de ve lopment in susceptible individuals This pro v ides an intere s ting new st rat egy for re searc h in the field of autoimmunity in that tl1e study of c ritical diff e rences in glandular hom eos tasis could be as in1portant to our under stand ing of autoinwunity as tl1e s tudy of immunological abnorn1alities Since exocrine gland abno1 n1alities encompassed such a wide range of cellular processes, it would be difficult to i so late a single event which underlies these ph ys iologicaJ cl1anges Instead, it appears that the entire balance of ex ocrine l1om eos tasis is di s turbed in

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1 33 aging NOD mi ce Wl1ile tl1e work in this dissertatio11 serves 111ainly to outline tl1e pre se nce of unique glandular cl1anges, it also ur1covers several clues as to how tl1ese processes n1ay occur One suc l1 clue is the re-e xp re ssio n of PSP i11 tl1e submandibular glands During subn1andibular gland developm e nt, PSP is expressed until S days of age by a transiti o 11 progenitor cell type wl1ich spawns mature s ubmandibular aci11i The re-e x pre ssion of PSP in aging NOD mice suggests a potential d e differentiation of the submandibular aciru This may add itionally explain the alt e red and disorganized appearance of aci nar tissue as seen in histological sections of aging NOD and NOD-scid nlice Future s tudies will be necessary to determine whether other character istics of tt1es e c e ll types are sin1ilar as well While acinar cell loss was expected in NOD 1nice, tt1e findi11g of acinar cell loss in NOD sci d mice was surprising The finding of increased c ysteine protease activity in NOD-scid mice was s imilarly unexpected W11ile direct detection of acinar cell apoptosis in NOD-scid tissues using l1i sto logical assays l1as not yet been examined, increased cysteine prot ease activity is highly suggestive that apoptotic cell death is takin g place Since apoptosis is considered an active, energy-driven process detection of apoptotic activity in NOD-scid mice indicates that non-l y n1pl1ocytic components are driving submandibular acinar cell death While trus does not discount a potential role of natural killer or antigen pre se nting cells in trus process, it is intriguing to speculate that the actual apoptotic trigger may reside in tl1e tissue itself Therefore future studies need to focus on the mechanisms leading to apoptotic cell death i11 NOD-scid exocri11e tissues Both NOD and N OD-s ci d saliva and salivary gland lysates contain a u ni que matrix-metallo pro teinase activity that is not present in control mice Developmental expression of extracellular matrix components or the degradation of existing extracellular

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134 1natrix h e lps to dri ve tl1e process of cellular differe11tiation and glandular re111odeling in the sa li vary gla11ds It tallows tl1at, s in1ilar to dedifferentiation ob se rved i11 cell culture, dege11eration of the extracellular n1atrix in adult exo cr ine tis s ues co uld lead to the di s ruption of existing mature cell types Tl1erefore, it is plausible that degradation of the EC~1 by novel MMP activity in NOD exocrine ti ss ues could lea d to acinar ce ll deditfere11tiation or cell death If this hypothesis were true it would provide a valid explanation for the ex1ensive nature of the exocrine gland abnor1nalities such as novel gene transcription, proteolytic cleavage of PSP, increa se d acinar cell death, and ductal l1yperproliferation Future studies need to focus proteolytic activity in both NOD and nor1nal co ntrol strains, s in c e tl1e absence of necessary Mrv1P activity may be as disruptive to glandular homeo s tasis as the expression of novel MMP activity It has been spec ulated that protein alterations such as proteolytic cleavage could expose novel, immunogenic se lf-p ep tides to the imn1une system In tl1is way thymic t o l era nce would n o t eliminate tl1e g e neration of a self -re sponse against tlus type of '' cryptic '' antigen Aberrant gene expressio11 and prote o lytic cleavage of PSP in the subm andibular glands and the presence of tlus protein in the lacrimal gland as well rai s es the possibility that PSP may represent a putative autoantigen PSP has yet to be found in human exocri11e tissues and difficulties in obtaining and expre ss ing large quantities of purified PSP have left this question unansw ere d However, the loss of tolerance to a protein antigen does not necessarily lead to the loss of glandular tolerance Moreover the extensive abnom1alities in the exocrine tissues of NOD mice and the unique proteolytic activity would indicate that, similar to diabetes in these mice, numerous putative autoantigens would be present In trus regard this work has largely focused on the use of

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1 35 PSP as a marker of glandular cl1anges in NOD ti ss ues Futt1re studies will inve s tigate the spec ific proteases in vo lved in PSP cleavage as well as in vestigate a pote11tial role of PSP as an autoantigen. Lastly, a pron1ising future lies ahead for the application of congenic NOD strains used to s tudy diabetes susceptibility intervals to tl1e study of autoin1111une sicca synd r on1e Recent research in our group has show11 secretory dysfu11ction in NOD B 1 OH-2b mice which do not develop insulitis or diabetes This work has allowed for the developn1ent of the first proposed muri11e model for primary Sjogren's Syndrome Additionally, NOD 1nice containing the Bl O PSP gene contained on the idd-13 gene interval will be s tudied to determine whether subm andibular gland expression of PSP is due to differences in the NOD PSP gene itself, or caused by additional factors Tt1ese s tudies, as well as tl1o se described above, will serve to provide a co1nprehensive ar1alysis of the mechanis1ns leading to secretory dysfunction in the NOD rnouse with future in1plications for our long -ter m goal of understanding tl1e pathogenesis of l1um a n Sjogren s Syndror11e

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37 Kay R Hay E and Dyer, P An abnor1nal T cell repertoire in hypergam1naglobuli11aemic primary Sjogren's Syndrome C/i11. '
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149 of the 111etl1od for titration of the binding proteins a11d co1npetetive binding studies A11 a l Bioc/1e111 1986~ 154 : 138-143 152 lsberg, R R Dis c rin1i11atio11 between intracellular uptake ar1d surface adl1esion of bacterial pathogens Scie11 c e 1991 ~ 252 : 934-938 153 Minato, T Wang, J Akasaka., K Okada, T Suzuki, N and Kataoka, T Quantitative analysis of mutually competitive binding of human Raf-1 and yeast adenylyl cyclase to Ras protein J Biol. C /1e111 1994~ 269 : 2 0 845-20851 154. Mengqud, J., Ohayon, H Gounon, P Mege, R M., and Cossart, P. cadherin is the receptor for intemalin, a surface protein required for entry of L. monocytogenes into epithelial cells Ce// 1996~ 84 : 923-932 155 Mikkelsen, T Brandt, J., Larsen, J Larsen B Poulsen, K I11gersslev, J Din, N and Hjorth, P Tissue specific expressio11 in the salivary gla11ds of transgeruc 11uce N11 c Acids Res 1992 ~ 20:2249-2255 156. Bedi, G Rapid radioimmunoassay for inducible rat submandibular gland cysteine proteinase inhibitor ( cystatin). Jn111111110/. I,,, est 1990~ 19 : 199-208 15 7 Barret, A New assay for Catl1epsin B and other th i ol proteases A11al. BiocJ1en1 1972~ 47 : 280-293 158. Henkins Y ., Van der Vellden, U Veerman, E and Arnerongen, A Protein, albun1i11 ar1d cystatin concentrations in saliva of l1ealtl1y subjects a11d of patients with gingivitis or periodontitis J Periodo11t. Res 1993 28 : 43-48.

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BIOGRAPHICAL SKETCH Christopher Paul Robinson was born in Nortl1ampton, MA on March 25 1970, tl1e youngest of three cl1ildren born to Paul and Wesley Robinson He con1pleted his undergraduate education in molecular biology at Lehigh University in 1 992. In Jan u ary of 1993 he moved to Gai11esviJle, FL, to begin working toward his Ph D at the University of Florida, D epart me11t of Pathology and Laboratory Medicine under the mentorship of Dr Ammon Peck His re se arch goals led him to undertake a joint pr ojec t with the Department of Oral Biology with the comentorsllip of Dr Michael Humphrey s-Be her. On June 19, 1994 Chris married his college sweetheart, Meryl Weiss in Woodbu l) NY To date he has authored five articles wl1ich l1ave been subnutted or publisl1ed in peer reviewed journals, and h as appeared a s a coauthor on several additional publications In addition, l1e has presented his findings at the 5' International Meeting on Sjogren s Syndrome in Amsterdam the Second International Conference on the Lacrimal Gland, Tear Film, and Dry Eye Syndromes in Bertnuda and the 25 th Annual Meeting of the American Association of Dental Research, where he received the Edward H Hatton Award for excellence in predoctoral re s earch Cl1ris plans to continue his work in the field of autoimmune Sjogren's Syndrome in collaboration with his mentors at the University of Florida and Dr Roland Jonsson in Bergen, Norway. 150

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I certify that I have read this study and that in 111y opinio11 it conforn1s to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dis sertat ion for the degree of Doctor of Philosopl1y \ c atr of essor of Patl1ology and Laboratory Medicine I certify tl1at I have re ad this study and that i11 my opinion it confonns to acceptable standards of scl1olarly prese11tatio11 and is fully adequate, in scope and quality as a dissertation for the degree of Doctor of Philosophy. Mchael G. Humplireys-Beher, Professor of Oral Bi o logy I certify that I have r ead this study a11d that in my opinio11 it coriforn1s to acceptable sta11dards of scholarly preser1tation and is fully adequate, in scope and quality, as a dissertation for the degree of Do cto r of Philo sop hy Micl1ae Cla -Salzle Assistant Professor o Laboratory Medicine I ology and I certify t l1a t I l1ave read this study and tl1at i11 111y opinion it confom1s to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a di ssertatio n for tl1 e degree of Do ctor of Pllilo sop hy Jo Schiffen auer 1 ociate Professor o f Molecular Genetics and Mcrobiology I certify that I have read thi s study and that in my opinion it conforms to acceptable sta ndards of scholarly presentation and is fully adequate, in scope and quality, as a di sserta tion for the de gree of Doctor of Philosophy. L Linda L Bri ... n.,ey Professor of Orthodontics

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This dissertation was subn1itted to the Graduate Faculty of tl1e College of Medicir1e and to tl1e Graduate Scl1ool and was accepted for partial fulfillment of the requiren1ents for the degree of Doctor of Pl1ilo so phy May 1997 De a n, College of Medicine Dean, Graduate Scl100I

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UNIVERSITY OF FLORIDA 111 11 11111 1 1111 1 111 1 111 1 111 1 1 / ll ~ I I I II II I I II I II I I II I I Ill l l lll I I 3 1262 08554 8997


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