Citation
Role of Complement, B Cells, and Genetics in Development of Sjogren's-Like Autoimmune Exocrinopathy

Material Information

Title:
Role of Complement, B Cells, and Genetics in Development of Sjogren's-Like Autoimmune Exocrinopathy
Creator:
NGUYEN, CUONG QUOC
Copyright Date:
2008

Subjects

Subjects / Keywords:
Antibodies ( jstor )
Autoantibodies ( jstor )
B lymphocytes ( jstor )
Diseases ( jstor )
Lacrimal apparatus ( jstor )
Mice ( jstor )
Receptors ( jstor )
Saliva ( jstor )
Salivary glands ( jstor )
Sjogrens syndrome ( jstor )
City of Gainesville ( local )

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Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright Cuong Quoc Nguyen. Permission granted to University of Florida to digitize and display 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.
Embargo Date:
8/31/2006

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Full Text












ROLE OF COMPLEMENT, B CELLS, AND GENETICS IN THE DEVELOPMENT
OF SJOGREN'S-LIKE AUTOIMMUNE EXOCRINOPATHY















By

CUONG QUOC NGUYEN


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


2006



























Copyright 2006

by

Cuong Quoc Nguyen


































I would like to dedicate this dissertation to my mother and my daughter, Be Thuong
(Hanna). My may's endless sacrifice, devoted love, and inspirational struggle inspire me
to pursue this degree. My daughter's smile and the way she says "Daddy" motivate me
complete it.















ACKNOWLEDGMENTS

I would like to thank all the members of my supervisory committee for their

continued support and encouragement, which include Dr. Ammon Peck, Dr. Sally

Litherland, Dr. Edward Chan, Dr. Westley Reeves, and Dr. John Aris. I would especially

like to thank my mentor, Dr. Peck, for his exemplary scientific and academic guidance.

His hardworking attitude and his enthusiasm for science always inspire me to be a better

student. I am sincerely indebted to his epitome of a gentleman with great personal and

scientific value and integrity.

I would like to thank my past and present lab members who have made my

graduate school experience in the laboratory educational and enjoyable, which include

Dr. Seunghee Cha, Dr. Smruti Killedar, Dr. Jeuhua Gao, Dr. Maire Doyle, Lori Boggs,

Woosuk Jang, Eric Singson, Brian Alverado, Jin Wang, Angie Kim, Marievic Bulosan,

and especially Mrs. Janet Cornelius who has always been the heartbeat of the laboratory.

Her willingness to help, knowledge of science, and high expectation have helped many

students like me graduate with a sense of accomplishment and pride. I would like to also

thank my friend, Monique Lara, for all her patience and wonderful editorial support.

Most importantly, I would like to thank my family members, Ma, Anh Hai, Anh

Ba, Anh Tu, Anh Phap, Chi My, Chi Ha, Chi Huong, Em Teo Em, Be Dao, Em Cu, Be

Le and the cutest, craziest, but phien-est baby in the whole wide world, Hanna, for their

unconditional love and endless support. I would not be the person that I am today

without them in my life. I would like to show my deepest appreciation and respect to my









father who is only a prayer away and who has given me the most unforgettable and

wonderful childhood. Last but not least, I am grateful for all my nephews and nieces,

Chau Vyvy, Chau Huu, Chau Helena, Chau Bac, Chau Cecilia, Chau Ethan, and Chau

Mancy, for bringing so much laughter and love to my life.
















TABLE OF CONTENTS


page

A CK N OW LED GM EN TS ..................................................... .............. iv

LIST OF TABLES.. .. ....... ......... ................. ........................ .x

LIST OF FIGURES ........................ ............ .. .... ............. xi

ABSTRACT........................................... ............xiii

CHAPTER

1 BACKGROUND AND SIGNIFICANCE.........................................................

Sjogren's Syndrom e (SjS) in H um ans....................................................................1
Secretory Function of the Exocrine Gland .....................................................1
Clinical Characteristics of Sj S ................................... ............... ...............3
Genetic Predisposition........................ .......5
Diagnostic Criteria/Technique................ .......................6
Mouse Models of Sj S-like Autoimmune Exocrinopathy....................................7
Sj S-like Autoimmune Exocrinopathy of Mice ........................7
Genetic Predisposition of Sj S-like Autoimmune Exocrinopathy ............... 11.....11
The Role of B Lymphocytes in the Development of Sj S-like Autoimmune
Exocrinopathy in M ice.......................................................................... 12
Development of Germinal Center (GC)-like Lymphocytic Foci in the
Exocrine Glands................................. .................... 12
Development and Characteristics of B Cells in Sj S ........................ ...............14
The Role of Marginal zone (MZ) and B-l B Cells in SjS................................15
Expression of Restricted Repertoire of the B Cell Receptor in Sj S Disease.......17
Signal Transduction of the B Cell Receptor................................ ... ................19
Intimate Interaction of the B Cell Receptor and B cell Co-Receptors .............20
The Role of Autoantibodies in the Development of Sj S ................. ........................22
Anti-Muscarinic Acetylcholine Type-3 Receptor Autoantibodies The
Effectors of Glandular Dysfunction............... ... .............25

2 EFFECT OF COBRA VENON FACTOR (CVF) ON COMPLEMENT AND B
LYMPHOCYTES IN THE NOD.B10.H2B MICE................................................35

Introduction ......................... ................................................. .......................35
Materials and Methods ................................................... ........36










Animals.................................. ... .......... ........ 36
Complement Depletion by Cobra Venom Factor..............................................37
M easurem ent of Salivary R ates.................................................. ................ .37
Histology and Immunofluorescent Staining for B and T Lymphocytes........ 38
Flow Cytom etry ................... ..... ... .. ........................... ...... .. .... ...... .. 38
Detection of Anti-Nuclear Autoantibodies in the Sera...............................39
Results..................................... .. .........................................................39
Effects of CVF Treatment on the Sj S-like Disease Profile in NOD.B10-H2b
M ice ....................... ... ........... ...................................................39
Effects of CVF Treatment on B cell Sub-Populations ......................................42
Discussion............................ ................................... 42

3 EFFECT OF KNOCKING OUT COMPLEMENT COMPONENT 3 ON
COMPLEMENT AND B LYMPHOCYTES IN THE DEVELOPMENT OF SJS-
LIKE AU TOIM M U N E D ISEA SE ........................................................................ 52

Introduction ...................................... .......... ........... .52
Materials and Methods ..............................................................54
Generation of C57BL/6.NOD-AecJAec2.C3- Mouse............... ...............54
Proteolysis of Parotid Secretory Protein (PSP) .................................................54
Detection of Cleaved Caspase-3 in the Submandibular Glands.........................55
Salivary Protein Concentration and Salivary Amylase Activity ......................55
Histological Examination of Submandibular and Lacrimal Glands....................56
Detection of Anti-Nuclear Autoantibody in Sera........... ...........................56
Detection of Immunoglobulin Specific Muscarinic Type III Receptor
Autoantibody.......................... ..... ..... ......... 57
Flow Cytometry for Subpopulations of B cells................................ .........57
Measurement of Stimulated Saliva Secretion.......... ......................................58
Statistical Analysis ...................... ........ ........ .... ...58
Results.............. ......... ......... ............ .. ...............58
Profiling of Phase I of Sj S-like Autoimmune Exocrinopathy of
C57BL/6.NOD-AecJAec2.C3X-1 Mice ............................. .................58
Profiling of Phase II of Sj S-like Autoimmune Exocrinopathy of
C57BL/6.NOD-AecJ.Aec2.C3X-1 Mice ...........................61
Profiling of Phase III of Sj S-like Autoimmune Exocrinopathy of
C57BL/6.NOD-AecJAec2.C3X-1 Mice ....................... ..... .......................63
Characterization of the changing dynamics in the subpopulation of B
lymphocytes: Marginal Zone (MZ) and Follicular (FO) B Cells ................64
Discussion ...................................... ................... .....................66

4 INVOLVEMENT OF STAT6 IN THE IL4 SIGNALING PATHWAY DURING
THE CLINICAL PHASE OF SJOGREN-LIKE SYNDROME.............................83

Introduction...................................... ................................. ......... 83
M material and M methods ................................................................ ......... 85
Animals.........................................85
Histology ........................................ ........85









Immunofluorescent Staining for B and T Lymphocytes ....................................86
Flow Cytometry ............... ......... .. ........... .. ..................86
Proteolysis of Parotid Secretory Protein........................................ .................87
Detection of Anti-Nuclear Autoantibodies in the Sera................... ....................87
M easurement of Salivary Flow Rates............................................ 88
Profile of Im m unoglobulin in Serum .............................................. ...................88
Immunoglobulin Specific M3R Autoantibodies Detection Using
Im m unofl ourescence .................................................................................. 88
Statistical Analysis ................................................. .........89
Results ................................................ ........ .............................89
Generation of NOD.B10-H2b.C-Stat6+, NOD.B 10-H2b.C-Stat",
NOD.BO1-H2b.C-Stat6" ............................................................... .....89
Pathophysiological Examination of NOD.B 10-H2b.C-Stat6? +, NOD.B 10-
H2b.C-Stat6l"andNOD.B10-H2b.C-Stat6- Mice.................. .. ..... 90
Leukocytic Infiltrations in the Submandibular and Lacrimal Glands in Both
the NOD.B 10-H2b.C-Stat6 and NOD.B 10-H2b.C-Stat~ Mice ...............91
Detection of Anti-Nuclear Antigens in the Sera of NOD.B 10-H2b.C-
Stat l and NOD.B l0-H2b.C-StatC M ice................................................92
Determination of Immunoglobulin Subclass Levels in Sera of NOD.B 10-
H2b.C-Stat6 land NOD.B10-H2b.C-Stat6"- Mice..........................................93
Detection of Specific Immunoglobulin against the Muscarinic Type 3
Receptor Autoantibody ..................... ........ ...............94
M easurement of Salivary Flow Rates......................... ................ 95
Discussion ............... ....................... .......... .......... 96

5 SJOGREN'S SYNDROME-LIKE DISEASE OF C57BL/6.NOD-AEC7AEC2
MICE: GENDER DIFFERENCES IN KERATOCONJUNCTIVITIS SICCA
DEFINED BY A CROSS-OVER IN THE CHROMOSOME 3 AEC] LOCUS......109

Introduction................... ................. ....... ...... ......... 109
M materials & M methods .................. ..................................... ...... .... ............... I
Animals....................... ............ ...............111
Histology ........................................................ ...... ..... ........ 112
M easurem ent of Saliva and Tear Flow Rates....................................................112
Saliva Protein Concentration and Salivary Amylase Activity ..........................113
Detection of Caspase-3 Activity........................ .........................................114
Proteolysis of Parotid Secretory Protein (PSP) .................................................114
Detection of Anti-Nuclear Autoantibodies (ANAs) in the Sera........................114
Detection of Immunoglobulin-Specific Anti-M3R Autoantibodies ..................114
Statistical Analyses............................... .. .. ...... ...... ..... ........ ..115
R esults.................. ......... .. ......... .. .. ..................................................... 116
Genetic Profile of Recombinant Inbred Line C57BL/6.NOD-AecJRJAec2
M ice .............. ... ....... .... .. ............ ...... 116
Phase 1 Profile of Sj S-Like Disease in C57BL/6.NOD-AeclRJAec2 Mice .....117
Phase 2 Profile of Sj S-Like Disease in C57BL/6.NOD-AeclRJAec2 Mice .....118
Phase 3 Profile of Sj S-Like Disease in C57BL/6.NOD-AeclRJAec2 Mice .....119
Discussion ............... ....................... ......... .......... 121









6 OVERALL CONCLUSION........................................................136

Focus of the D issertation ................................... ............................. 137
The Role of Complement in the Pathogenesis of Sj S-like Autoimmune
Exocrinopathy ........................... .... ... .... ........ ........ .... ....137
The Role of STAT6 of IL4 Signaling Pathway in the Pathogenesis of Sj S-like
A utoim m une Exocrinopathy ..................................................................... 138
Genetics of Sj S-like Autoimmune Exocrinopathy ............ ... ......... 139
The Clinical Implications in Translational Research..................................... 141

L IST O F R E F E R E N C E S ............................................................................................ 143

BIOGRAPHICAL SKETCH .............................................................................162
















LIST OF TABLES


Table page

1-1 M house strains used in the study of Sjogren's syndrome ..........................................29

1-2 Comparison of general symptoms of Sjogren's syndrome patients and NOD mice 32

2.1 Changes in the splenic CD19-positive B cell phenotypes. Each group included
eight female mice. ........................... .............. ......... 51

3-1 Changes in CD 19+ splenic B cell populations............................ ........... ....82

5-1 Caspase-3 activity in the salivary and lacrimal glands of
C57BL6.NOD.Aec]R]Aec2 mice. ............... ............................ .......... 135
















LIST OF FIGURES


Figure page

1-1 Model for the progression of Sj S-like autoimmune exocrinopathy ......................33

1-2 Dynamic cellular composition oflymphocytic infiltration in submandibular
glands of NOD mouse. .......................................................34

2-1 C3 levels in the plasma of NOD.B10-H2b mice following treatment with cobra
venom factor (CVF) or PBS................ ....................... 46

2-2 Salivary flow rates of NOD.B10-H2b mice treated with CVF or PBS. ...................47

2-3 Histological examination of the exocrine glands of NOD.B10--H2b mice treated
with CVF or PBS............................... ...... ....................48

2-4 Detection of anti-nuclear autoantibodies (ANAs) in NOD.B 10-H2b mice treated
with CVF or PB S .............. ....... ................ ...............49

2-5 Reduced expression of CD19 on splenic B lymphocytes of NOD.B10-H2b mice
treated with CVF.. ................................................. ........ 50

3-1 Proteolysis of parotid secretary protein. ..........................................................71

3-2 Examination of apoptosis by the presence of cleaved Capase-3 in the
submandibular glands... .. .................................................... ......... ......... ........72

3-3 Histological examination of the exocrine glands. .................................................73

3-4 Detection of anti-nuclear autoantibodies (ANAs) using Hep2 cells as subtrate. ....74

3-5 Detection of anti-muscarinic acetylcholine type-3 receptor antibodies. ..........75

3-6 Detection of anti-muscarinic acetylcholine type-3 receptor antibodies. ................76

3-7 Stimulated saliva flow of fem ale animals. ................ .................. ........ ........ 77

3-8 Stimulated saliva flow of male animals. ..................... ......... ...............78

3-9 A m ylase activity in saliva.. ........................................................... 79

3-10 Salivary protein concentration. ......................................................... 80









3-11 An representation of an approach to delineate marginal zone (MZ) and follicular
(FO) .................................................81

4-1 Generation of NOD.B10.H2b.C.STAT6- mouse........... ......... ..............101

4-2 Detection of proteolytic activity against PSP in saliva ............... .............102

4-3 Histological examination of the exocrine glands. .................................................103

4-4 Detection of anti-nuclear autoantibodies (ANAs)............................104

4-5 Amount of immunoglobulin isotypes present in sera of female .........................105

4-6 Detection of M3R isotypic autoantibodies by immunoflourescence. .................106

4-7 Salivary flow rates of female animals. ...............................107

4-8 Salivary flow rates of m ale anim als. .....................................................................108

5-1 Generation of the C57BL/6.NOD-AeclRIAec2 mouse line. .............................127

5-2 Detection of proteolytic activity against PSP in the salivary gland tissues of
C57BL/6.NOD-AecJRJAec2 mice. ....................... ...................... 128

5-3 Histological characterization of sialadenitis and dacryoadenitis of male
C57BL/6.NOD-AecJRJAec2 mice. ............ .............................................129

5-4 Histological characterization of sialadenitis and dacryoadenitis of female
C57BL/6.NOD-AecJRJAec2 mice. ...................................................... 130

5-5 Detection of ANAs in sera of C57BL/6.NOD-AeclR]Aec2 mice.........................131

5-6 Temporal loss of secretary function in C57BL16.NOD-AeclR]Aec2 mice. .........132

5-7 Detection of anti-muscarinic acetylcholine type-3 receptor antibodies in the sera
of C57BL/6.NOD-AeclRIAec2 mice. .............................133

5-8 Time-dependent loss of amylase activity in the saliva of C57BL/6.NOD-
AeclR]Aec2 mice. ............. ....... ............ ..................... 134
















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

ROLE OF COMPLEMENT, B CELLS, AND GENETICS IN THE DEVELOPMENT
OF SJOGREN'S-LIKE AUTOIMMUNE EXOCRINOPATHY

By

Cuong Quoc Nguyen

August 2006

Chair: Ammon Peck
Major Department: Medical Sciences-Immunology and Microbiology

Sjogren's syndrome (Sj S) is a human autoimmune disease characterized by the loss

of exocrine function as a result of a chronic immune attack directed primarily against the

salivary and lacrimal glands leading to xerostomia (dry mouth) and xerophthalmia (dry

eyes). Based on various animal models, the progression of SjS can be separated into

three distinct and consecutive phases. Phase I is involved in the initiation of glandular

pathophysiological changes associated with aberrant mRNA transcription, protein

expression and acinar cell apoptosis. During Phase II, initiation of acinar cell death and

immunological responses against cryptic peptide presentation results in leukocytic

infiltrations of the targeted glands expressing pro-inflammatory cytokines. In Phase III,

the onset of clinical manifestation occurs, characterized by the loss of exocrine secretary

function, and loss of important secretary proteins. Glandular dysfunction which

commences at this phase is thought to be mediated by activity of autoantibodies produced

by B lymphocytes.









The main focus of this dissertation is to elucidate genetic elements regulating the

development of Sj S-like autoimmune exocrinopathy and to identify potential approaches

that could interrupt any of the phases of the autoimmune process to prevent the onset of

the clinical disease. Results showed that the depletion or genetic elimination of

complement component C3 prevented onset of Sj S-like disease in genetically pre-

disposed pathology associated with both Phase II and III. In addition, the elimination of

IgGI isotypic antibody against the muscarinic acetylcholine type III receptor by

genetically knocking out of the STAT6 gene appeared to affect only phase III of the

disease, resulting in retention of normal salivary flow rates in the animals. Genetic

analysis of Sj S-like disease has redefined smaller genetic regions that contain genes

regulating the events of Phase 2. Narrowing down the Aecl region to a centromeric

stretch less than 20 cM is providing important potential candidate genes regulating the

development, onset and underlying cause of Sj S.














CHAPTER 1
BACKGROUND AND SIGNIFICANCE

Sj6gren's Syndrome (SjS) in Humans

Sjogren's syndrome is a human disease characterized by exocrine gland

dysfunction resulting from the consequences of an autoimmune response (1-10). Primary

Sj S is characterized generally by a chronic autoimmune attack against both the lacrimal

and salivary glands, while secondary Sj S is marked by an autoimmune attack against the

lacrimal and/or salivary glands in the presence of another autoimmune disease, most

often a connective tissue disease like scleroderma, rheumatoid arthritis (RA) or systemic

lupus erythematosus (SLE) (1, 11). In addition to the apparent primary sites of Sj S, i.e.,

the lacrimal and salivary glands, other tissues that may become affected include the entire

GI tract, skin, the lungs, the vasculature, kidneys, bladder and the vagina. Involvement of

the musculature often leads to fibromyalgia-like symptoms and chronic fatigue. As with

many autoimmune connective tissue diseases, there exists a sexual dimorphism in Sj S

with women affected 10- to 20-times more frequently than men, suggesting a role for sex

hormones in disease susceptibility, possibly related to the relative balance between

estrogen and androgen (12-16).

Secretory Function of the Exocrine Gland

Secretory mechanism of the exocrine glands is a highly regulated and intricately

controlled process. The major product of the salivary glands is saliva. Proteins such as

amylase and lingual lipase contained in saliva assist in the digestion of food by breaking

down the carbohydrates (starch) in the oral cavity and lipid hydrolysis respectively. The









mucus and water in saliva moisten the oral mucosa, aiding in swallowing and dissolving

of food. Lysozyme, lactoferrin, and IgA found in saliva also provide protection against

bacterial infection and control bacterial flora in the oral cavity, thus providing a very

important innate immune defense mechanism in the mouth. Saliva contains high

concentrations of calcium and phosphate that help in the development of new teeth and

repair of enamel lesions. Therefore, the loss of salivary flow can facilitate the

development of candidiasis, a fungal infection caused by Candida albicans, painful

ulcers, rampant dental caries and extreme difficulty in swallowing (17).

The salivary glands include the submandibular gland, sublingual gland and the

parotid gland as well as numerous minor salivary glands. The production of saliva is

under the control of the autonomic nervous system. The salivary gland is innervated by

both sympathetic and parasympathetic nerves. Upon neural stimulation, the

parasympathetic nerves utilize the muscarinic type III receptor (M3R) to induce the

secretion of water-rich saliva in the salivary glands, while the sympathetic nerves use the

P-adrenergic receptor to stimulate the release of protein-rich saliva. Acinar cells take

advantage of different ion transporters and co-transporters allowing the influx of Cl ions

into the acinar lumen. The accumulation of CYl ions generates a negative electrical

difference which initiates the passive movement of Na ions through the acinar tight

junctions. Higher concentration of ions in the acinar lumen creates an osmotic gradient

that favors the movement of water molecules from the surrounding blood capillaries

across tight junctions and water channels such as the Aquaporin 5 channel. This results

in the formation of isotonic primary saliva. The lumen of acinar cells collects all the

secretary products which are transported by long intercalated ducts to the striated ducts.









In the striated ducts, NaCl is reabsorbed back to the blood capillaries and saliva becomes

hypo-osmotic. Kallikrein, a serine protease secreted by the epithelial cells of the striated

duct, processes the proline-rich proteins and cystatins in the saliva. In addition, plasma

cells secrete IgA which reaches the lumen of the acinus and striated duct by transcytosis.

The final saliva contains a complex of proteins with antimicrobial activity and with

digestive function amylasee). Bicarbonate, the primary buffering agent of the saliva, is

produced in the striated duct (17).

Clinical Characteristics of SjS

While keratoconjunctivitis sicca (dry eyes) and stomatitis sicca (dry mouth) are

assessed by specific tests for changes in exocrine gland flow rates and

biochemical/enzymatic changes in protein composition and function, the recently adopted

European-American Consensus Group criteria for diagnosis of SjS recommends detection

of infiltrating lymphocytes within a minor salivary gland, determined by a

histopathological analysis of a labial gland lip biopsy. However, lip biopsy is not

required if anti-Ro or anti-La autoantibodies are detected (18). Less often used is an

analysis of a lacrimal gland biopsy (19). Nevertheless, based on studies in both humans

and animal models, infiltrates appear generally as peri-ductal foci within the glandular

architecture of the lacrimal and salivary glands consisting of CD4+ T cells, CD8+ T cells,

B cells and macrophages. The T cells exhibit a preferential antigen receptor repertoire ,

while the overall infiltrating cells express various cytokines (including IL-1p, IL-6, IL-

10, TNFac, and IFNy) whose significance in the autoimmune pathology has yet to be

determined (20-23). Serological evaluations have shown the presence of rheumatoid

factor, elevated immunoglobulin levels (hypergammaglobulinemia) and anti-nuclear









autoantibodies, especially anti-SS-A/Ro and anti-SS-B/La antibodies. Additional

autoantibodies are present which react with numerous cellular components of the

exocrine glands, probably representing spreading epitopes (24-35). Of late, intense

interest has revolved around the presence of antibodies to the muscarinic acetylcholine

type-3 receptor (M3R) (36-44).

Sj S is only one of many dry mouth/dry eye diseases, but considered one of the

more severe forms of these conditions. Xerostomia and keratoconjunctivitis sicca result

respectively from basic changes in the saliva and tear flow rates, the composition of

saliva and tears, and/or combinations thereof. Underlying causes of xerostomia include

the natural aging process, use of medications, asthma and mouth breathing,

chemotherapy, radiation therapy, autoimmune attack against secretary tissues/glands of

the mouth, thyroid dysfunction, kidney dialysis and/or stroke. Likewise, underlying

causes of xerophthalmia include the natural aging process, physical injury, surgical

procedures, meibomian gland dysfunction and/or autoimmune attack against one or more

of the multiple secretary tissues/glands of the eye. Because saliva is a critical factor in

oral health, patients with dry mouth can present with increased caries, increased oral

microbial infections, halitosis, cracked lips and bleeding gums, taste disturbances,

difficulty in eating, swallowing, and talking. In addition, patients can suffer from

esophageal dysphagia, epigastric pain and dyspepsia due in part to decreased levels of

epidermal growth factor (EGF) in saliva, as well as poor nutritional uptake. Moreover,

between 4-10% of patients with SjS will develop non-Hodgkin's malignant B cell

lymphomas (45-48). While considerable emphasis is placed on manifestations resulting

from xerostomia, the manifestations from xerophthalmia brought on by decreased tear









fluid secretion in conjunction with an increase in tear fluid evaporation are just as

debilitating. Complaints from patients with dry eyes include burning, grittiness, itching,

fatigue, blurred vision and, paradoxically, watery eyes resulting from increased reflex

tear secretions. Over time there is eye surface deterioration and ulceration, leading to

small red-appearing eyes with crusts in the ciliae, debris in the tear film, meibomitis,

mucus strands adhering to the corneal surfaces, reduced light reflectivity and irregular

blinking. In general, these manifestations of dry mouth and dry eyes, especially in SjS,

appear to correlate with a loss of exocrine cell mass, an onset of exocrine cell senescence

or refractivity, and loss of neural regulation of ocular secretary function (19).

Genetic Predisposition

Sj S shows a weak tendency toward familial aggregation which, together with the

presence of common autoantibodies in Sj S patients, suggests that genetic factors are

operative in disease susceptibility (49). While environmental triggers responsible for

initiating Sj S are unclear, intrinsic genes contributing to disease susceptibility are thought

to be critical potentiators for development of autoimmunity. Although the most probable

hereditary markers in disease susceptibility are those encoded by genes of the major

histocompatibility complex (MHC), previous associations with HLA-DQ are most likely

reflecting the presence of anti-nuclear SS-A/Ro and SS-B/La autoantibodies (50).

Furthermore, studies from different ethnic groups have yielded inconsistent results

suggesting only weak MHC class II associations for SjS (51). More importantly, family

studies suggest autosomal genes not linked to HLA and/or immunoglobulin genes are an

important element of autoimmune exocrinopathy susceptibility (52), a conclusion

consistent with studies in NOD mice, an animal model of Sj S, where non-MHC genes

clearly control susceptibility (53-55).









Diagnostic Criteria/Technique

While diagnosis of SjS is based, in part, on subjective patient symptoms, a number

of specific clinical tests are also critical. For xerostomia, these include (a) desiccated

buccal epithelium, (b) reduced production of either stimulated or unstimulated saliva

flow, (c) reduced amylase activity, (d) reduced EGF levels, and (e) detection of anti-

nuclear autoantibodies (ANAs), especially anti-SS-A/Ro and anti-SS-B/La, and (f)

presence of leukocyte foci (>50 lymphocytic cells per 4 mm2 is defined as a focus) within

minor salivary gland biopsies. For keratoconjunctivitis sicca, these include (a) break-up

time test which measures the ability of pre-corneal tear films to maintain their integrity,

(b) Schirmer-1 test which measures tear flow rates, (c) Rose-Bengal (or lissamine green)

dye test which shows staining of desiccated epithelial cells lacking mucous protection,

(d) lysozyme and lactoferrin enzyme activity measurements, and rarely (e) a minor

salivary gland biopsy to determine the presence ofleukocyte infiltrates. Despite this

battery of tests, both the diagnosis and the underlying mechanisms of autoimmune

exocrinopathy are difficult to assess because Sj S patients usually present when the

autoimmune process is at or near its end-stages. Thus, at the present time, a direct cause

of Sj S remains elusive and correlative, and this limits development of pre-disease

biomarkers, intervention therapies, as well as an ultimate prevention and cure for the

illness. As a result, it has been important to turn to animal models of Sj S, not only to

define the pathophysiological processes, but also to identify appropriate biomarkers

predictive of Sj S.









Mouse Models of SjS-like Autoimmune Exocrinopathy

SjS-like Autoimmune Exocrinopathy of Mice

Over the past two decades, a variety of mouse models exhibiting various aspects of

Sj S, whether spontaneously appearing or experimentally induced, have been intensively

investigated in an attempt to identify the nature of this autoimmune disease. Typically,

these mouse models show lymphocyte infiltration of the exocrine glands, increased

expressions of pro-inflammatory cytokines, generation of unique autoantibodies

(especially ANAs, anti-a-fodrin, and anti-muscarinic acetylcholine type-3 receptor

(M3R) antibodies), and eventually decreased saliva flow rates. Strains that have been

extensively studied include NZB/NZW Fl-hybrids, MRL/lpr, NOD/LtJ and NFS/sld.

More recently, several new strains have been added; including the Id3 gene knock-out

(KO) mouse, the aromatase gene KO mouse, the Baff gene knock-in (KI) mouse, as well

as the IQI/Jic mouse and C57BL/6.NOD-AeclAec2 congenic line. A listing of these

various models along with their general disease profiles is presented in Table 1-1. While

each strain has been reported to resemble features of Sj S in human patients, none

recapitulate completely the pathological characteristics of the human disease.

One of the more interesting and well-studied models of Sj S is the NOD mouse

which closely mimics the human disease (see Table 1-2). A major strength of this model

has been the ability to study a large number of congenic partner gene KO strains, e.g.,

NOD-scid, NOD.Ifny NOD.IL2 NOD.IL4 NOD.IL]O NOD.Igu and

NOD.Stat6~ permitting investigations into the role of individual genes in the

development and onset of Sj S-like disease in this model (56-59). Taken together, these

studies suggested that the disease could be divided into three distinct consecutive but

continuous phases (59-62). In phase 1 (initiation of glandular pathology), a number of









aberrant genetic, physiological and biochemical activities associated with retarded

salivary gland organogenesis and increased acinar cell apoptosis occur sequentially prior

to and independent of detectable autoimmunity (62). In phase 2 (onset of autoimmunity

believed to result from the acinar cell apoptosis), leukocytes expressing pro-inflammatory

cytokines infiltrate the exocrine glands, establishing lymphocytic foci, first of T cell

clusters followed by recruitment of B lymphocytes (63, 64). In phase 3 (onset of clinical

disease), loss of salivary and lacrimal gland secretary functions occur, most likely the

result of (auto)-antibodies reactive with the M3Rs (37, 56, 65, 66). These three phases

are portrayed in Figure 1-1. Although the onset of Sj S-like disease in NOD mice is

independent of the production of ANAs (anti-nuclear antibodies), Scofield et al. (67)

have recently shown that immunization of BALB/c mice with Ro antigen induces a SjS-

like disease, pointing to the importance of individual antibodies in the onset of exocrine

gland dysfunction. Interestingly, the recently derived IQI/Jic mouse line appears to

mimic the disease profile of the NOD mouse (68).

A critical observation derived from studies with NOD mice is the important role of

B lymphocytes in the development and onset of disease. First, NOD.Igpu'~ mice lacking

mature B cells fail to develop glandular dysfunction despite exhibiting peripheral T cell

activation and T cell infiltrations of the salivary and lacrimal glands (64). Second,

NOD.IL4^ mice lacking the ability to produce the cytokine IL-4 also fail to develop

glandular dysfunction despite exhibiting both T and B cell activation in the periphery, T

and B cell infiltration of the salivary and lacrimal glands, and the production of ANAs

(58). Third, NOD.IL4-1 mice fail to produce IgGI subclass antibodies reactive with the

muscarinic acetylcholine receptors, a deficiency that appears to be circumvented









following an adoptive transfer of T cells isolated from NOD.JIgyu mice or injections of

recombinant IL-4 cytokine protein (58). Considering these observations, it is not

surprising that passive transfer of serum IgG from human Sj S patients or from diseased

NOD mice, but not healthy human subjects or pre-diseased animals, into NOD.Igu/'- mice

results in a temporary loss of saliva secretion (66). Furthermore, serum IgG fractions

from human Sj S patients, but not healthy human subjects, can competitively inhibit the

binding of the muscarinic receptor agonist, [3H]-quinuclidinyl benzilate, to salivary gland

membranes (66).

The disease profile observed in NOD.IL4 mice raises several questions pertinent

to the role of the B lymphocyte in development and onset of Sj S-like disease. First, do B

lymphocytes act as antigen-presenting cells (APCs) for initiation of the Sj S-like

autoimmune response, as has been proposed for initiation of type 1 diabetes in NOD

mice? Second, do the B lymphocyte populations in NOD mice respond abnormally to the

presence of IL-4 resulting in circumvention of normal homeostatic mechanisms that

would prevent over-proliferation, survival and escape from negative selective pressures?

Third, is there an autoimmune B cell population present in NOD mice that is activated by

IL-4 to produce a specific subclass of IgG antibody that can inhibit normal acinar cell

function? Whereas sorting out the possibility that the B cell population can act as APCs

might be difficult due to the fact that the autoimmunity is generally characterized as a

hypersensitivity type 2 (or antibody-mediated) immunological response, recent studies

indicate that IL-4 may be important in both survival of M3R (auto)-reactive B cells and

in their IgG isotype switching to promote production of IgGI anti-M3R autoantibody

(58). This latter concept is supported by observations from studies using BAFF-









transgenic mice in which there is an over-production of BAFF. These BAFF-transgenic

mice exhibit enhanced B cell proliferation and survival, as well as altered differentiation

patterns, and develop an autoimmune condition resembling SLE (69). Interestingly, by 4

months of age, these mice develop a secondary pathology reminiscent of Sj S

characterized by severe sialadenitis, decreased saliva production, and destruction of the

submaxillary glands. Infiltrates within the salivary glands of BAFF-transgenic mice

appear to be a marginal zone (MZ) B cell population, a population known to be increased

in the spleens of BAFF-transgenic mice and thought to participate in the maintenance of

germinal centers in the target tissue and subsequent antibody production in Sj S (70).

A role for aberrant lymphocyte survival is also supported by studies using the

MRL/lpr mouse model (71). MRL/lpr mice carry a mutation in the 1pr gene that causes a

defect in the Fas protein involved in the Fas/FasL apoptotic pathway. This defect leads to

impairment of normal lymphocyte apoptosis, resulting in an abnormal proliferation and

survival of lymphocytes, especially B cells (72). While the MRL/lpr mouse was

developed for the study of SLE, it manifests an autoantibody pattern found in part in SjS,

including anti-dsDNA, anti-ssDNA, ANA and rheumatoid factor (73). In addition, nearly

30% of mice develop anti-52 KDa SS-A/Ro antibodies, 6% develop anti-60 KDa SS-

A/Ro antibodies, and 6% develop anti-SS-B/La antibodies (74), however other studies

have not been able to detect similar frequency of these antibodies in the MRL/lpr mouse.

MRL/lpr mice also develop sialadenitis of the submandibular, parotid and lingual glands

and dacryoadenitis of the lacrimal glands (75). The infiltrates are generally comprised of

CD4+ T cells, with lesser numbers of CD8+ T cells and B cells. In addition, there are

scattered macrophages and dendritic cells (76). However, the infiltrates appear diffuse









and not as compact, tightly packed foci found in NOD mice. Although lymphocyte

infiltrations may cause destruction of exocrine gland tissues, perhaps by iNOS / nitric

oxide (NO) (77), tumor necrosis factor (TNF)-a and/or various cytokines (78), anti-M3R

antibodies have not been observed in MRL/lpr mice, thus loss of secretary function is not

detected (79). To put these observations in perspective, it is important to note the fact

that, in the SjS-like disease process of NOD mice, a high rate of acinar cell apoptosis

occurs in the submandibular glands around 2 months of age, or approximately 2-4 weeks

prior to onset of detectable leukocytic infiltration of the salivary glands, and this process

is associated with an up-regulation of Fas/Fas-ligand expression by acinar cells (62).

Genetic Predisposition of SjS-like Autoimmune Exocrinopathy

Genetic manipulations of a variety of mice can either result in the appearance of

various disease traits observed in Sj S patients or delay / prevent development and onset

of pre-clinical and clinical disease. This is obvious in such mouse lines as MRL/lpr,

NFS/sld, the Baff-KI transgenic, the Id3-KO transgenic, and Aromatase-KO transgenic.

A remaining question, however, is whether we can identify those genetic regions of the

mouse genome that predispose mice to develop Sj S-like disease. The NOD mouse

provides an excellent model to investigate this issue. A large collection of congenic

NOD mice are available defining the diabetes susceptibility (Idcd) loci that predispose

these mice to autoimmune type 1 diabetes (T1D). Unlike the genetic predisposition for

T1D in both humans and NOD mice which is dependent on specific genes mapping to the

major histocompatibility complex (MHC), the genetic predisposition for Sj S-like disease

in NOD mice appears independent of, or only weakly dependent on, MHC-associated

genes, thus mimicking SjS in humans. The first indication involved the studies of the

congenic strain, NOD.B10-H2b, in which the NOD MHC I-A7 Iddl T1D susceptibility









locus is replaced by MHC I-Ab (53). These mice, while failing to exhibit insulitis and

development of diabetes, continue to show a complete Sj S-like syndrome including

salivary and lacrimal gland dysfunction. Thus, NOD.B 10-H2b mice were advanced as

the first naturally-occurring model for primary Sj S (53).

Replacing other diabetes susceptibility loci in the NOD mouse (e.g., IddlO, Idd9,

Iddl3, and so forth), while lowering the incidence of insulitis and diabetes, proved to

have little effect on its Sj S-like disease. However, when both the Idd3 and Idd5 loci were

replaced with the corresponding genetic intervals derived from C57BL/6 mice, the

severity of the biological markers of epithelial cell pathology was reduced and the loss of

secretary function reversed (31, 32). In a reverse approach, introducing both the Idd3

and Idd5 genetic regions derived from NOD mice into the Sj S non-susceptible C57BL/6

mouse resulted in the appearance of Sj S-like disease, confirming the contributions of

these two genetic loci to development and onset of disease (54, 55).

The Role of B Lymphocytes in the Development of Sj S-like Autoimmune
Exocrinopathy in Mice

Development of Germinal Center (GC)-like Lymphocytic Foci in the Exocrine
Glands

Considerable attention has been focused on attempts to define the organization of

the immune cell infiltrates, referred to as lymphocytic foci, which appear in salivary and

lacrimal glands with the onset of disease. We and others have reported that these

lymphocyte infiltrations within the submandibular and lacrimal glands of NOD mice are

composed predominantly of CD4+ T cells with lesser numbers of CD8+ T cells and B

cells, a fact generally accepted universally (63). Subsequently, we have found that these

findings appear to be misleading due to the procedures used to isolate the infiltrating cells

from the exocrine tissues for flow cytometric analyses. We now believe that vigorous









digestion and subsequent purification steps may account for the lost of many fragile cells,

thereby leading to possible erroneous conclusions. Recently, we have revisited this

question using immunofluorescent staining of paraffin-embedded salivary and lacrimal

tissues that not only maintain the native structure of the glands but retain the cellular

organizations. Results strongly indicate that lymphocytic foci are dynamic entities whose

cellular compositions and organizations change dramatically as they mature. Thus, to

state that the major cell type is a CD4+ T cell appears to over-simplify the real situation.

As presented in Figure 1-2, Immunofluorescent staining of paraffin-embedded

sections of glandular tissues freshly explanted from NOD mice of different ages depicts a

rapidly changing cellular composition of the lymphocytic foci. During the early stages of

lymphocyte infiltration into the salivary glands, small densely packed foci appear that are

mostly composed of CD3+ T cells virtually free of detectable B cells. As the foci mature,

an increasing number of B220+ B cells are detected which histologically appear to

surround T cell cores. Over time, the number of T cells decrease relative to the B cells

and become more dispersed throughout the B cell areas.

An important observation from studies using NOD mice, first reported by van

Blokland and colleagues (76), is the fact that dendritic cells (DCs) and possibly

macrophages appear to be the first cells to migrate into the glands, resulting in the

activation of epithelial cells to express adhesion molecules and produce chemokines that

attract T cells, and subsequently additional macrophages and B cells. Most BM8+

macrophages are found to occupy areas around the periphery of the focus and T cells

regions (76).









We have recently shown by immunohistochemistry that F4/80 macrophage are also

scattered throughout the foci (unpublished data). However, careful examination of these

well-defined lymphoepithelial sialadenitis-like foci does not appear to mimic ectopic

germinal centers. It is still uncertain if these ectopic lymphocytic foci have any

biological significance in terms of the clinical manifestation of SjS, but their absence

generally results in loss of progression of disease into the clinical phase. It has been

reported that the lymphocytic infiltrates are made up of clonally expanded T cells with

the TCR repertoire of V08.1,2, V06, and Vj4 (80). However, it is also unknown if there

is localized or systemic activations that result in effector molecules for the clinical

development of Sj S. Consistently, histological analysis of salivary and lacrimal glands in

human Sj S patients revealed the presence of leukocytic infiltrations which consists

mostly of T cells and significantly less B cells. However, our findings have clearly

demonstrated that the infiltrates are mostly B220+ B cells (75%) and a smaller

percentage of CD3+ T cells when the disease appears to be most progressive; however, to

fully understand the nature of the organization of these lymphocytic foci, additional work

needs to clarify what subpopulations of B220+ B cells and T cells are present in the

infiltrates.

Development and Characteristics of B Cells in SjS

The life-history of B lymphocytes is now a well-studied and well-documented area

on which many reviews have been written (81-84). While it is recognized that T cells

play an important role in regulating B cell activation, maturation and antibody

production, considerable attention has recently been focused on the role of B

lymphocytes in the development and onset of Sj S in humans and Sj S-like disease in mice.

This is due, in part, to reports documenting a number of apparent innate developmental









abnormalities in B cells for both patients and mouse models. However, identifying the

stages) at which this abnormal B cell behavior is induced and/or how it is maintained

remains unknown and probably represents a multi-factorial process, since multiple

environmental factors dictate the fate and behavior of B cells..

The Role of Marginal zone (MZ) and B-1 B Cells in SjS

As previously discussed, mice over-expressing BAFF develop SLE-like disease

with significant increases in the number ofMZ B cells (69). When these mice are aged to

about 16-18 months, they exhibit a SjS-like disease with a subset of B cells in the

salivary glands that resemble MZ B cells (70). In Grave's disease, MZ cells are found to

infiltrate the thyroid gland (85), and NOD mice have been reported to also have

infiltrations in their thyroid glands (86). While it is not known if these MZ B cells play

any role in the pathogenesis of autoimmunity, they may provide insight into how chronic

stimulation of auto-reactive B lymphocytes within a tissue eventually transform to

lymphomas. Hyperproliferation of MZ B cells is hypothesized to be the cause and source

of transformed B cells that often arise in Sj S patients and are capable of developing into

non-Hodgkin B cell lymphomas (87). Dependent on location, MZ B cells can be induced

to transform into splenic MZ lymphomas, nodal MZ lymphomas, and/or extra-nodal

MALT lymphomas (87). Therefore, this particular subset of B cells appears to be an

important connection between autoimmunity and tumorigenesis in Sj S patients.

For B-l cells, a vast majority is found in the peritoneal and pleural cavities of the mouse,

thus only 5% of this B cell population is present in the spleen and virtually none are

present in the peripheral lymph nodes (88). B-i cells can be further separated into B-la

cells that express membrane CD5 molecules or B-lb cells that are CD5 negative. The

development and survival of B-l cells are critically dependent on the strength of B cell









receptor signaling. Numerous studies in mice involving the genetic disruption (either by

deletion or over-expression) of molecules that contribute to the signal transduction

pathways of the BCR greatly effects the development of B-1 cell populations. For

example, genetic alterations that enhance BCR signaling, e.g., deletion of CD22 or CD72,

and over-expression of CD19, lead to significant increases in the number of B-I cells. In

contrast, genetic manipulations that lower BCR signaling, e.g., deletion of CD19,

CD21/35 or Vav-1, reduce the number of B-I cells (89). Therefore, these observations

indicate the importance of maintaining homeostasis of BCR signaling to ensure

appropriate development, survival or even expansion of this B cell population.

B-l cells manifest many unique characteristics important to proper B cell function,

including longevity of survival with high potential for self-renewal, refractory to

activation by ligand binding to BCRs, lack of somatic mutation, and restriction of N

insertions that result in limited immunoglobulin gene repertoires (90). Since B-I cells

arise from fetal precursors, they express limited Ig gene repertoires, mostly restricted to

"natural" IgM, where the term "natural" indicates the production of IgM subclass of

antibody in the absence of stimulation by exogenous antigens (91). Therefore, B-I cells

produce antibodies that are predominantly autoreactive, but highly polyreactive with

numerous naturally or evolutionarily conserved pathogen-associated carbohydrate

antigens, such as phosphorycholine (PC) (92), phosphatidyl choline (PtC) (93) and

lippolysaccharide (LPS) (94). The specificities of these B-i produced IgM antibodies

suggest that B-l cells are critical for protection against bacterial infection (95), acute

septic peritonitis (96), vascular diseases such as atherosclerosis (97), and viral infections

(in particular, influenza) (98).









A correlation between the functions of B-1 cells and systemic rheumatic autoimmune

diseases has drawn considerable attention. Several reports have demonstrated that in

both humans and mice there is a significant correlation between the frequency of B-1

cells and the eventual development of autoimmune diseases (99, 100). Interestingly,

dramatic increases in the number of B-1 cells are found in human patients with Sj S (101)

and RA (102). While B-I cells are present in minor labial glands of SjS patients (103), it

is not certain whether B-l cells can initiate autoimmunity or provide protection against it.

In the latter case, it is important to note the similarity between B-l cells and anergic B

cells, including failure of BCR-antigen stimulation to induce Ca+ influx (104), the

expression of low levels of CD5 (105), and the expression of high levels of the

transcription factor NFATc (106, 107). Therefore, it has been speculated that B-I cells

may induce tolerance of potentially autoreactive B cells rendering them anergic or

nonpathogenic.

On the other hand, B-l cells are often auto-reactive, producing autoantibodies with

low affinity compared to conventional B cells, thereby possibly involved in initiating

autoimmunity (90). A smaller number of this B-l cell population, however, can migrate

to and reside in the germinal centers where they will receive signal from helper T cells,

the consequence of which is an induction of class switching and somatic hypermutation

(108). This newly emerged set of B-i cells can then give rise to high affinity

autoantibodies that are potentially pathogenic.

Expression of Restricted Repertoire of the B Cell Receptor in SjS Disease

An interesting hallmark feature of B cells in Sj S is the relatively restricted B cell

receptor (BCR) repertoires found on peripheral B cells and exocrine gland-infiltrating B

cells, as well as the MALT-associated B cell lymphomas that develop in a subset of Sj S









patients. In a recent study by Kaschner et al. (109) involving three patients, a significant

over-representation of specific VLS was observed: for Vx, four genes (2A2, 2B2, 2C and

7A) represented 56% of all functional Vxs. In the productive VK repertoires, three genes

(L12, 012/02, and B2) represented 43% of all VKJKS. VA27, a gene frequently found on

autoantibodies, rheumatoid factor and lymphomas in Sj S patients, was identified at an

increased frequency of 29% in the parotid gland compared to only 8% in the peripheral

blood. In addition, significant enrichment of VKA19 and Vx2E, specifically the clonal

expansion of VKA27-JK5 and VKA19-JK2 has also been reported in the parotid glands of

SjS patients (110). Since B cells take advantage of receptor editing to escape apoptosis

and prevent recognition of self-antigen, and because there is a marked decrease in

receptor editing observed in primary Sj S patients, this restricted BCR V-region usage

may be a result of a defect in receptor editing. However, also influencing this restricted

V-region usage may be the reported depletion of memory B cells from the peripheral

blood with a concomitant elevated level of antigen-activated B cells in the parotid glands

(109), an observation implying a defect in selection. This peripheral memory B cell

population, characterized as CD19+CD27+, was found to have a mutational frequency of

8.6% in Sj S patients in the immunoglobulin VH transcripts compared to 4.3% in normal

healthy controls, as well as an elevated mutational frequency in Cut transcripts for

multiple Ig heavy chain isotypes (111). Not addressed by these data is whether the

restricted use of BCR repertoires is due to general innate selection mechanisms for B cell

survival or an antigen-driven expansion and survival of a restricted receptor-bearing B

cell population.









Signal Transduction of the B Cell Receptor

The BCR complex is made up of membrane-bound immunoglobulin (Ig) associated with

CD79a and CD79b, two heterodimers of Iga and Igo. Following interaction of the BCR

with a ligand, protein tyrosine kinases (PTKs) including Syk and Btk of the Tec family

and Lyn, Fyn, Blk, and Lck of the Src family, are activated. In addition, the BCR is fine-

tuned by crosstalk between PTKs, protein tyrosine phosphatases and adapter proteins

such as B cell linker (BLNK) and BAM32 (B lymphocyte adapter molecule of 32 kDa)

(112). Activated Src-family PTKs phosphorylate the immunoreceptor tyrosine-based

activation motifs (ITAM) in the cytoplasmic tails of the Iga/Ig3 heterodimers, resulting in

recruitment of Syk and Btk through their tandem Src homology 2 (Src 2) domains.

Activated Syk, through BLNK, can activate PLCy2 (phospholipase Cy2) by tyrosine

phosphorylation. PLCy2 can hydrolyze PIP2 (phosphatidylinositol 4, 5-bisphosphate), a

membrane phospholipids, to produce DAG (diacylglycerol) and IP3 inositoll 1, 4, 5-

trisphosphate). IP3 then triggers the release of intracellular Ca2+. Together, DAG and

Ca2+ activate PKC (protein kinase C). Activated PKC is required for the activation of

mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinase

(ERK), c-JUN NH2-terminal kinase (JNK), p38 MAPK, and eventually the transcription

factors, nuclear factor-KB (NF-KB) and nuclear factor of activated T cells (NFAT).

Signals from activated Syk can also phosphorylate IK-B which results in the release of

transcription factor NF-KB into the nucleus. Furthermore, activated Syk, through BCR

ligation, can trigger the activation of the Ras pathway. Activated Syk phosphorylates and

activates Ras. Activated Ras will in turn activate RAF1 by tyrosine phosphorylation, and

ultimately activate effector molecules similar to the PKC/Ca+ pathway (112).









Intimate Interaction of the B Cell Receptor and B cell Co-Receptors

Activation of BCR-associated signaling pathways is critical to the generation of

humoral immunity as these signaling pathways determine B cell proliferation,

differentiation, selection, survival and eventually function. B cell function, however, is

also regulated by B cell surface co-receptor molecules. These co-receptors modulate the

intensity, quality, and duration of the BCR signal transduction pathways. These co-

receptor molecules, which include CD19, CD21, and CD22 and complement component

C3d, modify the intrinsic intracellular signal transduction threshold by adjusting the

strength of the signals needed to initiate BCR-mediated activation. CD19, CD21 and

CD22, are functionally linked with Lyn, Vav, and SHP1 in a common signal transduction

pathway that is initiated by BCR binding to its ligand (113).

CD19 is a 95 KDa transmembrane glycoprotein of the Ig superfamily expressed by

all B cells from the late pro-B cell to the plasma cell (114). The cytoplasmic domain

contains nine highly conserved tyrosine residues that recognize active SH2 (Src

Homology 2) domain motifs of regulatory molecules. The CD19 tyrosine residue is

phosphorylated constitutively in splenic B cells at low level and completed constitutively

with Lyn and Vav. When BCR binds its ligand, a local increase in the number of

activated Lyn molecules permits phosphorylation of the Immunoreceptor Tyrosine-based

Activation Motifs (ITAMs) of Iga/Ig3 heterodimers. Lyn also phosphorylates the

tyrosine residue at position 513, plus a number of other tyrosine residues on CD19.

Phosphorylation of CD19 at tyrosine position 391 recruits Vav to bind at this site, and

due to the close proximity with Lyn, Vav is also phosphorylated by Lyn. This process

functions to activate MAPK and induce a prolonged Ca2+ influx, which initiates

additional downstream signaling pathways, including cytoskeletal reorganization (113).









CD19 also interacts with phosphatidylinositol-3 kinase (PI3K) by providing a docking

site for the p85 subunit of PI3K when the tyrosine residues are phosphorylated by Lyn.

Activated PI3K facilitates Ca2+ mobilization and activation of the serine/threonine kinase

AKT pathway (112). Once Lyn has performed this function, it loses its affinity for the

SH2 domain.

Despite all, the ligand for CD19 remains undefined. However, CD19 can form a

non-covalent quaternary complex on the surface that includes CD21, CD81 and CD225.

CD81 is a member of tetra-spans family that is involved in regulation of cell growth,

mobility and signaling. CD81 is physically associated with CD225, whose precise

function is unknown but thought to be involved in anti-proliferative activity regulated by

interferon and B cell growth (113). CD21 is the receptor for C3d, a cleavage product of

C3 that forms covalent bonds with foreign antigen and immune complexes. It is also a

receptor for Epstein Bar Virus (EBV). The ability of C3d-antigen complexes to crosslink

the CD19/CD21/BCR complex provides a possible link between innate and adaptive

immunity (115). Functioning as positive regulators of B cell activation, signals generated

by the co-ligation of CD19/CD21 and BCR are additive. Co-ligation of BCR and

CD19/CD21 by a C3d-antigen complex lowers the threshold for B cell activation, thus

lowering the amount of antigen required to activate B cells (116). Currently, there are at

least two models for the CD19/CD21 complex in its function as a regulator of

transmembrane signals. The "co-stimulatory" model postulates that transmembrane

signaling results from cross-linking between the BCR and CD19/CD21 by the

simultaneous binding of C3d-antigen complexes. The "response regulator" model

postulates that the BCR independently transmits the transmembrane signal, whereas









CD19 merely regulates intrinsic levels of Lyn and Vav phosphorylation and activation.

In this model, an association between CD21 and C3d-antigen complexes promotes the

intrinsic functions of CD19 (113).

In opposition to CD19/21, CD22 plays an important inhibitory role in B cell

activation. The cytoplasmic domain of CD22 contains six tyrosines located in the three

Immunoreceptor Tyrosine-based Inactivation Motifs (ITIMs) and two ITAMs, suggesting

the potential for both negative and positive signaling. Phosphorylated CD22 recruits the

phosphotyrosine phosphatases, SHP1 and SHIP, to limit BCR signaling (117), thus,

CD19/CD21 and CD22 are reciprocally regulated by each other. Only limited numbers

of Lyn and Vav proteins are available in this system and must be shared by BCRs,

CD19/CD21 and CD22. The phosphorylations of CD19 by Lyn and Vav also results in

the phosphorylation of CD22 that, in turn, recruits SHP-1 and SHIP, bringing these

molecules in close proximity to CD19/CD21 and BCRs. SHP-1 and SHIP, when bound

to CD22, dephosphorylates CD19 and BCRs thereby down-regulating CD19 and BCR

function. In addition, SHIP can convert PIP3 to PI3,4-P2 which down-regulates the

PI3K-dependent pathway (118).

The Role of Autoantibodies in the Development of SjS

In recent years, the nature of numerous (auto)-antigens in connective tissue

disorders have been identified using various molecular approaches. Many autoantigens

have proven to be intracellular enzymes and regulatory factors required for cellular

function, e.g., gene replication, transcription, RNA processing and protein synthesis

(119). Antibody against autoantigen such as anti-Ro IgG antibody can cross the placenta

and cause neonatal lupus (120). However, limited data are available indicating that

antibodies directed against these molecules have any direct effect in eliciting a









pathological consequence, for example, loss of fluid secretion by exocrine glands.

Nevertheless, antibodies targeting nuclear proteins (ANAs) in Sj S, especially the

ribonuclear proteins Ro/SS-A and La/SS-B (121), have long been used as a diagnostic

marker of disease in both humans and animal models. Different immuno-fluorescence

patterns of ANAs have, over time, led to the identification of other nuclear proteins

targeted by autoantibodies, such as Sm, dsDNA, the nuclear mitotic apparatus (NuMA),

proteasomes, mitotic chromosomal autoantigens (MCAs), and poly-(ADP-ribose)

polymerase (33).

Autoantibodies detected in Sj S patients and various animal models are not limited

to targeting nuclear proteins. One well-studied autoantigen is the intracellular

cytoskeletal protein a-fodrin, targeted by proteolytic enzymes in the salivary glands of

both Sj S patients and NFS/sld mice (122). In addition, as NFS/sld mice age, they

develop autoantibodies against ssDNA, IgGI and IgG2a subclasses, rheumatoid factor

and type II collagen (123). Other examples of intracellular autoantigens are tissue

kallikrein-1 (Kik-1) and kallikrein-13 (Kik-13) as defined by detectable autoantibodies in

sera of IQI/Jic mice affected with SjS (12 week and older). However, only Kik-13 was

shown to induce a proliferative response by splenic T cells (124). IQI/Jic is an inbred

strain that originated, like the NOD strain, from the ICR mouse colony in Japan and

exhibits spontaneous SjS-like disease that mimics that of NOD mice (68). The fact that

Kik-13 is highly expressed in the salivary gland ductal cells may explain target tissue

specificity of Sj S as it is consistent with periductal infiltration of immune cells.

Furthermore, since only reduced forms of Kik-13 are recognized by sera of IQI/Jic mice,

one might conclude that cryptic epitopes are involved, not unlike the case proposed for









the potential development of autoantibody responses to PSP in NOD mice (60). Lastly,

both Sj S patients and NOD mice form autoantibodies reactive with islet cell autoantigen-

69 (ICA-69) expressed in pancreatic islets, the brain and both salivary and lacrimal

glands (125). Disruption of the ICA69 locus in the NOD mouse prevents lacrimal gland

disease and greatly reduces salivary gland, suggesting that immunoreactivity against

ICA-69 might play a role in disease progression (125).

An intriguing question in autoimmune diseases like Sj S pertains to how

intracellular components, i.e., self-proteins, become recognized and presented as

dominant neo-antigens by immune cells. The recent reviews and journal articles by

Rosen et al. (126-128) advance cellular apoptosis as an initial event. In this work, Rosen

and colleagues describe how molecules within the subcelluar compartment are

redistributed in apoptotic cells. Small membrane blebs could be shown to contain 52-

kDa Ro and other molecules, such as calreticulin, normally present within the ER lumen.

Nuclear antigens also exhibit redistribution during apoptosis, showing an increase in

localization of 60-kDa Ro/SS-A, La/SS-B, the snRNPs, Ku and PARP as a rim around the

condensing chromatin (128, 129). Such clustering of potential autoantigens occurs during

apoptosis, but not during necrosis (130, 131). Although the degree of acinar cell

apoptosis in the salivary and lacrimal glands is not well-established or universally

accepted, proteolysis, phosphorylation, glutathiolation, transglutamination, citrullination,

and/or formation of novel protein-protein or protein-nucleic acid complexes probably

play a role in the alteration of molecular structures permitting exposure of neo- or cryptic

epitopes to the immune system (130, 132, 133).









The importance of T lymphocytes in the activation, proliferation and differentiation

of antigen-reactive B cells in autoimmune-prone mice is nicely demonstrated in the Id3-

gene KO mouse (134). The Id proteins bind basic helix-loop-helix transcription factors

and function as dominant negative inhibitors of gene expression. Id3 is an immediate

early-response gene regulating growth and is involved directly in TCR-mediated T cell

selection during T cell development in the thymus. Id3-deficient mice with various

genetic backgrounds develop a Sj S-like disease, including the synthesis of anti-Ro/SS-A

and anti-La/SS-B antibodies at approximately one year of age (135). In the absence of T

cells, these Id3-deficient mice failed to exhibit development of a Sj S-like disease. One

fascinating observation in this mouse model is the appearance of secretary dysfunction as

early as 6 weeks of age, a time point prior to other visible disease symptoms. This raises

the possibility that organogenesis of the salivary glands may be impaired in this Md3-gene

KO mouse, resulting in aberrant antigen presentation and autoreactive T cell activation,

as Id3 is known to be a Smad4-dependent TGF-P responsive gene whose pathway is

important for salivary gland development (136). We have speculated that delayed

organogenesis of the salivary glands in NOD mice is critical for subsequent development

of Sj S-like disease in the NOD mouse, as well (57).

Anti-Muscarinic Acetylcholine Type-3 Receptor Autoantibodies The Effectors of
Glandular Dysfunction

Although the loss of saliva and tear flow in Sj S was initially believed to be a

consequence of acinar cell apoptosis elicited by cytotoxic T lymphocytes, an interesting

paradigm shift has occurred based on studies showing the requirement for B lymphocytes

and immunoglobulin. First, B cell deficient NOD.Igu""" mice fail to develop secretary

dysfunction, and second, IgG from Sj S patients can induce a reversible stimulation or









inhibition of salivary function when infused into NOD-scid mice (66). Accumulating

evidence suggests that disturbances in lymphocyte homeostasis, including ectopic

germinal center formation in the target tissue and/or aberrations of cellular signaling

regulated by B cell activating factor (BAFF), are present in SjS (70, 111, 137). As shown

in both transgenic mice over-expressing BAFF and patients with Sj S, B cell hyperactivity

may lead to excessive immunoglobulin production and prolonged B cell expansion that

eventually lead to the monoclonal expansion of B cells and transformation to B-cell

lymphomas in a subset of patients (46, 47). Additionally, intrinsic defects in the B cell

compartment associated with SjS may play a role in the generation of Sj S autoantibodies

with diversified prevalence and specificity, as evidenced by the wide array of

autoantigens targeted. Thus, identification of autoantibodies that directly cause dryness

in Sj S patients is essential for understanding the pathogenesis and onset of clinical

disease.

Of the many autoantibodies identified in SjS patients to date, the association

between anti-M3R autoantibodies and secretary dysfunction seems most intuitive since

the M3R is the major receptor mediating secretion in the salivary and lacrimal glands in

response to parasympathetic stimuli. Studies strongly indicate that serum or purified IgG

from Sj S patients down-regulate carbachol-evoked bladder muscle contraction by 50 %,

while anti-idiotypic antibodies neutralize this inhibition of cholinergic neurotransmission

(36, 39, 138). Furthermore, anti-idiotypic antibodies were able to neutralize

autoantibodies that inhibit cholinergic neurotransmission (138). Similarly, studies using

the human salivary gland ductal cells, HSG, showed that pretreatment of the cells with









Sj S IgG for 12 or 24 hours reduced the magnitude of subsequent carbachol-induced

intracellular calcium release (38).

In a recent study, Cha et al.(37) reported that M3R desensitization occurs in mice

with anti-M3R autoantibodies, as revealed in a comparison of carbachol-evoked

responses in NOD mice >20 weeks of age versus either age-matched C57BL/6 or

antibody-negative 8-10 week old NOD mice. These observations, therefore, would be

consistent with the hypothesis that chronic stimulation by anti-M3R antibody induces an

inhibitory affect on M3Rs. Importantly, NOD mice with overt Sj S-like disease initially

responded well to pilocarpine-induced stimulation; however, this stimulation was down-

regulated following chronic injections. We currently interpret these results as an

augmented desensitization of M3Rs in the presence of anti-M3R autoantibodies.

Extrapolating these data to a clinical setting, chronic intake of pilocarpine might enhance

saliva secretion initially, yet may induce eventually a more rapid desensitization in

human patients positive for anti-M3R autoantibodies. The effect of prolong usage of

pilocarpine on desensitization of M3Rs might need further investigation to elucidate the

mechanism and other potential clinical consequence.

Furthermore, mixed response profiles were seen when bladder strips isolated from

normal, healthy C57BL/ 6 mice were incubated with sera from a number of different Sj S

patients. While a few sera enhanced smooth muscle contraction in comparison with

either normal sera or Krebs physiological buffer, other sera inhibited the contractions.

This could represent effects of different antibody titers, length of incubation time, and/or

different titers of a pathogenic subset of anti-M3R autoantibodies. In light of recent

studies discussed below, SjS-like disease may be dependent on anti-M3R autoantibodies






28


of a specific isotype (56). If the effects are dependent on a specific IgG subclass, then it

will be necessary to determine if its inhibitory activity on acinar cell secretion is

dependent on the constant region of an immunoglobulin subclass or relies on a variable

region whose specificity is shaped by the constant region.












Table 1-1. Mouse strains used in the study of Sjogren's syndrome

Mouse strains Characteristics Disease Manifestations / Phenotypes

NZB/W [(NZB x Naturally occurring mouse Lacrimal gland involvement
NZW)F1 hybrid] model by crossing NZB and A greater percentage of B cells compared with MRL/lpr mouse
NZW (139)
MRL/lpr Mutation in Ipr that encodes Diffuse lymphocytic infiltration
Fas protein(140) Autoantibodies (against ssDNA, RNPs, IgG)
No loss of secretion
No detection of anti-M3R Abs

NOD/LtJ / NOD- Sj S-like disease phenotype; Loss of secretion, anti-M3R Abs,
congenics focal lymphocytic infiltration

NOD/LtJ Spontaneous insulitis and Disease phenotype w/ diabetes
diabetes(141)

NOD.B 10-H2b NOD with H-2b from Disease phenotype w/o diabetes
C57BL/10
(142)
NOD-scid Homogygous mutation in scid No disease phenotype but abnormal organogenesis
locus (no functional
lymphocytes)
(143)
NOD.Igt -/ No functional B-lymphocytes Disease phenotype w/ normal flow
(66)

NOD.IL4- Cytokine IL-4 gene knockout Disease phenotype w/ normal flow
(56, 58)












Table 1-1. Continued

Mouse strains Characteristics Disease Manifestations / Phenotypes

NOD.IFNy-' IFN gamma KO Neither disease phenotype nor abnormal organogenesis
NOD.IFNyR IFN gamma receptor KO observed

NOD-scid.IFNy7- No functional lymphocytes in Neither disease phenotype nor abnormal organogenesis
IFN gamma KO observed
(57)

NOD.Q NOD with H2q from C3H.Q Severity of sialadenitis greater than that of NOD

NOD.P NOD with H2p from C3H.NB Severity of sialadenitis lesser than that of NOD
(144)
C57BL/6.NOD- C57BL/6 carrying Aecl (Idd3) Sj S-like disease phenotype in a C57BL/6 genetic background
Aec]Aec2 genetic region on Chr 3 and
Aec2 (Idd5) genetic region on
Chr 1
(55)
IQI/Jic Inbred strain originating from Anti-kallikrein 1 and -13 Abs
ICR (68)

NFS/sld Autosomal recessive gene with Anti-alpha fodrin Abs
sublingual gland
differentiation arrest
(145)
Id3 gene KO Id3; basic helix-loop-helix Impaired TCR-mediated T cell selection
transcription factor, a Loss of secretion
dominant negative inhibitor of Anti-Ro and anti-La Abs
gene expression (135)












Table 1-1. Continued





Estrogen deficiency due to
Aromatase gene KO absence of enzyme catalyzing B cell hyperplasia in the BM and spleen
the conversion of testosterone Anti-alpha fodrin Abs
to estradiol
(146)
Alymphoplasia Homozygous mutation in aly Conserved CDR3 in TCR of infiltrating T cells in the lacrimal
(aly/aly) (alymphoplasia) glands, salivary glands and kidneys
(147)
GVHD (graft-vs-host GVHD induced by the Lymphocytic infiltration w/ majority of T cells
disease) injection of DBA/2 spleen
cells into nonirradiated
(C57BL/6 x DBA/2) Fl mice
(148)
BAFF transgenic Transgenic for B-cell survival Loss of secretion by 15-17 months of age. Lymphocytic
factor infiltration w/ majority of B cells
(69)



BALB/c Immunized with short peptides Loss of secretion, anti-Ro, lymphocytic infiltration
)f 60 kDa Ro antigen
(67)




















Table 1-2. Comparison of general symptoms of Sjogren's syndrome patients and NOD mice


Characteristic


Sjogren's Syndromed NOD micee


Dacryoadentitis
Sialadenitis
Decreased tear & saliva flow rates
Altered proteins in tears & saliva

Pro-inflammatory cytokine production


Autoantibodies
Anti-Ro/SS-A, Anti-La/SS-B
Anti-DNA (ANAs)
Anti-a-fodrin
Anti-p-adrenergic receptor
Anti-type-3 muscarinic ACh receptor

Keratoconjunctivitis sicca
Ocular epithelium dessication (Rose-Bengal Dye)
Break-up time testing
Lysozyme & Lactoferrin activity

Stomatitis sicca
Buccal epithelium dessication
Serine protease activity against PSP
Amylase & EGF activity

a Biopsies of lacrimal glands not often performed
b Data presented under Research & Methods Section
SPossibly detected as part of ANAs


Yes
Yes
Yes
Yes
Yes

Yes
Yes
Decreased
Decreased

Yes
Yes
(?)
Decreased


Probably not0
Yes
Yes
Yes
Yes

(?)
Yes b
(?)
Decreased

Yes
(?)
Yes
Decreased b


(Yes) a
Yes
Yes
Yes


Yes b
Yes b
Variable b
Yes b

Yes












Wk: 0


Phase I


Delayed
organogenesis Leuk
of ex
Aberrant gene &
protein expression

SMX expression of parotid
secretary protein (PSP)

Acinar cell apoptosis


Phase II


Phase III


ocyte infiltration
ocrine glands


Loss of secretary
function


Autoantibody production

PSP-proteolysis ( marker of SjS)


Detection of anti-M3R antibody


Figure 1-1. Model for the progression of Sj S-like autoimmune exocrinopathy


I --nn


>24























Figure 1-2. Dynamic cellular composition oflymphocytic infiltration in submandibular
glands of NOD mouse. Green: CD3.FITC. Red: B220-Texas Red. A) 8-10
weeks of age. 400X magnification. B) 12-16 wks of age. 200X
magnification. C) 20-24 weeks of age. 100X magnification.














CHAPTER 2
EFFECT OF COBRA VENON FACTOR (CVF) ON COMPLEMENT AND B
LYMPHOCYTES IN THE NOD.B10.H2b MICE

Introduction

Sjogren's syndrome (Sj S) is a human autoimmune disease characterized by loss of

exocrine function as a result of a chronic immune attack directed primarily against the

salivary and lacrimal glands leading to xerostomia (dry mouth) and xerophthalmia (dry

eyes) (1, 3, 11, 18). Although the underlying cause of Sj S remains elusive, a number of

studies using the NOD mouse model of Sj S have led us to conclude that autoimmune

exocrinopathy progresses in three consecutive phases (61, 149). In phase 1, a number of

genetically predetermined physiological and biochemical activities associated with

retarded salivary gland organogenesis occur prior to and independent of initiation of

autoimmunity (59). In phase 2, leukocytes infiltrate the exocrine glands with a

concomitant increase in the expression of pro-inflammatory cytokines. In phase 3,

secretary dysfunction of the salivary and lacrimal glands occurs, most likely, the result of

production of autoantibodies (39, 56, 65, 66, 150, 151). Interruption within any one of

these three phases can prevent onset of clinical Sj S-like disease.

Sj S is considered a lymphoproliferative B cell disorder, thus, the clinical phase of

disease fails to develop in the absence of either B cells or autoantibodies. Although B cell

development is stringently regulated, B lymphocytes in Sj S are hyper-proliferative,

capable of evading apoptosis and overly sensitive to activation and maturation,

suggesting escape from tolerance-inducing mechanisms. One mechanism controlling









activation, survival and proliferation of B cells is through a cross-linking between B cell

receptors and their co-receptors CD 19, CD21 and CD22. While the ligand for CD19

remains unknown, CD19 can form a non-covalent quaternary complex that includes

CD21, CD81 and CD225 (113). CD21, in turn, is the receptor for C3d, a cleavage

product of C3 that forms covalent bonds with foreign antigen or immune complexes.

Signals generated by the co-ligation of CD19/CD21 and BCR by C3d fragment act as

positive regulators of B cell activation. Co-ligation of BCR and CD19/CD21 by a C3d;

Ag complex lowers the threshold for B cell activation, possibly contributing to the hyper-

proliferative and hyper-active properties of autoreactive B cells (116). A number of

studies using anti-complement treatment have shown that complement plays an important

role in several autoimmune diseases, including rheumatoid arthritis, multiple sclerosis,

myasthenia gravis and systemic lupus erythematosus (152). However, little attention has

focused on the role of complement, especially complement component C3, in Sj S. In the

present study, we have examined whether C3 plays a role in the pathophysiological

aspects of SjS-like disease ofNOD.B10-H2b mice.

Materials and Methods

Animals

NOD.B10-H2b and BALB/cJ mice were bred and maintained under specific

pathogen-free conditions within the mouse facility of the Department of Pathology,

Immunology and Laboratory Medicine at the University of Florida, Gainesville, FL.

Breeder pairs of both strains were purchased from the Jackson Laboratories (Bar Harbor,

ME). All mice received water and food ad libitum.









Complement Depletion by Cobra Venom Factor

Cobra venom factor (Naja naja kaouthia) was purchased from Calbiochem

(#233552, stated to be >95% pure by SDS-PAGE) (La Jolla, CA). Starting at 10 weeks of

age, mice received intraperitoneal injections of either 200 [l CVF (one unit diluted in

sterile PBS) or 200 ul of sterile PBS. The injections were carried out twice a week over

the course of the study (14 weeks). Bloods were collected from the tail veins once each

week, the sera prepared and tested for levels of complement by ELISA. In brief, goat IgG

anti-mouse C3 antibody (#55463, Cappel, ICN Pharmaceuticals Inc., Aurora, OH) diluted

1:250 was used as the capture antibody. A peroxidase-conjugated goat IgG anti-mouse

C3 antibody (#55557, Cappel-ICN Pharmaceuticals Inc.) diluted 1:500 was used as the

detection antibody. ELISAs were run in triplicate for each serum sample diluted 1:12,500

in PBS. For a standard, mouse complement provided by Cappel, ICN Pharmaceuticals

Inc. was used and diluted over the range from 1:100 to 1:1,562,500. Color was developed

with TMB (Sigma, St. Louis, MO) for approximately 10 min and stopped by adding 50 ul

of 2N H2SO4; OD readings were determined at 450 nm.

Measurement of Salivary Rates

To measure stimulated flow rates of saliva, individual mice were weighed and

given an intraperitoneal (i.p.) injection of a cocktail containing isoproterenol

(0.2 mg/100 g body weight) and pilocarpine (0.05 mg/100 g body weight) dissolved in

PBS. Saliva was collected for 10 min from the oral cavity of individual mice using a

micropipette starting 1 min after the injection of the secretagogue. The total volumes of

saliva sample were measured.









Histology and Immunofluorescent Staining for B and T Lymphocytes

Submandibular glands were surgically removed at time of euthanasia (24 weeks of

age), placed in 10% phosphate-buffered formalin for 24 h, then embedded in paraffin and

sectioned at 5 [m thickness. Paraffin-embedded slides were de-paraffinized by

immersing in xylene, followed by dehydrating in ethanol. Tissue sections were stained

with H&E dye (Gainesville Service Tech., Gainesville, FL) and observed at 100x

magnification.

For immunohistochemical staining, the tissue sections were washed in PBS for

5 min, then incubated 1 h with blocking solution containing normal rabbit serum diluted

1:50 in PBS. Each section was incubated with rat anti-mouse B220 (BD Biosciences-

Pharmagen, San Diego, CA) diluted 1:10 and goat anti-mouse CD3 (Santa Cruz

Biotechnology, Santa Cruz, CA) diluted 1:50 for 1 h at 25 'C. The slides were washed

three times with PBS for 5 min per wash followed by a 1 h incubation with Texas Red-

conjugated rabbit anti-rat IgG (Biomeda, Foster City, CA) diluted 1:25 and FITC-

conjugated rabbit anti-goat IgG (Sigma Chemicals, St. Louis, MO) diluted 1:100 at

25 'C. The slides were washed thoroughly with PBS, treated with Vectashield DAPI-

mounting medium (Vector Laboratory, Burlingame, CA) and overlayed with glass

coverslips. Stained sections were visualized at 200x magnification.

Flow Cytometry

Splenic leukocytes were prepared for flow cytometric analyses as detailed

elsewhere. Aliquots of each cell preparation containing 1 x 105 cells were incubated

45 min with either R-PE-conjugated rat anti-mouse CD19 monoclonal antibody

(#557399), FITC-conjugated rat anti-mouse CD19 monoclonal antibody (#557398),

FITC-conjugated rat anti-mouse CD21 monoclonal antibody (#553818) or PE-conjugated









mouse anti-mouse CD22.2 monoclonal antibody (#5533384) (BD Biosciences-

Pharmingen, San Diego, CA), washed in FACS buffer, then analyzed for fluorescence

staining on a FACScan (BD Biosciences, San Jose, CA).

Detection of Anti-Nuclear Autoantibodies in the Sera

ANA in the sera of mice were detected using the Sigma ANA screening kit (Sigma

Chemicals, St. Louis). HEp-2-fixed substrate slides were overlaid with the appropriate

mouse serum diluted 1:50. Slides were incubated for 3 h at room temperature in a

humidified chamber. After three washes for 5 min with PBS, the substrate slides were

covered with FITC-conjugated goat anti-mouse IgG (Sigma, St. Louis) diluted 1:50 for

1 h at room temperature. After three washes, nuclear fluorescence was detected by

fluorescence microscopy at 100x magnification.

Results

Effects of CVF Treatment on the SjS-like Disease Profile in NOD.B10-H2b Mice

SjS-like disease in NOD.B10-H2b mice, similar to SjS in humans, is characterized

by high levels of B cell survival and proliferation, plus hyper-gammaglobulinemia with

production of autoantibodies(53). One mechanism postulated to regulate B cell survival

involves the cross-linking of the BCR with the CD19/CD21 complex via complement

component C3d. In a series of studies using either the NOD or NOD.B10-H2b mouse

models of SjS, we have shown that the critical time point to delay or prevent onset of

clinical disease is between 12 and 16 weeks of age, a time when B cell hyper-activity and

appearance of anti-acinar cell autoantibodies are occurring. Based on these two

independent observations, we examined the effects of depleting systemically complement

component C3 from NOD.B10-H2b mice by routine injections of CVF starting at 10

weeks of age. CVF resembles C3 functionally and binds to factor Bb, a proteolytic









product of factor B x factor D. CVF-Bb, however, is resistant to the complement

regulator proteins; thus, the persistence of CVF-Bb consumes plasma C3 resulting in the

depletion or inactivation of C3(153). Taking advantage of this property, CVF was

injected intraperitoneally into NOD.B 10-H2b mice twice weekly to block the production

of C3d. For comparative controls, age- and sex-matched NOD.B10-H2b mice were

injected with PBS. Beginning 2 days prior to the first injections and at weekly intervals

thereafter, the individual mice were bled, their sera prepared and pooled and the C3 levels

in the pooled sera determined by ELISA. As presented in figure 2-1, NOD.B10-H2 b mice

treated with CVF showed a rapid, but temporary decrease in the levels of detectable C3.

In contrast, NOD.B10-H2b mice treated with PBS maintained constant levels of serum

C3.

An early marker predictive of subsequent onset of SjS-like disease in NOD and

NOD.B10-H2b mice is the activation of a serine protease capable of proteolytic digestion

of parotid secretary protein (PSP), a zinc-dependent anti-bacterial protein secreted into

saliva(60). Measurements of serine protease activity in the salivas of PBS and CVF-

treated NOD.B10-H2b mice revealed that all NOD.B10-H2b mice at 7 weeks of age failed

to show detectable serine protease activity, but exhibited positive serine protease activity

by 24 weeks of age (data not presented). These data indicate that treatment with CVF did

not alter this acinar cell-associated pathophysiological manifestation.

The unique characteristic of NOD and NOD.B 10-H2b mice as models for Sj S is the

gradual decrease in salivary flow rates starting around 12-14 weeks of age concomitant

with the appearance of leukocyte infiltrates within the exocrine glands. To measure

changes in salivary flow rates, each mouse was weighed and injected with isopreterenol









and pilocarpine. Saliva flow rates were measured 1 day prior to treatment and every 2

weeks thereafter. NOD.B10-H2b mice treated with PBS showed a 20% decrease in saliva

secretion over the first 12 weeks of treatment, as depicted in figure 2-2. In contrast, the

NOD.B10-H2b mice treated with CVF showed nearly a 70% average increase in their

salivary flow rates during the same period, consistent with their increasing age and size.

At 24 weeks of age or 14 weeks after initiating treatment, the mice were euthanized

and their submandibular glands explanted for histological analysis. As presented in figure

2-3A, NOD.B10-H2b mice treated with PBS showed multiple areas of leukocyte

infiltration, having an average of 13 foci per histological section. In contrast, NOD.B10-

H2b mice treated with CVF showed fewer areas of leukocyte infiltration, having an

average of only two foci per section (Figure 2-3C). In addition, the foci in the CVF-

treated mice appeared to be smaller. Furthermore, when immunostained using anti-B220

and anti-CD3 antibodies to detect B and T cells, respectively, the number of B cells

within the foci of the CVF-treated animals was significantly reduced (Figure 2-3B and 2-

3D).

Several clinical manifestations of SjS are mediated by autoantibodies, thus, the

detection of such autoantibodies are important in the diagnosis of disease. The presence

of ANA in sera of human patients, as well as NOD and NOD.B10-H2b mice, indicates

development of the clinical phase. Thus, at time of euthanization, blood was collected

from each animal and the sera prepared and pooled within each group. The two-pooled

sera were assayed for the presence of ANA using HEp-2 cells. As expected, NOD.B10-

H2b mice treated with PBS were strongly positive for ANA in their serum (Figure 2-4C),

while NOD.B 10-H2b mice treated with CVF proved to be nearly negative (Figure 2-4D).









Effects of CVF Treatment on B cell Sub-Populations

NOD.B10O-H2b mice injected with CVF or PBS were euthanized at 24 weeks of

age, their splenocytes collected and examined for different sub-populations of B cells

based on expressions of the BCR's co-receptors CD19, CD21 and CD22. Mice injected

with CVF had a two-fold decrease in the number of B cells within unfractionated total

spleen cell preparations that expressed CD19 molecules on their surface when compared

to normal BALB/c mice or NOD.B10-H2b mice treated with PBS (Figure 2-5C versus

Figure 2-5A and B, respectively; Table 2-1). In addition to the altered distribution of

CD19-positive B cells in animals treated with CVF, there was also a concomitant

reduction in the MFI of CD19 expression on the surface of the B cells, as shown in

Figure 2-5D. This shift in MFI was observed for both the CD19hi and CD191o" B cells

populations.

Similar to the altered expression of CD19 on B cells from CVF-treated mice,

expression of CD21, a receptor for the C3d molecule, was also reduced as compared to

the CD21 expression on B cells from PBS-treated mice. As presented in Table 2-1, the

numbers of CD19-positive B cells expressing high levels of CD21 nearly disappeared

(0.1% for CVF-treated mice versus 41.0% for the PBS-treated control mice).

Interestingly, expression of CD22 on CD19-positive B cells, a negative regulator of B

cell responses, proved similar for splenic B cells isolated from either PBS- or CVF-

treated mice (Table 2-1).

Discussion

In the present study, we have investigated the possible involvement of complement

component C3 in the development of SjS-like disease of NOD.B10-H2b mice by

temporally reducing C3 during the early stage of disease by treatment with CVF. The









NOD.B10-H2b mouse is a model of primary SjS exhibiting many of the immunological

manifestations observed in SjS patients(53), including leukocyte infiltration of the

exocrine glands, hyper-reactive B cells, hyper-gammaglobulinemia and production of

autoantibodies thought to be the effectors of clinical disease. Recent studies of both

humans and animal models have suggested that B cells and pathogenic autoantibodies

play major roles in SjS and SLE due in part to a breakdown in B lymphocyte self-

tolerance, possibly from a cross-linking of BCRs with co-receptors CD19 and CD21(114,

154). One mechanism involved in survival, activation and proliferation of B cells is

through such cross-linking involving C3d. We reasoned, therefore, that inactivation of C3

at the time of disease onset (12 2 weeks of age) might prevent this over-reactivity of B

cells and reduce the severity of the SjS-like disease in NOD.B10-H2b mice.

Results of the present study indicate that CVF-treatment of NOD.B10-H2b mice

starting at 10 weeks of age, while having little or no effect on the aberrant physiological

activities (e.g., activation of unique serine proteases), reduced the severity of lymphocyte

infiltration into the salivary glands, decreased the production of ANAs and prevented the

onset of xerostomia sicca. Interestingly, this reduction in disease severity correlated with

significant reductions in both the numbers of splenic B cells expressing CD19 and a

concomitant reduction in the mean fluorescent intensity (MFI) of CD19 expression in the

absence of any major changes in CD22 expression levels. Stronger cross-linking of the

CD19/CD21 complex to the BCR is known to be mediated by complement-tagged

antigen (155, 156), the effects of which is a greatly lowered signaling threshold, a

prolonged BCR signaling, sustained tyrosine phosphorylation of proteins and enhanced

responses of B cells to antigen.









Based on the present findings, we would postulate that the importance of C3 in Sj S-

like disease of NOD.B10-H2b mice involves C3d cross-linking BCRs with the co-

receptors CD19/CD21 molecules or CD21/CD35 through CR1/CR2 to provide powerful

secondary signals within B lymphocytes. Although we were able to find direct

correlations between C3-depletion, loss of CD19hi/CD2lhi B cell sub-populations and

reduced autoimmunity in CVF-treated NOD.B10-H2b mice that could suggest one mode

of action might be the loss of C3d cross-linking of the BCR and its co-receptors, a

number of other explanations are also possible. First, however, it may be possible to rule

out any involvement of membrane-attack complex formation since the NOD mouse is a

C5-deficient animal (157). Nevertheless, the importance of C3 in both innate and

adaptive inflammatory responses cannot be underestimated. C3a is a critical mediator of

inflammatory responses, especially in recruiting monocytes/macrophages to the site of

cell injury. Furthermore, C3 products can bind to or form complexes with antigens to

facilitate inflammatory and immunological responses, in part through binding to specific

complement receptors, such as CR1/CR2 present on FDCs. Such localization of antigen

on FDCs in secondary lymphoid tissues promotes germinal center formation, B cell

retention, survival and activation within germinal centers, as well as subsequent antibody

formation (158-160). Thus, reduced germinal center formation in the salivary glands of

CVF-treated animals could be due to reduced levels of functional C3 affecting FDC

secretion of chemokines that would normally recruit B lymphocytes to the germinal

centers.

The role of complement in either development or severity of Sj S disease in humans

has not been easily defined, resulting in conflicting reports appearing over the past two









decades. Molina et al. (161) reported that complement levels in SjS patients with

neutrophilic inflammatory vascular disease were decreased, while Thomsen et al.(162)

reported increased levels in primary Sj S patients. Both of these reports, however, differed

from an earlier study by Fishbach et al. (163) indicating that SjS patients exhibited

normal levels of complement. Recently, renewed interest in this area has surfaced with

reports that hypocomplementaemia (specifically, reduced levels of C3 and/or C4) is

closely associated with B cell lymphoma development in Sj S and increased pathogenicity

(164). Considered together, levels of complement may vary during progression and

activity of SjS; however, the role of complement components, like C3, appears to be

essential for prolonged hyper-activity of B cells. Understanding the mode of action for

C3 and its active components could be useful in designing highly specific intervention

therapies in treating SjS patients, assuming individuals predisposed to develop SjS can be

identified early enough in the disease state.













1200





e 60


L 4 .+--PBS Trealed
20
R-- --- CVF Trealed

-5 0 5 10 15 20 25 30 35 40 45 50 55 60 65
Day(s) after initial Injection

Figure 2-1.C3 levels in the plasma of NOD.BI10-H2b mice following treatment with cobra
venom factor (CVF) or PBS. Starting at 10 weeks of age and continuing for
14 weeks, mice received i.p. injections twice weekly of either CVF or sterile
PBS. Bloods were collected, the sera prepared and the levels of C3 were
determined using ELISA. The star (*) indicates time of the first injections.















12 P<0 05

10ns
0











= Day -1 Day +83 Day -1 Day +83


Figure 2-2. Salivary flow rates ofNOD.B10-H2b mice treated with CVF or PBS.
NOD.B10-H2b mice treated either with PBS (open bars) (n=8) or CVF
(striped bars) (n=8) were given an i.p. injection of isoproterenol and
pilocarpine on the day prior to (day -1) and 7 weeks after (day 83) treatment.
Saliva was collected for 10 min from the oral cavity of individual mice using a
micropipette starting 1 min after the injection of the secretagogue. The volume
of each saliva sample was measured and standardized against the weight of
the individual mice. Data are the means of eight animals per group + S.D.
Difference in salivary flow rate was determined by Student-Newman-Keuls
test. p<0.05 was considered significant.
test. p< 0. 05 was considered significant.




































Figure 2-3. Histological examination of the exocrine glands of NOD.B10-H2b mice
treated with CVF or PBS. Submandibular glands, removed from each mouse
at 24 weeks of age and fixed in 10% formalin, were stained with Mayer's
H&E dye. Stained sections from glands of NOD.B10-H2b mice treated with
PBS (A) or CVF (B) were observed at 40x magnification for glandular
structure and leukocyte infiltrations. Lymphocyte foci are indicated with
arrows. The distribution of the T and B cells within the foci were determined
by staining first with rat anti-mouse B220 or goat anti-mouse CD3 antibodies,
followed by an incubation in Texas Red-conjugated rabbit anti-rat IgG- or
FITC-conjugated rabbit anti-goat IgG antibodies, respectively. The slides
were treated with Vectashield DAPI-mounting medium and overlayed with
glass coverslips. Stained sections containing a focus from a PBS (C) or CVF
(D) treated animal were visualized at 200x magnification. T cells stain green,
B cells stain red and cell nuclei stain blue. All individual figures are
representative.


A B









C D








































Figure 2-4. Detection of anti-nuclear autoantibodies (ANAs) in NOD.B 10 -H2b mice
treated with CVF or PBS. ANAs in a positive control serum (A), a negative
BALB/c serum (B) or the sera of NOD.B10-H2b mice treated with PBS (C) or
CVF (D) mice were detected using the ANA screening kit supplied by Sigma
Chemicals. HEp-2-fixed substrate slides were incubated with the appropriate
mouse serum for 3 h, followed by development with FITC-conjugated goat
anti-mouse IgG secondary antibody for 1 hour. Nuclear fluorescence was
detected by fluorescence microscopy at 100x magnification. All individual
figures are representative.







50
















High High
High High
1^~~ K--
SLow Lo -


10 10 10 10 10 1,
Fluorescent intensity hanelFL2 CD19PE Fluoresent intensity channel FL2- CDI9PE

Figure D19 on splenic B lymphocytes of NOD.B -H2



treated with eit High PBS (B) or CVF (C), prepared as s ige-cell suspensions
antibody. Each cell preparation was analyzed by fLow cytometry for




fluorescentintensitchainingel FL2 CD1 identifyPE CD19uhi oresenCD in19esic expressing aelB cells. The1PE


Figure 2-5. Reduced expression of CD19 on splenic B lymphocytes of NOD.B10 miH2
mice treated with CVF. Spleens were freshly explanted from normal,
untreated BALB/c (A) as well as NOD.B10-H2 mice at 24 weeks of age
treated with either PBS (B) or CVF (C), prepared as single-cell suspensions
and incubated with R-PE-conjugated rat anti-mouse CD19 monoclonal
antibody. Each cell preparation was analyzed by flow cytometry for
fluorescence staining to identify CD1911 or CD1910 expressing B cells. The
fluorescent intensities of CD19 in splenic B cells ofNOD.B1O-H2b mice
treated with CVF (dark solid line) vs. PBS (dotted line) and BALB/c (light
solid line) are shown in the histogram of panel D. All individual plots are
representative.





















Table 2.1. Changes in the splenic CD19-positive B
eight female mice.


FA


cell phenotypes. Each group included


%CD19 B cells
(in spleen)


%CD19Lo

%CD19Th


%CD21Lo

%CD21I"t

%CD21 I


%CD22LO

%CD22mI


CVF

18.3+0.7


1.5+0.1 (8.2+0.9)

16.8+0.8 (91.8+0.92)


PBS

37.0+0.6


3.1+0.1 (8.3+0.5)

33.9+0.71 (91.7+0.5)


0.4+0.1 (2.6+0.7) 1.9+0.1 (5.8+1.0)

14.5+0.7 (97.3+0.7) 17.7+0.8 (52.8+0.1)

0.02+0.01 (0.1+0.1) 14.0+0.6 (41.4+1.2)


1.2+0.1 (7.3+0.5)

14.9+0.7 (92.7+0.5)


2.7+0.2 (7.9+0.8)

30.1+0.8 (92.1+0.8)


a % of cells within the total spleen cell population, followed in parenthesis by % of the CD19-positive B cell population.














CHAPTER 3
EFFECT OF KNOCKING OUT COMPLEMENT COMPONENT 3 ON
COMPLEMENT AND B LYMPHOCYTES IN THE DEVELOPMENT OF SJS-LIKE
AUTOIMMUNE DISEASE

Introduction

Sjogren's syndrome is a chronic human autoimmune disease in which the clinical

symptom is highly dependent on the activity of B lymphocytes (1-10) One mechanism

that controls survival, activation and proliferation of B cells is through cross-linking of B

cell receptors and their co-receptors, especially CD19 and CD21. Cross-linking of BCRs

and co-receptors that result in the hyperproliferation of B cells involves C3d (165). To

investigate this issue in greater detail, I turned to the NOD.B10-H2b mouse, our model of

primary Sj S exhibiting many of the immunological manifestations observed in Sj S

patients (53), including hyper-reactivity of B cells, hypergammaglobulinemia, and

production of numerous autoantibodies. We have shown that NOD.B10-H2b mice

exhibited reduced apoptosis, hyper-proliferation and over-activation of B lymphocytes

starting around 10 weeks of age. The result is the production of both organ-specific and

organ-nonspecific autoantibodies.

In chapter 2, I reasoned that inactivation of complement component C3 might

prevent this over-reactivity of B cells and subsequently reduce the severity of the Sj S-like

disease in NOD.B10-H2b mice by preventing production of C3d, thereby reducing the

cross-linking of BCRs and their CD19/CD21 co-receptors. Treatment of NOD.B 10-H2b

mice with cobra venom factor (CVF), known to deplete C3 from circulation, while not

preventing the aberrant physiological activities of pre-clinical disease (e.g., activation of









unique serine protease), reduced the severity of lymphocyte infiltration into the salivary

glands, the production of autoantibodies, and the degree of salivary gland dysfunction.

Interestingly, this reduction in clinical disease severity correlated with significant

reductions in the co-expressions of CD19/CD21 on the B cell subpopulations. No major

changes were noted in the CD22 expression levels of CD19-positive B cell

subpopulations (166).

These initial findings suggested a direct correlation between C3 depletion, loss of

CD19h'/CD2lhi B cell subpopulations and reduced autoimmunity in CVF-treated

NOD.B10O-H2b mice (166). However, the usage of CVF to deplete C3 might manifest

other unexpected immunological consequences. The action of CVF on complement

appeared to last approximately 2 weeks, due to the fact that the immune system reacts to

the CVF by making antibodies against it, rendering CVF ineffective (153). It is unknown

whether immune response that results in antibodies against CVF has any consequences

on the on-going autoimmune process. Study has shown that autoimmunity can be

prevented if the immunological response is deviated by exposing the immune system to

stimulating antigen prior to the initiation of autoimmunity (167). In addition to the

known immunological effect, the purity of CVF resolved by SDS-PAGE is estimated to

be 95% as described in the manufacture product sheet. The effect of the undetermined

portion of CVF (-5%) remains unknown. To eliminate any unforeseeable consequences

of CVF, it is critical to examine the role of C3 by genetically knocking out C3 gene in a

newly described mouse referred to as C57BL6.NOD-AecJAec2 or double congenic

(DC/DC).









In this study I have reassessed the critical role of C3 in the pathogenesis of Sj S by

examining Sj S-like disease in a C3 gene KO model predisposed to this autoimmune

disease. In this chapter, the effect manifested by C3 on the severity of autoimmune

exocrinopathy and its impact on the development of B lymphocytes is presented.

Materials and Methods

Generation of C57BL/6.NOD-AeclAec2. C3' Mouse

Previously, we have generated a mouse strain designated as C57BL/6.NOD-

Aec]Aec2 or DC/DC that contained both the Idd3 and Idd5 chromosomal intervals from

the NOD in the non-autoimmune C57BL/6 mice genetic background. This animal

manifested all the phenotypes of human Sjogren's syndrome. To generate the C3 KO

mice, C57BL/6.NOD-Aec]Aec2 female mice were bred with B6.129S4-C3tmlcrr/J male

purchased from The Jackson Laboratory (Bar Harbor, ME). Fl generation offspring

were interbred. F2 generation offspring were screened by genotyping for homozygosity

for Aecl locus, Aec2 locus, and C3 gene knockout. Additional mit markers were used

during the genotyping process to select for non-crossover by the C57BL/6 background at

the Aecl and Aec2 loci of the NOD mice. All the animals used in this experiment were

bred and maintained at the Animal Care Services at University of Florida.

Proteolysis of Parotid Secretory Protein (PSP)

Detection of PSP proteolysis was carried out by incubating whole saliva specimens

with a synthesized oligopeptide corresponding to amino acids 20 through 34 of the

published sequence for mouse PSP. This oligopeptide contains the proteolytic site

(NLNL) for a serine kinase present in salivary glands during development and onset of

Sj S-like disease in the NOD mouse (unpublished data). Eight [l of saliva collected from

individual mice were mixed with 42 [l of the PSP oligopeptide (2.5mg/ml) and incubated









at 42C for 12 hrs. Following incubation, 50 pl Tris-HCI buffer (50 mM, pH 8.0) was

added and the mixture centrifuged through Micro-spin filter tubes at 14,000 rpm for 10

min. The filtrates were analyzed by HPLC (Dionex Systems, Sunnyvale, CA) for

proteolytic products. Control samples consisted of 50 [l of the PSP oligopeptide.

Detection of Cleaved Caspase-3 in the Submandibular Glands

Submandibular glands were surgically removed at time of euthanasia (4-7 and 24-

27 weeks of age), placed in 10% phosphate-buffered formalin for 24 h, then embedded in

paraffin and sectioned at 5 [tm thickness. Paraffin-embedded slides were de-paraffinized

by immersing in xylene, followed by dehydrating in ethanol. The tissue sections were

washed in PBS for 5 min, and then incubated 15 min at RT in Sniper blocking solution

(Biocare Medical Cat# BT967H, Concord, CA). Each section was incubated with Rabbit

anti-Cleaved Caspase-3 diluted at 1:400 (BioCare Medical #CP229B, Concord, CA)

overnight at RT. The slides were washed three times with PBS for 5 min per washed

followed by 30 min incubation at RT with Mach-2 goat anti-rabbit HRP polymer

secondary antibody (BioCare Medical #RHRP520, Concord, CA). The slides were

washed thoroughly with PBS, and then stained for Cardassian DAB chromagen (Biocare

Medical Cat# DBC859L10, Concord, CA). Rinsed in deionized water, and

counterstained for methyl green (DakoCytomation Cat# S1962, Clostrup, Denmark).

Stained sections were visualized at 200X magnification. For each whole submandibular

gland of the animals, number of cleaved Caspase-3 positive cells was identified and

counted

Salivary Protein Concentration and Salivary Amylase Activity

Total protein content was determined using the Bradford method. Amylase activity

in saliva was determined using the InfinityTM Liquid Amylase Kit (Thermo Trace









Electron Corp. Waltham, MA) in which starch was the substrate. Saliva samples were

diluted 250-fold with deionized water and added to 1 ml of the InfinityTM Amylase

Liquid Stable Reagent. Following 1 min and 2 min incubators at 370C, absorbance was

measured at a wavelength of 405 nm. Amylase activity was calculated according to the

manufacture's instructions using the formula: Amylase activity (U/L) = A A/2 x 5140 x

400 (sample dilution).

Histological Examination of Submandibular and Lacrimal Glands

The animals were euthanized at 5, 9, 13, 17, and 25 weeks of age. Submandibular

and lacrimal glands were surgically removed from each mouse and placed in 10%

phosphate buffered formalin for 24 hrs. Fixed tissues were embedded in paraffin and

sectioned at 5 [im thickness. Paraffin-embedded slides were de-paraffinized by

immersing in xylene, followed by dehydrating in ethanol. The tissue sections were

stained with H&E dye (Gainesville Service Tech, Gainesville, FL). Stained sections

were observed at 100X and 200X magnifications for glandular structure and leukocyte

infiltration.

Detection of Anti-Nuclear Autoantibody in Sera

ANA in the sera of mice were detected using ANA screening kit (Immunoconcepts,

USA). HEp-2 fixed substrate slides were overlaid with the appropriate mouse serum

diluted 1:40. Slides were incubated for 30min at room temperature in a humidified

chamber. After three washes for five minutes with PBS, the substrate slides were

covered with FITC-conjugated goat anti-mouse IgG (Immunoconcepts) diluted 1:50 for

30 min at room temperature. After three washes, nuclear fluorescence was detected by

fluorescence microscopy at 200X magnification.









Detection of Immunoglobulin Specific Muscarinic Type III Receptor Autoantibody

Anti-M3R antibodies in sera of C57BL/6.NOD-AeclAec2 mice were determined as

described in detail elsewhere (44). In brief, Flp-In CHO cells transfected with mM3R

were collected from growing cultures, washed once with phosphate-buffered saline

(PBS), and resuspended in FACS buffer (PBS, 0.5% BSA, 0.07%NaN3). Aliquots of

cells at a density of 1 x 106 cells/0.1 ml were incubated 2 hrs at 40C with 10 tl sera from

individual mice or pooled from appropriate groups. Cells were washed once with FACS

buffer, resuspended in 50 [l of FACS buffer and incubated for 30 min at 40C with either

FITC-conjugated goat anti-mouse IgGI, IgG2b, IgG2c, IgG3, IgM (Southern

Biotechnology Associates, Inc., Birmingham AL). After a final wash with FACS buffer,

the cells were resuspended in FACS buffer and analyzed using a FACScan cytometer

equipped with Cell Quest software (Becton Dickinson, Mountain View, CA). Control

reactions included cells incubated with secondary antibody alone or an appropriate

isotype control. An increase in fluorescence intensity compared to secondary antibody

alone was considered a positive reaction.

Flow Cytometry for Subpopulations of B cells

Spleens were freshly explanted from euthanized mice and gently minced through a

steel sieve. Following a single wash with PBS, the red blood cells were lysed by a 7 min

exposure to 0.84% NH4C1. The resulting cell suspensions were washed two times in

PBS, counted and resuspended in FACS buffer (PBS supplemented to 2% ABS and

0.010/oNaN3) to lxlO8cells/ml. Aliquots of each cell preparation containing 1 x 105 cells

were incubated 45 min with either R-PE-conjugated rat anti-mouse CD19 monoclonal

antibody (#557399), FITC-conjugated rat anti-mouse CD19 monoclonal antibody









(#557398), FITC-conjugated rat anti-mouse CD21 monoclonal antibody (#553818), PE-

conjugated mouse anti-mouse CD22.2 monoclonal antibody (#5533384), or PE-

conjugated goat anti-mouse CD23 (#X) (BD Biosciences Pharmingen, San Diego, CA),

washed in FACS buffer, then analyzed for fluorescence staining on a FACScan (BD

Biosciences, San Jose, CA).

Measurement of Stimulated Saliva Secretion

To measure stimulated flow rates of saliva, individual mice were weighed and

given an intraperitoneal (ip) injection of a cocktail containing isopreterenol (0.2 mg / 100

gm body weight) and pilocarpine (0.05 mg / 100 gm body weight) dissolved in PBS.

Saliva was collected for 10 min from the oral cavity of individual mice using a

micropipette starting one min after the injection of the secretagogue. The volume of

saliva sample was measured. The saliva samples were then frozen at -800C until

analyzed.

Statistical Analysis

Differences in cleaved Caspase-3, stimulated saliva volume, amylase activity, or

salivary protein concentration were analyzed with the Student-Newman-Keuls test.

p<0.05 was considered significant.

Results

Profiling of Phase I of SjS-like Autoimmune Exocrinopathy of C57BL/6.NOD-
AeclAec2.C3' Mice

The salivary glands include the submandibular gland, sublingual gland and the parotid

gland as well as numerous minor salivary glands. It is innervated by both sympathetic

and parasympathetic nerves. The parasympathetic nerves utilize the muscarinic type III

receptor (M3R)to regulate the fluid secretion in the salivary gland while the sympathetic









nerves use the P-adrenergic receptor to modulate the protein secretion (17). In Sj S, the

pathway which regulates fluid secretion is thought to be dysfunctional due to the activity

of the M3R autoantibody binding to its receptor (10). In addition, acinar cells made up of

mucous cells (release mucins) and serious cells (secrete kallikrein proteins, enzymes such

as amylase, peroxidases, lysozyme, lactoferrin, cystatins, and histatins) are sporadically

destroyed (59). Therefore, Phase I of this autoimmune response is often characterized by

the loss of fluid secretion resulting in an elevated concentration of salivary proteins.

Because of the sporadic cellular destruction, this phase is mainly accompanied by an

increase in activity of Caspase-3 and expression of cysteine protease that cleaves PSP.

For the past few decades, tremendous amounts of research devoted to delineate the

progression of Sj S using animal models. It proposed that Sj S-like autoimmune disease

progresses through three different but slightly overlapped phases. At Phase I from 4-8

weeks of age, the animals undergo delay in organogenesis allowing the delay expression

of acinar cell antigens, and consequently, the lack of clonal deletion of autoreactive

lymphocytes escaping into the periphery. In addition, the constant remodeling of the

cellular physiology results in expression of aberrant or incorrect proteins or enzymes,

leading the loss of cellular homeostasis at Phase I of the disease. Furthermore, the

cellular dynamic of the animal is accompanied by the initiation of acinar cell apoptosis

resulting in the loss of glandular mass (59).

As presented in chapter 2, depletion of C3 did not change the intrinsic properties of

the acinar cells of the animal which are mainly regulated by the genetic elements of the

animal. It further supported the observation that depletion of C3 did not change the

ability of the cysteine protease to cleave parotid secretary protein (PSP). Consistently,









this study has shown that knocking out C3 also did not affect the activity of cysteine

protease in degrading PSP in saliva samples (Figure 3-1). As expected, PSP cleavage

occurred as early as 5 weeks of age in the disease control animals, C57BL/6.NOD-

Aec].Aec2, predisposed to SjS-like disease.

concurrent with the aberrant expression of PSP cleavage product at Phase I, apoptosis

can also be observed at this phase. Apoptosis is the mechanism in which cells are

programmed to undergo cell death in response to detrimental trigger. Caspases are a

family of proteases known to mediate programmed cell death. Apoptosis of exocrine cells

has been suggesting to be critical during the early phase of the development of

autoimmune exocrinopathy (60). The controlled cell death is thought to be involved in

exposing and releasing of cryptic antigens that the immune system has never recognized

previously. As the result of recognizing newly exposed antigens, when sufficient level of

antigens are being presented, the immune system reacts by possibly recruiting

inflammatory cells to the site of injury such as the exocrine glands in Sj S. One of the

ways to identify apoptosis is by using expression of cleaved caspase-3 to detect

endogenous levels of the large fragment (17/19 kDa) of activated caspase-3. As

anticipated, C57BL/6 animals serving as disease-free normal control mice showed little

positivity for cleaved caspase-3 in the submandibular glands from 4-7 to 24-27 weeks of

age, while C57BL/6.NOD-Aec].Aec2 mice with Sj S exhibited a significant increase of

approximately five-fold in the number of cleaved caspase-3 positive cells in the

submandibular glands at 4-7 weeks of age and decreased to a normal level at 24-27

weeks of age. Interestingly, C57BL/6.NOD-AecJAec2.C3- mice displayed a similar

level of apoptosis or a similar number of cleaved caspase-3 positive cells to C57BL/6









disease-free control mice, and a five-fold decrease compared to C57BL/6.NOD-

Aecl.Aec2 at 4-7 weeks of age. However, at 24-27 weeks of age, C57BL/6.NOD-

AeclAec2.C3- mice showed no significant increase in cleaved caspase-3 compared to

C57BL/6 and C57BL/6.NOD-AeclAec2 mice (Figures 3-2a, 3-2b).

Profiling of Phase II of SjS-like Autoimmune Exocrinopathy of C57BL/6.NOD-
Aecl.Aec2. C3' Mice

In response to the pathophysiological abnormality that occurred during Phase I of

Sj S-like autoimmune exocrinopathy, the animal initiated an immunological reaction to

changes which signify Phase II of the disease. The immunological response includes

leukocyte infiltrations in submandibular and lacrimal glands that may play an important

role of glandular destruction by occupying the space that once resided by acinar and

ductal cells of the glands. In addition, the Phase II of Sj S is characterized by presence of

autoantibodies produced by autoreactive B lymphocytes that escaped developmental

selection (61). Many studies have now indicated that the clinical manifestation of Sj S

which are involved in the decline of salivary flow rates, is possibly mediated by

autoantibody against M3R (56, 65, 150). Other autoantibodies such as the presence of

ANAs, anti-Ro and anti-La, found in sera of human patients and SjS animal models,

indicate the advancement of Sj S to a clinical phase involving secretary dysfunction (121).

To examine the leukocytic infiltrations in the exocrine glands, mice were

euthanized at 5, 9, 13, 17, and 25-27 weeks of age. The submandibular and lacrimal

glands were explanted for histological examination by routine H&E staining.

Interestingly, meticulous examination of the submandibular and lacrimal glands revealed

the complete absence of lymphocytic infiltrations in the C57BL16.NOD-AeclAec2.C3-"

mice. As expected, there was a tremendous number of infiltrates in the exocrine glands









including the submandibular and lacrimal glands of the diseased control mice in

C57BL/6.NOD-AeclAec2 while the C57BL/6 normal control mice exhibited no

lymphocytic infiltrations (Figure 3-3).

Concurrently, sera of the mice were collected prior to their euthanization. The

expression level of ANAs was performed using collected mice sera. Sera were incubated

on the Hep2 cells and visualized by staining with goat anti-mouse whole IgG-FITC

conjugated secondary antibodies. Expression of ANAs can be detected as early as 13

weeks of age in males and females of C57BL/6.NOD-AeclAec2 mice with higher

frequency and intensity at older ages suggesting the advancement of the autoimmune

disease process. Interestingly, ANAs were not seen at a 1:40 dilution in C57BL/6.NOD-

AeclAec2.C3~-~ mice even at 27 weeks of age in both male and female animals. C57BL/6

serving as normal control showed the absence of ANAs level when aged to 27 weeks old

(Figure 3-4).

One of the most critical autoantibodies believed to be involved in the shutdown of

salivary flow rate is anti-M3R. Previous studies using the NOD.IL4-/- and

NOD.B1O.H2b.IL4-' animals indicated that IgGI specific anti-M3R is the most important

effector autoantibody that is required for the secretary dysfunction of submandibular

glands. In this study, to determine the presence of anti-M3R, sera were incubated with

CHO cells expressing M3R on their surface. Sera that are positive for M3R will bind to

the cells and fluorescent at different intensity depending on the antibody titers using

isotypic fluorochrome conjugated secondary antibodies by flow cytometry. As shown

in Figure 3-5, pooled sera from C57BL/6.NOD-AeclAec2 mice were exhibited positivity

for IgGI-M3R, IgG2b-M3R, and IgG2c-M3R autoantibodies, while C57BL/6.NOD-









Aec]Aec2.C3- mice pooled sera failed to produce any IgGl-M3R and IgG2b-M3R

autoantibodies, or showed a decreasing level of IgG2c-M3R compared to the wild type

mice. Pooled sera from all the mice were positive for IgM-M3R, and were negative or

express at the same level for IgD-M3R and IgA-M3R respectively (Figure 3-6).

Profiling of Phase III of SjS-like Autoimmune Exocrinopathy of C57BL/6.NOD-
AeclAec2.C31 Mice

Phase III of SjS-like autoimmune exocrinopathy is characterized by glandular

secretary dysfunction exemplified by the loss of amylase activity due to the destruction

of the acinar cells and temporal decrease in stimulated salivary flow rates concomitant

with the appearance of leukocyte infiltrates within the exocrine glands occurred during

Phase II of the disease. To measure changes in secreted saliva volume, each mouse was

weighed and injected with a secretogue containing both isopreterenol and pilocarpine.

Stimulated saliva secretion was collected and measured. Analysis was performed

separately for male and female animals due to the gender dichotomy existing between the

two sexes in terms of the severity of the autoimmune process. Sj S-like diseased controls

male and female C57BL/6-NOD.AecJAec2 mice displayed a 30% loss of stimulated

saliva volume being secreted from 4 to 25 weeks of age. Interestingly, male and female

C57BL/6-NOD.AecJAec2. C3-- mice exhibited no loss of stimulated saliva volume over

time similar to the non-diseased C57BL/6 control mice. C57BL/6-NOD.AecJAec2.C3X-1

male and female mice actually showed a significant increase in secreted saliva volume

from 4 to 25 weeks of age (Figures 3-7 and 3-8).

Alpha-amylase activity was measured by the ability to degrade the substrate,

etylidenepNP-G7 using whole saliva samples collected from C57BL/6, C57BL/6.NOD-

Aec]Aec2, and C57BL/6.NOD-Aec]Aec2.C3- mice at 4, 8, 12, 16, and 24-27 weeks of









age. As shown Figure 3-9, C57BL/6 mice serving as normal control exhibited no loss in

a-amylase activity from 4 to 24-27 weeks of age, while the SjS-like diseased control

animals, C57BL/6.NOD-AecJAec2, exhibited significant decrease in a-amylase activity

over the same period of time, indicating the loss of acinar cell function in the salivary

gland. Interestingly, C57BL/6.NOD-AecJAec2.C3~-~ mice with no functional C3 gene

showed a slight increase in a-amylase activity from 4 to 24-27 weeks of age. No loss of

a-amylase activity of C57BL/6.NOD-AecJAec2.C3X-1 mice could be directly contributed

by down-regulation of cellular apoptosis, or possibly is the result of regeneration of

acinar cells in the absence of C3. Normal a-amylase activity over time found in

C57BL/6.NOD-AeclAec2. C3 mice is also accompanied by constant concentration of

salivary proteins while the disease control animal exhibited an elevated level of salivary

protein concentration over time (Figure 3-10).

Characterization of the changing dynamics in the subpopulation of B lymphocytes:
Marginal Zone (MZ) and Follicular (FO) B Cells

The clinical manifestation of Sj S is thought be highly dependent on the activity of

autoantibodies produced by B lymphocytes. Most evidence supports the notion that these

autoantibodies must be produced by autoreactive B lymphocytes(90). However, it

remains speculative which subpopulation ofB cells contributes more significantly to the

autoimmune process. Adding to this complexity, sex hormones play a critical role in the

changing dynamic of different B cell populations (168). Therefore, it is essential that

analysis of these B cell populations must be determined based on the difference in male

and female sexes of the animals. Splenocytes collected from male and female of

C57BL/6, C57BL/6.NOD-AecJAec2, and C57BL/6.NOD-AecJAec2.C3- mice were

analyzed by flow cytometry. Separation of MZ and FO B cells is based on the









fluorescent intensity of CD21 and CD23 markers. MZ B cells are CD23+CD21hi while

FO B cells are CD23+CD211nt. Figure 3-11 presents a representative finding for male

C57BL/6, C57BL/6.NOD-AeclAec2, and

C57BL/6.NOD-AeclAec2. C3-- experimental models, and similar analysis were done for

female from each of the three experimental mouse models (data not shown). Data for

both sexes are compared in Table 3.1.

As presented in Table 3.1, comparing the male among the three different mouse

models, C57BL/6, C57BL/6.NOD-AecJAec2, and C57BL/6.NOD-AeclAec2.C3-", of the

CD19+ splenic cells, the percent of MZ B cells found in the spleens of all three models

appeared to be very similar, while both male C57BL/6 and C57BL/6.NOD-AeclAec2

mice have similar percentage of splenic FO B cells at 61.70% and 64.63% respectively.

However, there is a reduction of approximately 9% of FO B cells found in the spleen of

male C57BL/6.NOD-AeclAec2.C3-1" mice compare to C57BL/6.NOD-AeclAec2 male

mice. In the three female models, the number of MZ and FO B cells found to be very

interesting. Of the female mouse CD19+ splenic B cells, the percent of splenic MZ B

cells appeared to be at 7.33, but increase up to 13.0 % in the C57BL/6.NOD-AeclAec2

female mouse. However, only 7.3% of CD19+ MZ B cells are found in C57BL/6.NOD-

Aec]Aec2.C3" mice which is similar to female C57BL/6 mice, possibly indicating the

decrease in hyperproliferation of the MZ B cells in C3KO female mice and returning it to

a normal number. Interestingly, the increase in the percentage of female splenic CD 19+

MZ B cells in the C57BL/6.NOD-AeclAec2 mice is accompanied by the decrease in the

percentage of splenic CD19+ FO B cells which found to be at 57.94%, while there are

65.60% and 66.70% of splenic CD19+ FO B cells are found in C57BL/6 and









C57BL/6.NOD-AecJAec2. C3-- mice respectively. Therefore, changing dynamic in the

populations of MZ and FO B cells is appeared to regulated and driven by both C3 and the

sex differences among the three animal models.

Discussion

In the present studies, I have extended our understanding on the role of

complement during the development of autoimmune exocrinopathy by using an animal

model in which C3 gene was knocked out in the animal model predisposed to Sj S-like

disease, referred to as C57BL/6-NOD.Aec]Aec2. Results from the current study have

clearly indicated the critical requirement of C3 for the full initiation of the Sj S. By

eliminating C3 gene using speed congenic breeding in the C57BL/6.NOD-Aec]Aec2

animals, the clinical manifestations of SjS-like disease were diminished. These included

significant reduction of cellular apoptosis, the absence of lymphocytic foci within the

exocrine glands, lack of ANAs production, and elimination of IgGI-M3R autoantibody

with decreasing level of other IgG isotypic M3R autoantibodies. Most importantly,

C57BL/6-NOD.AecJAec2. C3-- mice restored normal saliva secretion in both female and

male animals. In addition, alterations of splenic B cell subpopulations were identified.

In the three male models, C57BL/6, C57BL/6.NOD-AeclAec2, and C57BL/6.NOD-

Aec]Aec2.C3 -, of the CD19+ splenic cells, the percent of MZ B cells is similar among

the three models while splenic CD19+ FO B cells of the C57BL/6.NOD-AeclAec2.C3'-1

mouse showed decline of close to 9% compared to C57BL/6 and C57BL/6.NOD-

Aec]Aec2 mice. However, in the three female models, the number of splenic CD19+ MZ

and FO B cells appeared to be similar between the C57BL/6 and C57BL/6.NOD-

Aec]Aec2.C3- mice while there is an increase of 8% in the percent of MZ B cells and

decrease of 9% in the percent of FO B cells found in C57BL/6.NOD-AeclAec2 mice









compare to C57BL/6 and C57BL/6.NOD-Aec]Aec2.C3- -. The trends in the alterations in

various B cells populations among the three strains of mice were very interesting.

However, because of the small number of experimental animals in each strain, the final

output results performed by Student-Newman-Keuls test appeared to be statistically

insignificant

The previous study has suggested an important role of C3 during the development

of SjS in the NOD.B10.H2b, the animal model of primary SjS (166). Data presented in

chapter 2 indicated that when the level of serum C3 was temporally reduced by

intraperitoneally injecting 10-weeks old NOD.B10.H2b mice with CVF, the mice showed

reduced severity of lymphocytic infiltrations in the submandibular glands, decreased

production of ANAs and retention of normal salivary flow rates. Therefore, the SjS

phenotypes were greatly improved when C3 was depleted in serum at 10 weeks old

animals. However, the side effect of CVF on the biological and immunological reactions

on administered animals has never been determined and its role on autoimmune process

is unknown. Furthermore, the impurity of the C3 preparation raised questions about its

mechanism of action. Therefore, in this study, we focused our attention in investigating

the role C3 has on the pathogenesis of Sj S by using genetic knockout out of the C3 gene

in a mouse model that is predisposed to Sj S-like autoimmune disease.

SjS is one of the connective tissue autoimmune diseases in which the membrane

attack complex (MAC) is not involved in the cellular destruction in animal models and

human patients (169). In this context, NOD mice and NOD congenic strains are

consistent with that conclusion because they are deficient in C5 but still manifest the full

phenotypes of SjS. Saliva and salivary glands of SjS patients were found to contain or









express elevated levels of complement regulatory proteins compared to healthy subjects.

These complement regulatory proteins include protection (CD59), decay accelerating

factor (CD55), membrane cofactor protein (CD46) and clusterin (SP-40, 40), which

function to prevent complement mediated destruction on the exocrine glands in patients

(169). Therefore, it is possible to eliminate the possibility of complement mediated lysis

of acinar tissues in the glands. In this study, the complete absence of leukocyte infiltrate

in the exocrine glands in the C57BL/6-NOD.AecJAec2.C3-- mice was quite striking

because of the fact that C5 is still active in these animals. One of the cleavage product of

C5, C5a, is the most potent inflammatory mediator of the complement system (114). It is

clear that C5 is not required for the initiation of infiltrations into the injured tissues.

Additionally, C5a directly regulates the functions of PMNs by increasing their adherence

to vessels walls and their migration to site of tissue destruction (114). Interestingly, the

presence of PMNs has never been detected in salivary glands of human patients and

animals, but was found to be circulating in sera of patients with pSjS (170, 171). These

studies suggest the irrelevant role of PMNs in the development of SjS in term of

occupying the exocrine gland as part of leukocytic focus to carry out their destructive

function, but they reinforced the biological requirement and importance of C3 in SjS

autoimmune process.

Based on our previous study, the importance of C3 in adaptive immunity of SjS

must be addressed. Sj S appears to be a lymphoproliferative B cell disorder in which the

clinical phase of disease fails to develop in the absence of either B cells or

autoantibodies. B lymphocytes in SjS are believed to be hyper-proliferative, capable of

evading apoptosis, and overly sensitive to activation and maturation. C3 plays an









important role in the adaptive immunity by controlling activation, survival and

proliferation of B cells by cross-linking B cell receptors and their co-receptors CD19,

CD21 and CD22. Cross-linking of BCRs and B cell co-receptors by a cleavage product of

C3, C3b, can modulate the strength, intensity and duration of the signal generated by

BCR (113). Signals generated by the co-ligation of CD19/CD21 and BCR by the C3d

fragment act as positive regulators of B cell activation, resulting in the lowering of the

threshold for B cell activation, possibly contributing to the hyper-proliferative and hyper-

active properties of autoreactive B cells (165).

Activation of B lymphocytes requires secondary signals that mediated by C3d

cross-linking BCRs with the co-receptors CD19/CD21 molecules. In addition, C3

products can bind to or form complexes with antigens to facilitate inflammatory and

immunological responses, in part through binding to specific complement receptors, such

as CR1/CR2 present on FDCs. Such localization of antigen on FDCs in secondary

lymphoid tissues promotes germinal center formation, B cell retention, survival and

activation within germinal centers, as well as subsequent antibody formation (158, 159).

Therefore, elimination of C3 could prevent formation of functional germinal center in

secondary lymphoid tissues of the animals and formation of ectopic germinal centers-like

foci, often found in the exocrine glands of human and Sj S-like animal models.

The role of complement is well established in the etiology of other autoimmune

diseases such as SLE and rheumatoid arthritis. However its role in the pathogenesis of

SjS has remained elusive. Early studies have shown a correlation in the deposition of

immune of complex in the form of IgA or IgM with the clinical manifestations of SjS

(172). As mentioned previously, Cuida et al. (169) has demonstrated the presence of









complement regulatory proteins in saliva and salivary glands of Sj S patients serving to

prevent activation of complement on the tissues. Recent reports have demonstrated that

hypocomplementaemia (specifically, reduced levels of C3 and/or C4) is closely

associated with B cell lymphoma development in Sj S and increased pathogenicity (164).

Since all of the above studies using Sj S patients were examined only at Phase III or the

clinical phase of the disease in which complement activation may not be relevant or

required to the development of Sj S at this stage. Each patient behaved slightly differently,

which may explain the inconsistency in the interpretation of the data collected. With our

animal models, we were able to show that complement is required at Phase II of the

disease, not for the complement mediated lysis destruction but instead for activation,

proliferation, and survival of B cells. The low level of C3 found in sera of patients in the

study previous mentioned may be due to lack of circulating C3 in the periphery, but it is

probably the result of B cells taken up all the circulating C3 molecules for their

hyperactivity and hyperproliferation.

This study reinforces the importance of C3 in the pathogenesis of SjS. Its

implication in translational research may prove to be very effective and beneficial.

Further data is required to determine if C3d has any preferences of binding to self or non-

self antigens, and if reversal of disease can occur once C3 is infused back into the C3KO

mice.











PSP peptide

K


I~cwA IlinoaB -


PSP peptide
\K~


I C iNf D






II 1





Figure 3-1.Proteolysis of parotid secretary protein. A. PSP peptide alone. B. 24 wks old
C57B/6 (n=2). C. 24 wks old C57BL/6-NOD.Aec].Aec2 (n=2). D. 24 wks
old C57BL/6-NOD.AecJ.Aec2.C3- (n=5). All individual figures are
representative.


,, ,,


.1 i 1 4 I .- I I II : I I I


I, 1 1 I .


.. I I I 0 S I 111 11I


I .1 1I 3 1I 11 |












A




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il -" ^' W f *
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-*s .*-r *.% :


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U


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e: ** '.
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*.:". \ ,->.
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t
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*


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.~4.

fr .4

f*, it ..
Lr .2 w !


a


4-7 weeks 24-27 weeks
' LL rJ hifrl i bDCDC DCOC..C3-'


Figure 3-2.Examination of apoptosis by the presence of cleaved Capase-3 in the
submandibular glands. (a) Immunohistochemistry staining for Caspase-3. A.
4-7 weeks old C57BL/6 (n=4). B. 24-27 weeks C57BL/6. (n=4). C. 4-7
weeks old DC.DC (C57BL/6-NOD.Aec]Aec2). (n=4). D. 24-27 weeks old
DC.DC. (n=4). E. 4-7 weeks old DC.DC.C3- (C57BL/6-
NOD.AecJAec2.C3- -). (n=2). F. 24-27 weeks old DC.DC.C3"--. (n=2) All
individual figures are representative. (b) Number of apoptotic cells or
Caspase-3 positive cells in the submandibular glands determined by counting
total number of cells stained positive for cleaved Caspase-3 per whole
histological gland from 4-7 weeks old to 24-27 weeks old mice. Statistical
analysis was performed by Student-Newman-Keuls test. p<0.05 was
considered significant.




















B D F


Figure 3-3. Histological examination of the exocrine glands. Submandibular and lacrimal
glands were removed from each mouse approximately 24-27 weeks of age and
fixed in 10% formalin, were stained with Mayer's H&E dye. Stained section
of C57BL/6, lacrimal glands (A) submandibular glands (B). Stained section
of C57BL/6-NOD.AeclAec2, lacrimal glands (C) submandibular glands (D).
Stainined section of C57BL/6-NOD.AecJAec2.C3-", lacrimal glands (E)
submandibular glands (F). All individual figures are representative























Figure 3-4. Detection of anti-nuclear autoantibodies (ANAs) using Hep2 cells as subtrate.
24 weeks old C57BL/6 serum as negative control (A). 24 weeks old
C57BL/6-NOD.AeclAec2 serum as positive control (B). 24 weeks old
C57BL/6-NOD.AecJAec2.C3- serum (C). All individual figures are
representative















IgG1
200-

-150- .

101 .
d I' I



id' id id 10
FL1

S200-

C151 -.

10( -

0


i( id irF 1


1 Id 1i 1i6
FL1


IgG2b
200-

150-

10C. -

5(.


id' id id 10
FL1

200 1


FL1


200

150- .

10C.

lO:C


i id 10 1i
FL1


IgG2c
200-

150-

10' I





5d. Id i6 i
FL1

200

150-

10





' I 1
FL1


id id 1 106
FL1


IgG3
200

150

10 -





iJ id id FL1
FL1


i1 id 10 1i
FL1

200

15n

10

5, I.


1 d1 1F 1
FL1


Figure 3-5. Detection of anti-muscarinic acetylcholine type-3 receptor antibodies. Pooled

sera collected from 27 wks old C57BL/6 (n=5), 30 wks old C57BL/6-

NOD.Aec]Aec2 (n=5), and 24 wks old C57BL/6-NOD.AecJAec2.C3- (n=5)

mice. Sera were incubated 2 hrs at 40C with either mouse-M3R transfected

Flp-In CHO cells or respective isotype controls. Cells were washed with

FACS buffer, resuspended in 50 pl of FACS buffer and incubated for 30 min

at 4oC with either FITC-conjugated goat anti-mouse IgGi, anti-mouse IgG2b,

anti-mouse IgG2c or anti-mouse IgG3 antibodies. After a final wash with

FACS buffer, the cells were resuspended in FACS buffer and analyzed using a

FACScan cytometer.












1gM


id ia ia


200 -

S150-

10C
r-







20 -

S150
0 150-
o
0


z









100

050-


Id Icd ib


IgD










id icF 10


200-

150

100

50

I"rI


IgA




Not Done


1d 1 10I
FL1











1l 10' 10
1I

id ic~ icd


Figure 3-6. Detection of anti-muscarinic acetylcholine type-3 receptor antibodies. Pooled
sera collected from 27 wks old C57BL/6 (n=5), 30 wks old C57BL/6-
NOD.AecJAec2(n=5), and 24 wks old C57BL/6-NOD.AecJAec2.C3X-1 (n=5)
mice. Sera were incubated 2 hrs at 40C with either mouse-M3R transfected
Flp-In CHO cells or respective isotype controls. Cells were washed with
FACS buffer, resuspended in 50 [l of FACS buffer and incubated for 30 min
at 4oC with either FITC-conjugated goat anti-mouse IgM, anti-mouse IgD, or
anti-mouse IgA antibodies. After a final wash with FACS buffer, the cells
were resuspended in FACS buffer and analyzed using a FACScan cytometer.


FL1


-






77



350- ** **

EL300

E 250


CU 200

in 150-

M 100





4 weeks 25 weeks

1 C57BL6(VWT) DC.DC DC.DC.C3'


Figure 3-7. Stimulated saliva flow of female animals. C57BL/6 (4 weeks, n=3, 25 weeks,
n=3), DC.DC (4 weeks, n=5, 25 weeks, n=5), and DC.DC.C3- (4 weeks, n=9,
25 weeks, n=6) were given an i.p. injection of isoproterenol and pilocarpine
and collected for 10 min from the oral cavity of individual mice using a
micropipette starting 1 min after the injection of the secretagogue. The volume
of saliva samples was measured. Statistical analysis was performed by
Student-Newman-Keuls test. **: p< 0.0], ***: p< 0.001.

























.***..


+:+*: -







4 weeks


I iC57BL6(WT)


EESDC.DC


DC.DC.C31


Figure 3-8. Stimulated saliva flow of male animals. C57BL/6 (4 weeks, n=4, 25 weeks,
n=4), DC.DC (4 weeks, n=8, 25 weeks, n=10), and DC.DC.C3 (4 weeks,
n=6, 25 weeks, n=9) were given an i.p. injection of isoproterenol and
pilocarpine and collected for 10 min from the oral cavity of individual mice
using a micropipette starting 1 min after the injection of the secretagogue. The
volume of saliva samples was measured. Statistical analysis was performed
by Student-Newman-Keuls test. *:p<0.05, ***:p<0.001.


-r


*-*


350-

S300-
a)
E
3 250-
O
u 200-

w 150-


'S

E 50-
U)


-F


25 weeks


0-1---






79



1.0106- *





> 6.0D105


4.0610-


E 2.0D10O



C57BLIS DCfDC DCIDC.C3--

= 14 wks M 24-27 wks

Figure 3-9. Amylase activity in saliva. Saliva of C57BL/6 {4 wks old (n=7), 24-27 wks
old (n=7)), DC/DC {4 wks old (n=13), 24-27 wks old (n= 11)}, and
DC/DC.C3" {4 wks old (n=10), 24-27 wks old (n=14)}were collected.
Amylase activity in saliva was determined using the InfinityTM Liquid
Amylase Kit. Saliva samples were diluted and added to InfinityTM Amylase
Liquid Stable Reagent. Absorbance was measured at a wavelength of 405 nm
after incubating at 370C. Statistical analysis was performed by Student-
Newman-Keuls test. *: p<0.05






80





o 8000 p


S 6000





a. 2000 .. . ..


I .I.I
0O C57BLI6 DCIDC DCIDC.C3"'

8 wks 24-27 wks


Figure 3-10.Salivary protein concentration. Saliva of C57BL/6 {8 wks old (n=7), 24-27
wks old (n=7)}, DC/DC {8 wks old (n=8), 24-27 wks old (n=8)}, and
DC/DC.C3-- {8 wks old (n=8), 24-27 wks old (n=8)) were collected. Total
salivary protein content was determined using the Bradford method.
Statistical analysis was performed by Student-Newman-Keuls test.








81



A. C57 BL6 Male 24-27 wks old


I. -


10 10 10




10 10 10
10 10 0 1 10 10 101 101l" 10 10 10 10i 10

B. C57 BLG-NO D.A ec Aec2 Male 24-27 wks old



10 10 10,- -







10 10 10
1 1 10 1 110 1 0 10 10 1 10 10


o10 10. 10 5

10 10 10


10 ** : 10 1 *. !
1 0 E ll1





S10 10 101 10i Io I' IQ id 1' 0o 10 10 10 10
CD23-PE w


Figure 3-11.An representation of an approach to delineate marginal zone (MZ) and
follicular (FO) B lymphocytes in male animals based on CD21 and CD23
markers. A. Male C57BL/6. B. Male C57BL/6-NOD.AecJAec. C.
C57BL/6-NOD.AeclAec2. C31-










Table 3-1.Changes in CD19+ splenic B cell populations

Experimental animals I animals Sex MZ B cells' FO B cells'

C57BLG6 3 M 6.680.36 61.702.10

3 F 7.33t0.69 65.601.20

BG-NOD.AeclAec2 3 M 8.001.00 64.631.40

3 F 13.00.70 57.94-5.00

B6-NOD.AecAec2.C3-'- 3 M 7.300.80 53.002.10
3 F 7.500.50 66.701.40

'Percentacle of CD1 9+ splenic B cells














CHAPTER 4
INVOLVEMENT OF STAT6 IN THE IL4 SIGNALING PATHWAY DURING THE
CLINICAL PHASE OF SJOGREN-LIKE SYNDROME

Introduction

Although the underlying cause of Sj ogren's syndrome (Sj S) remains elusive, a

number of studies using the NOD mouse model and numerous congenic strains have led

us to propose the concept that this autoimmune exocrinopathy progresses in three

consecutive phases (61, 149). In phase 1, a number of aberrant genetic, physiological

and biochemical activities associated with retarded salivary gland organogenesis occur

sequentially prior to and independent of initiation of an autoimmune attack. In phase 2,

leukocytes infiltrate the exocrine glands with a concomitant increase in the expression of

inflammatory cytokines. In phase 3, secretary dysfunction of the salivary and lacrimal

glands occurs, most likely the result of production of anti-muscarinic acetylcholine type-3

receptor (M3R) autoantibodies (4, 65). An interruption within any one of these three

phases prevents onset of clinical Sj S-like disease.

Sj S is an autoimmune disease in which B cells and autoantibodies play an

important role in the glandular dysfunction (66). Hyperproliferation and hyperactivity of

autoreactive B lymphocytes result in severe hypergammaglobulinemia that is often found

in patients with Sj S and NOD mice (53, 87). In addition, Sj S patients, as well as NOD

mice and some of its congenic partners, develop specific autoantibodies against nuclear

antigens, intracellular components, membrane proteins and secreted products of exocrine

tissues (27, 30, 32, 121, 173). Approximately 40-70% of Sj S patients' sera contain









autoantibodies that are reactive to SS-A/Ro and SS-B/La antigens. These two specific

anti-nuclear autoantibodies have been used as diagnostic markers of Sj S disease (18).

However, recent reports have focused considerable attention on anti-M3R. Preliminary

studies suggest that anti-M3R autoantibodies may be present in 80-100% of sera from

both Sj S patients and NOD mice (44, 65) and may be an important effector of glandular

dysfunction by blocking normal signal transduction pathways possibly leading to the

internalization of the antibody-receptor complex or desensitizing acinar cells to normal

neural stimulations (38, 39, 138). This concept is supported, in part, by study showing

that the IgG fractions of sera obtained from Sj S patients or NOD mice with disease can

suppress stimulated salivary flow rates when infused into healthy, normal mice (66).

The possibility that anti-M3R autoantibodies may be involved in development of

xerostomia and xerophthalmia in Sj S has led us to examine this issue in greater detail.

Preliminary studies utilizing a number of congenic partner strains of NOD and

NOD.B10-H2b with non-functional cytokine genes revealed that the IL-4 gene knockout

(KO) mice, NOD.IL4- and NOD.B10-H2b.IL4- -, failed to develop salivary gland

dysfunction despite severe leukocytic infiltration of the salivary glands, accompanied by

detectable increases in the expression of pro-inflammatory cytokines. Interestingly, both

NOD.IL4 and NOD.B10-H2b. L4 mice failed to produce M3R autoantibodies of the

IgGI isotype (56, 58). IL-4 is a pleiotropic cytokine involved in cell proliferation,

activation and differentiation. Function of this Th2 cytokine is known to be involved in

two different pathways, the IL4-IRS (Insulin Receptor Substrate) pathway responsible for

the cellular proliferation and activation while the IL4-STAT6 (Signal Transducers and

Activators of Transcription) pathway involved in isotype switching to IgGI and IgE by









specifically stimulating germline yl and F immunoglobulin gene transcription (174).

Even though IL-4 acting through the IRS pathway might be critical in activation and

proliferation that results in the survival and expansion of autoimmune T and/or B cells,

recent studies have pointed to the requirement of IgGI isotypic autoantibodies against

M3R in the secretary dysfunction in NOD mice (56, 58). In support of this concept,

adoptive transfer of T cells capable of producing IL4 into NOD.B 10-H2b.IL4- mice

incapable of producing IgGI/IgE resulted in shutdown of secretary function due to of

IgGl-M3R autoantibody (58). In the present study, I have investigated the biological

roles of IL-4-STAT6 pathway; specifically examining the role isotypic switching plays in

the development and onset of clinical Sj S-like disease.

Material and Methods

Animals

The following animals were used in this study: NOD.B1O.H2b, C.129S2-Stat6

tmlGru/J, NOD.B 1O-H2b.C-Stat6+, NOD.B 10-H2b.C-Stat6-, NOD.B 10-H2b.C-Stat6"

and Balb/C. All the mice were bred and maintained under specific pathogen-free

conditions within the mouse facility of the Department of Pathology, Immunology &

Laboratory Medicine at University of Florida, Gainesville, FL. Breeder pairs of

NOD.B10-H2b and C. 129S2-Stat6tmlGru/J mice were purchased from the Jackson

Laboratories (Bar Harbor, ME). All mice received water and food ad libitum.

Histology

Submandibular glands were surgically removed from each mouse at time of

euthanasia (24 weeks of age), and placed in 10% phosphate buffered formalin for 24 hrs.

Fixed tissues were embedded in paraffin and sectioned at 5 [im thickness. Paraffin-

embedded slides were de-paraffinized by immersing in xylene, followed by dehydrating









in ethanol. The tissue sections were stained with H&E dye (Gainesville Service Tech,

Gainesville, FL). Stained sections were observed at 100X magnification for glandular

structure and leukocyte infiltration.

Immunofluorescent Staining for B and T Lymphocytes

Paraffin-embedded tissues of the submandibular glands were sectioned and

mounted onto microscope slides. Slides were de-paraffinized by immersing in xylene,

then dehydrated in ethanol. Following a 5 minute wash with PBS at 250C, the sections

were incubated 1 hr with blocking solution containing normal rabbit serum diluted 1:50

in PBS. Each section was incubated with rat anti-mouse B220 (BD Biosciences-

Pharmagen, San Diego, CA) diluted 1:10 and goat anti-mouse CD3 (Santa Cruz

Biotechnology, Santa Cruz, CA) diluted 1:50 for 1 hr at 250C. The slides were washed

3X with PBS for 5 min per wash followed by a 1 hr incubation with Texas Red-

conjugated rabbit anti-rat IgG (Biomeda, Foster City, CA) diluted 1:25 and FITC-

conjugated rabbit anti-goat IgG (Sigma Chemicals, St. Louis, MO) diluted 1:100 at 250C.

The slides were washed thoroughly with PBS, treated with Vectashield DAPI-mounting

medium (Vector Laboratory, Burlingame, CA) and overlayed with glass coverslips.

Stained sections were visualized at 200X magnification.

Flow Cytometry

Spleens were freshly explanted from euthanized mice and gently minced through a

steel sieve. Following a single wash with PBS, the red blood cells were lysed by a 7 min

exposure to 0.84% NH4C1. The resulting cell suspensions were washed two times in

PBS, counted and resuspended in FACS buffer (PBS supplemented to 2% ABS and

0.010/oNaN3) to 1 x 108 cells/ml. Aliquots of each cell preparation containing 1 x 105

cells were incubated 45 min with either R-PE-conjugated rat anti-mouse CD19




Full Text

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ROLE OF COMPLEMENT, B CELLS, AN D GENETICS IN THE DEVELOPMENT OF SJOGRENÂ’S-LIKE AUTOIMMUNE EXOCRINOPATHY By CUONG QUOC NGUYEN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2006

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Copyright 2006 by Cuong Quoc Nguyen

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I would like to dedicate this dissertati on to my mother and my daughter, Bé Th ng (Hanna). My má’s endless sacrifice, devoted love, and inspirational struggle inspire me to pursue this degree. My daughter’s smile and the way she says “Daddy” motivate me complete it.

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iv ACKNOWLEDGMENTS I would like to thank all the members of my supervisory committee for their continued support and encouragement, wh ich include Dr. Ammon Peck, Dr. Sally Litherland, Dr. Edward Chan, Dr. Westley Reev es, and Dr. John Aris. I would especially like to thank my mentor, Dr. Peck, for his ex emplary scientific and academic guidance. His hardworking attitude and his enthusiasm for science always inspire me to be a better student. I am sincerely indebt ed to his epitome of a gen tleman with great personal and scientific value and integrity. I would like to thank my past and pr esent lab members who have made my graduate school experience in the laboratory educational and enjoyable, which include Dr. Seunghee Cha, Dr. Smruti Killedar, Dr. Jeuhua Gao, Dr. Maire Doyle, Lori Boggs, Woosuk Jang, Eric Singson, Brian Alverado, Jin Wang, Angie Kim, Marievic Bulosan, and especially Mrs. Janet Cornelius who has al ways been the heartbeat of the laboratory. Her willingness to he lp, knowledge of science, and hi gh expectation have helped many students like me graduate with a sense of accomp lishment and pride. I would like to also thank my friend, Monique Lara, for all her patience and wonderful editorial support. Most importantly, I would like to thank my family members, Má, Anh Hai, Anh Ba, Anh Tu, Anh Phap, Chi My, Chi Ha, Chi Huong, Em Teo Em, Be Dao, Em Cu, Be Le and the cutest, craziest, but phien-est baby in the whol e wide world, Hanna, for their unconditional love and endless support. I would not be the person that I am today without them in my life. I w ould like to show my deepest a ppreciation and respect to my

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v father who is only a prayer away and w ho has given me the most unforgettable and wonderful childhood. Last but not least, I am grateful fo r all my nephews and nieces, Chau Vyvy, Chau Huu, Chau Helena, Chau Bac, Chau Cecilia, Chau Ethan, and Chau Mancy, for bringing so much laughter and love to my life.

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vi TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES...............................................................................................................x LIST OF FIGURES...........................................................................................................xi ABSTRACT.....................................................................................................................xi ii CHAPTER 1 BACKGROUND AND SIGNIFICANCE....................................................................1 Sjögren’s Syndrome (SjS) in Humans..........................................................................1 Secretory Function of the Exocrine Gland............................................................1 Clinical Characteristics of SjS...............................................................................3 Genetic Predisposition...........................................................................................5 Diagnostic Criteria/Technique...............................................................................6 Mouse Models of SjS-like Autoimmune Exocrinopathy..............................................7 SjS-like Autoimmune Exocrinopathy of Mice......................................................7 Genetic Predisposition of SjSlike Autoimmune Exocrinopathy........................11 The Role of B Lymphocytes in the Development of SjS-like Autoimmune Exocrinopathy in Mice...........................................................................................12 Development of Germinal Center (G C)-like Lymphocytic Foci in the Exocrine Glands...............................................................................................12 Development and Characteristics of B Cells in SjS............................................14 The Role of Marginal zone (MZ) and B-1 B Cells in SjS...................................15 Expression of Restricted Repertoire of the B Cell Receptor in SjS Disease.......17 Signal Transduction of the B Cell Receptor........................................................19 Intimate Interaction of the B Cell Receptor and B cell Co-Receptors................20 The Role of Autoantibodies in the Development of SjS............................................22 Anti-Muscarinic Acetylcholine Type -3 Receptor Autoantibodies – The Effectors of Glandular Dysfunction.................................................................25 2 EFFECT OF COBRA VENON FACTOR (CVF) ON COMPLEMENT AND B LYMPHOCYTES IN THE NOD.B10.H2B MICE.....................................................35 Introduction.................................................................................................................35 Materials and Methods...............................................................................................36

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vii Animals................................................................................................................36 Complement Depletion by Cobra Venom Factor................................................37 Measurement of Salivary Rates...........................................................................37 Histology and Immunofluorescent St aining for B and T Lymphocytes..............38 Flow Cytometry...................................................................................................38 Detection of Anti-Nuclear Au toantibodies in the Sera........................................39 Results........................................................................................................................ .39 Effects of CVF Treatment on the Sj S-like Disease Profile in NOD.B10-H2b Mice.................................................................................................................39 Effects of CVF Treatment on B cell Sub-Populations........................................42 Discussion...................................................................................................................42 3 EFFECT OF KNOCKING OUT COMPLEMENT COMPONENT 3 ON COMPLEMENT AND B LYMPHOCYTES IN THE DEVELOPMENT OF SJSLIKE AUTOIMMUNE DISEASE.............................................................................52 Introduction.................................................................................................................52 Materials and Methods...............................................................................................54 Generation of C57BL/6.NODAec1Aec2.C3-/Mouse.......................................54 Proteolysis of Parotid S ecretory Protein (PSP)...................................................54 Detection of Cleaved Caspase-3 in the Submandibular Glands..........................55 Salivary Protein Concentration and Salivary Amylase Activity.........................55 Histological Examination of Subm andibular and Lacrimal Glands....................56 Detection of Anti-Nuclear Autoantibody in Sera................................................56 Detection of Immunoglobulin Specific Muscarinic Type III Receptor Autoantibody....................................................................................................57 Flow Cytometry for Sub populations of B cells...................................................57 Measurement of Stimulated Saliva Secretion......................................................58 Statistical Analysis..............................................................................................58 Results........................................................................................................................ .58 Profiling of Phase I of SjS-lik e Autoimmune Exocrinopathy of C57BL/6.NODAec1Aec2.C3-/Mice.............................................................58 Profiling of Phase II of SjS-lik e Autoimmune Exocrinopathy of C57BL/6.NODAec1.Aec2.C3-/Mice............................................................61 Profiling of Phase III of SjS-lik e Autoimmune Exocrinopathy of C57BL/6.NODAec1Aec2.C3-/Mice.............................................................63 Characterization of the changing dynamics in the subpopulation of B lymphocytes: Marginal Zone (M Z) and Follicular (FO) B Cells...................64 Discussion...................................................................................................................66 4 INVOLVEMENT OF STAT6 IN TH E IL4 SIGNALING PATHWAY DURING THE CLINICAL PHASE OF SJOGREN-LIKE SYNDROME.................................83 Introduction.................................................................................................................83 Material and Methods.................................................................................................85 Animals................................................................................................................85 Histology.............................................................................................................85

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viii Immunofluorescent Staining for B and T Lymphocytes.....................................86 Flow Cytometry...................................................................................................86 Proteolysis of Parotid Secretory Protein..............................................................87 Detection of Anti-Nuclear Au toantibodies in the Sera........................................87 Measurement of Salivary Flow Rates..................................................................88 Profile of Immunoglobulin in Serum..................................................................88 Immunoglobulin Specific M3R Au toantibodies Detection Using Immunoflourescence........................................................................................88 Statistical Analysis..............................................................................................89 Results........................................................................................................................ .89 Generation of NOD.B10H2b.CStat6+/+, NOD.B10H2b.CStat6+/-, NOD.B10H2b.CStat6-/-..................................................................................89 Pathophysiological Examination of NOD.B10H2b.CStat6+/+, NOD.B10H2b.CStat6+/-and NOD.B10H2b.CStat6-/Mice...........................................90 Leukocytic Infiltrations in the Submandi bular and Lacrimal Glands in Both the NOD.B10H2b.CStat6+/+ and NOD.B10H2b.CStat6-/Mice..................91 Detection of Anti-Nuclear Antigens in the Sera of NOD.B10H2b.CStat6+/+and NOD.B10H2b.CStat6-/Mice......................................................92 Determination of Immunoglobulin Subc lass Levels in Sera of NOD.B10H2b.CStat6+/+and NOD.B10H2b.CStat6-/Mice...........................................93 Detection of Specific Immunoglobulin against the Muscarinic Type 3 Receptor Autoantibody....................................................................................94 Measurement of Salivary Flow Rates..................................................................95 Discussion...................................................................................................................96 5 SJÖGREN’S SYNDROME-LIKE DISEASE OF C57BL/6.NODAEC1AEC2 MICE: GENDER DIFFERENCES IN KERATOCONJUNCTIVITIS SICCA DEFINED BY A CROSS-OVER IN THE CHROMOSOME 3 AEC1 LOCUS......109 Introduction...............................................................................................................109 Materials & Methods................................................................................................111 Animals..............................................................................................................111 Histology...........................................................................................................112 Measurement of Saliva and Tear Flow Rates....................................................112 Saliva Protein Concentration and Salivary Amylase Activity..........................113 Detection of Caspase-3 Activity........................................................................114 Proteolysis of Parotid S ecretory Protein (PSP).................................................114 Detection of Anti-Nuclear Autoan tibodies (ANAs) in the Sera........................114 Detection of Immunoglobulin-Speci fic Anti-M3R Autoantibodies..................114 Statistical Analyses............................................................................................115 Results.......................................................................................................................1 16 Genetic Profile of Recombinant Inbred Line C57BL/6.NODAec1R1Aec2 Mice...............................................................................................................116 Phase 1 Profile of SjS-Li ke Disease in C57BL/6.NODAec1R1Aec2 Mice.....117 Phase 2 Profile of SjS-Li ke Disease in C57BL/6.NODAec1R1Aec2 Mice.....118 Phase 3 Profile of SjS-Li ke Disease in C57BL/6.NODAec1R1Aec2 Mice.....119 Discussion.................................................................................................................121

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ix 6 OVERALL CONCLUSION.....................................................................................136 Focus of the Dissertation..........................................................................................137 The Role of Complement in the Pa thogenesis of SjS-like Autoimmune Exocrinopathy................................................................................................137 The Role of STAT6 of IL4 Signaling Path way in the Pathogenesis of SjS-like Autoimmune Exocrinopathy..........................................................................138 Genetics of SjS-like Auto immune Exocrinopathy............................................139 The Clinical Implications in Translational Research................................................141 LIST OF REFERENCES.................................................................................................143 BIOGRAPHICAL SKETCH...........................................................................................162

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x LIST OF TABLES Table page 1-1 Mouse strains used in the study of Sjögren’s syndrome..........................................29 1-2 Comparison of general symptoms of Sjögren's syndrome patients and NOD mice32 2.1 Changes in the splenic CD19-positive B cell phenotypes. Each group included eight female mice.....................................................................................................51 3-1 Changes in CD19+ splenic B cell populations.........................................................82 5-1 Caspase-3 activity in the sa livary and lacrimal glands of C57BL6.NOD. Aec1R1Aec2 mice..........................................................................135

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xi LIST OF FIGURES Figure page 1-1 Model for the progression of SjS-like autoimmune exocrinopathy.........................33 1-2 Dynamic cellular composition of lym phocytic infiltration in submandibular glands of NOD mouse. ............................................................................................34 2-1 C3 levels in the plasma of NOD.B10H2b mice following treatment with cobra venom factor (CVF) or PBS.....................................................................................46 2-2 Salivary flow rates of NOD.B10H2b mice treated with CVF or PBS....................47 2-3 Histological examination of th e exocrine gla nds of NOD.B10H2b mice treated with CVF or PBS......................................................................................................48 2-4 Detection of anti-nuclear autoantibodies (ANAs) in NOD.B10H2b mice treated with CVF or PBS......................................................................................................49 2-5 Reduced expression of CD19 on splenic B lymphocytes of NOD.B10H2b mice treated with CVF......................................................................................................50 3-1 Proteolysis of paro tid secretory protein...................................................................71 3-2 Examination of apoptosis by the pr esence of cleaved Capase-3 in the submandibular glands...............................................................................................72 3-3 Histological examination of the exocrine glands.....................................................73 3-4 Detection of anti-nuclear autoantibod ies (ANAs) using Hep2 cells as subtrate......74 3-5 Detection of anti-muscarinic acet ylcholine type-3 receptor antibodies...................75 3-6 Detection of anti-muscarinic acet ylcholine type-3 receptor antibodies. ................76 3-7 Stimulated saliva flow of female animals................................................................77 3-8 Stimulated saliva fl ow of male animals...................................................................78 3-9 Amylase activity in saliva.. ....................................................................................79 3-10 Salivary protein concentration.................................................................................80

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xii 3-11 An representation of an approach to de lineate marginal zone (MZ) and follicular (FO)..........................................................................................................................8 1 4-1 Generation of NOD.B10. H2b.C. STAT6-/mouse....................................................101 4-2 Detection of proteolytic activity against PSP in saliva . .........................................102 4-3 Histological examination of the exocrine glands...................................................103 4-4 Detection of anti-nucle ar autoantibodies (ANAs)..................................................104 4-5 Amount of immunoglobulin isotype s present in sera of female...........................105 4-6 Detection of M3R isotypic au toantibodies by immunoflourescence. ...................106 4-7 Salivary flow rates of female animals....................................................................107 4-8 Salivary flow rates of male animals.......................................................................108 5-1 Generation of the C57BL/ 6.NOD-Aec1R1Aec2 mouse line.................................127 5-2 Detection of proteolyti c activity against PSP in the salivary gland tissues of C57BL/6.NODAec1R1Aec2 mice. .......................................................................128 5-3 Histological characterization of sial adenitis and dacryoadenitis of male C57BL/6.NODAec1R1Aec2 mice. .......................................................................129 5-4 Histological characterization of sial adenitis and dacryoadenitis of female C57BL/6.NODAec1R1Aec2 mice.........................................................................130 5-5 Detection of ANAs in sera of C57BL/6.NODAec1R1Aec2 mice.........................131 5-6 Temporal loss of secretory function in C57BL/6.NODAec1R1Aec2 mice. .........132 5-7 Detection of anti-muscarinic acetylcho line type-3 receptor antibodies in the sera of C57BL/6.NOD-Aec1R1Aec2 mice...................................................................133 5-8 Time-dependent loss of amylase activity in the saliva of C57BL/6.NODAec1R1Aec2 mice...................................................................................................134

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xiii Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ROLE OF COMPLEMENT, B CELLS, AN D GENETICS IN THE DEVELOPMENT OF SJÖGREN’S-LIKE AUTOIMMUNE EXOCRINOPATHY By Cuong Quoc Nguyen August 2006 Chair: Ammon Peck Major Department: Medical Sc iences-Immunology and Microbiology Sjögren’s syndrome (SjS) is a human auto immune disease characterized by the loss of exocrine function as a result of a chronic immune attack directed primarily against the salivary and lacrimal glands l eading to xerostomia (dry mouth) and xerophthalmia (dry eyes). Based on various animal models, th e progression of SjS can be separated into three distinct and consecutive phases. Phase I is involved in the in itiation of glandular pathophysiological changes asso ciated with aberrant mRNA transcription, protein expression and acinar cell apoptosis. During Ph ase II, initiation of acinar cell death and immunological responses against cryptic pep tide presentation results in leukocytic infiltrations of the targeted glands expressing pro-inflammato ry cytokines. In Phase III, the onset of clinical manifestation occurs, ch aracterized by the loss of exocrine secretory function, and loss of important secretory proteins. Glandular dysfunction which commences at this phase is thought to be me diated by activity of autoantibodies produced by B lymphocytes.

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xiv The main focus of this dissertation is to elucidate genetic elements regulating the development of SjS-like autoimmune exocrinop athy and to identify potential approaches that could interrupt any of the phases of th e autoimmune process to prevent the onset of the clinical disease. Results showed that the depletion or ge netic elimination of complement component C3 prevented onset of SjS-like disease in genetically predisposed pathology associated w ith both Phase II and III. In addition, the elimination of IgG1 isotypic antibody against the muscar inic acetylcholine type III receptor by genetically knocking out of the STAT6 gene appeared to affect only phase III of the disease, resulting in retention of normal salivary flow rates in the animals. Genetic analysis of SjS-like disease has redefined smaller genetic regions that contain genes regulating the events of Ph ase 2. Narrowing down the Aec1 region to a centromeric stretch less than 20 cM is providing important potential candidate genes regulating the development, onset and unde rlying cause of SjS.

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1 CHAPTER 1 BACKGROUND AND SIGNIFICANCE Sjögren’s Syndrome (SjS) in Humans Sjögren’s syndrome is a human diseas e characterized by exocrine gland dysfunction resulting from the consequences of an autoimmune response (1-10). Primary SjS is characterized generally by a chronic au toimmune attack against both the lacrimal and salivary glands, while sec ondary SjS is marked by an au toimmune attack against the lacrimal and/or salivary glands in the presence of anothe r autoimmune disease, most often a connective tissue disease like scleroderma, rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE) (1, 11). In addition to the apparent primary sites of SjS, i.e., the lacrimal and salivary glands , other tissues that may become affected include the entire GI tract, skin, the lungs, the vasculature, ki dneys, bladder and the va gina. Involvement of the musculature often leads to fibromyalgia-l ike symptoms and chronic fatigue. As with many autoimmune connective tissue diseases, there exists a sexual dimorphism in SjS with women affected 10to 20-times more fr equently than men, suggesting a role for sex hormones in disease susceptib ility, possibly related to th e relative balance between estrogen and androgen (12-16). Secretory Function of the Exocrine Gland Secretory mechanism of the exocrine glands is a highl y regulated and intricately controlled process. The major pr oduct of the salivary glands is saliva. Proteins such as amylase and lingual lipase contained in saliva assist in the digestion of food by breaking down the carbohydrates (starch) in the oral cavity and lipid hydrolysis respectively. The

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2 mucus and water in saliva moisten the oral mucosa, aiding in swallowing and dissolving of food. Lysozyme, lactoferrin, and IgA found in saliva also provide protection against bacterial infection and control bacterial flor a in the oral cavity, thus providing a very important innate immune defense mechanism in the mouth. Saliva contains high concentrations of calcium and phosphate that help in the development of new teeth and repair of enamel lesions. Therefore, the loss of salivary flow can facilitate the development of candidiasis, a fungal infection caused by Candida albicans , painful ulcers, rampant dental caries and extr eme difficulty in swallowing (17). The salivary glands include the submandi bular gland, sublingual gland and the parotid gland as well as nume rous minor salivary glands. The production of saliva is under the control of the autonomic nervous syst em. The salivary gland is innervated by both sympathetic and parasympathetic ne rves. Upon neural stimulation, the parasympathetic nerves utilize the muscar inic type III receptor (M3R) to induce the secretion of water-rich saliva in the salivary glands, while the sympathetic nerves use the -adrenergic receptor to stimulate the release of protein-rich saliva. Acinar cells take advantage of different ion transporters a nd co-transporters allowing the influx of Clions into the acinar lumen. The accumulation of Clions generates a negative electrical difference which initiates the passive movement of Na+ ions through the acinar tight junctions. Higher concentration of ions in the acinar lumen creates an osmotic gradient that favors the movement of water mol ecules from the surrounding blood capillaries across tight junctions and water channels such as the Aquaporin 5 channel. This results in the formation of isotonic primary saliva. The lumen of acinar cells collects all the secretory products which are tran sported by long intercalated duc ts to the striated ducts.

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3 In the striated ducts, NaCl is reabsorbed back to the blood capillaries and saliva becomes hypo-osmotic. Kallikrein, a serine protease secret ed by the epithelial ce lls of the striated duct, processes the proline-rich proteins and cystatins in the saliva. In addition, plasma cells secrete IgA which reaches the lumen of the acinus and striated duct by transcytosis. The final saliva contains a complex of prot eins with antimicrobial activity and with digestive function (amylase). Bicarbonate, th e primary buffering agen t of the saliva, is produced in the striated duct (17). Clinical Characteristics of SjS While keratoconjunctivitis sicca (dry eyes) and stomatitis sicca (dry mouth) are assessed by specific tests for changes in exocrine gland flow rates and biochemical/enzymatic changes in protein co mposition and function, the recently adopted European-American Consensus Group criteria for diagnosis of SjS recommends detection of infiltrating lymphocytes within a minor salivary gland, determined by a histopathological analysis of a labial gland lip biopsy. However, lip biopsy is not required if anti-Ro or anti-La autoantibodies are detected (18). Less often used is an analysis of a lacrimal gland biopsy (19). Nevertheless, based on studies in both humans and animal models, infiltrates appear generally as peri-ductal foci within the glandular architecture of the lacrimal and salivary glands consisting of CD4+ T cells, CD8+ T cells, B cells and macrophages. The T cells exhibit a preferential antigen r eceptor repertoire , while the overall infiltrating cells ex press various cytoki nes (including IL-1 , IL-6, IL10, TNF , and IFN ) whose significance in the autoimmune pathology has yet to be determined (20-23). Serological evaluations have shown the presence of rheumatoid factor, elevated immunoglobulin levels (hypergammaglobulinemia) and anti-nuclear

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4 autoantibodies, especially anti-SS-A/Ro and anti-SS-B/La antibodies. Additional autoantibodies are present which react w ith numerous cellular components of the exocrine glands, probably representing spr eading epitopes (24-35). Of late, intense interest has revolved around the presence of antibodies to the muscarinic acetylcholine type-3 receptor (M3R) (36-44). SjS is only one of many dry mouth/dry ey e diseases, but considered one of the more severe forms of these conditions. Xe rostomia and keratoconjunctivitis sicca result respectively from basic changes in the saliva and tear flow rates, the composition of saliva and tears, and/or combinations thereo f. Underlying causes of xerostomia include the natural aging process, use of me dications, asthma a nd mouth breathing, chemotherapy, radiation therapy, autoimmune at tack against secretory tissues/glands of the mouth, thyroid dysfunction, kidney dialys is and/or stroke. Likewise, underlying causes of xerophthalmia include the natural aging process, physical injury, surgical procedures, meibomian gland dysfunction and/or autoimmune attack against one or more of the multiple secretory tissues/glands of the ey e. Because saliva is a critical factor in oral health, patients with dry mouth can pres ent with increased caries, increased oral microbial infections, halitosis, cracked lips and bleeding gums, taste disturbances, difficulty in eating, swallowing, and talking. In addition, patients can suffer from esophageal dysphagia, epigastric pain and dyspepsia due in pa rt to decreased levels of epidermal growth factor (EGF) in saliva, as well as poor nutritional uptake. Moreover, between 4-10% of patients with SjS w ill develop non-HodgkinÂ’s malignant B cell lymphomas (45-48). While considerable empha sis is placed on mani festations resulting from xerostomia, the manifestations from xerophthalmia brought on by decreased tear

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5 fluid secretion in conjunction with an increa se in tear fluid evaporation are just as debilitating. Complaints from patients with dry eyes incl ude burning, grittiness, itching, fatigue, blurred vision and, para doxically, watery eyes resulting from increased reflex tear secretions. Over time there is eye surface deterioration and ulceration, leading to small red-appearing eyes with crusts in the ciliae, debris in the tear film, meibomitis, mucus strands adhering to the corneal surface s, reduced light refl ectivity and irregular blinking. In general, these manifestations of dry mouth and dry eyes, especially in SjS, appear to correlate with a loss of exocrine cell mass, an onset of exocrine cell senescence or refractivity, and loss of neural regulation of ocular secretory function (19). Genetic Predisposition SjS shows a weak tendency toward familial aggregation which, together with the presence of common autoantibodies in SjS pa tients, suggests that genetic factors are operative in disease susceptib ility (49). While environmental triggers responsible for initiating SjS are unclear, intrinsic genes contri buting to disease suscep tibility are thought to be critical potentiators for development of autoimmunity. Although the most probable hereditary markers in disease susceptibil ity are those encoded by genes of the major histocompatibility complex (MHC), previous associations with HLA-DQ are most likely reflecting the presence of anti-nuclear SSA/Ro and SS-B/La autoantibodies (50). Furthermore, studies from different ethnic groups have yielded inconsistent results suggesting only weak MHC class II associations for SjS (51). More importantly, family studies suggest autosomal ge nes not linked to HLA and/or immunoglobulin genes are an important element of autoimmune exocri nopathy susceptibility (52), a conclusion consistent with studies in NOD mice, an animal model of SjS, where non-MHC genes clearly control susceptibility (53-55).

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6 Diagnostic Criteria/Technique While diagnosis of SjS is based, in part , on subjective patient symptoms, a number of specific clinical te sts are also critical. For xerost omia, these include (a) desiccated buccal epithelium, (b) reduced production of either stimulated or unstimulated saliva flow, (c) reduced amylase activity, (d) reduc ed EGF levels, and (e) detection of antinuclear autoantibodies (ANAs), especially anti-SS-A/Ro and anti-SS-B/La, and (f) presence of leukocyte foci ( 50 lymphocytic cells per 4 mm2 is defined as a focus) within minor salivary gland biopsies. For keratoconj unctivitis sicca, these include (a) break-up time test which measures the ability of pre-co rneal tear films to maintain their integrity, (b) Schirmer-1 test which measures tear flow rates, (c) Rose-Bengal (or lissamine green) dye test which shows staining of desiccated epithelial cells lacki ng mucous protection, (d) lysozyme and lactoferrin enzyme activ ity measurements, and rarely (e) a minor salivary gland biopsy to determine the presen ce of leukocyte infiltra tes. Despite this battery of tests, both the diagnosis and the underlying mechanisms of autoimmune exocrinopathy are difficult to assess because SjS patients usually present when the autoimmune process is at or ne ar its end-stages. Thus, at th e present time, a direct cause of SjS remains elusive and correlative, and this limits development of pre-disease biomarkers, intervention therapies, as well as an ultimate prevention and cure for the illness. As a result, it has been important to turn to animal models of SjS, not only to define the pathophysiological processes, but also to identify appropriate biomarkers predictive of SjS.

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7 Mouse Models of SjS-like Autoimmune Exocrinopathy SjS-like Autoimmune Exocrinopathy of Mice Over the past two decades, a variety of m ouse models exhibiting various aspects of SjS, whether spontaneously appearing or expe rimentally induced, have been intensively investigated in an attempt to identify the nature of this au toimmune disease. Typically, these mouse models show lymphocyte infilt ration of the exocrine glands, increased expressions of pro-inflammatory cytoki nes, generation of unique autoantibodies (especially ANAs, anti-fodrin, and anti-muscarinic ace tylcholine type-3 receptor (M3R) antibodies), and eventually decreased sa liva flow rates. Stra ins that have been extensively studied include NZB/NZW F1-hybrids, MRL/ lpr , NOD/LtJ and NFS/ sld . More recently, several new strains have been added; including the Id3 gene knock-out (KO) mouse, the aromatase gene KO mouse, the Baff gene knock-in (KI) mouse, as well as the IQI/Jic mouse and C57BL/6.NODAec1Aec2 congenic line. A listing of these various models along with their general diseas e profiles is presented in Table 1-1. While each strain has been reported to resemble features of SjS in human patients, none recapitulate completely the pathological char acteristics of the human disease. One of the more interesting and well-studied models of SjS is the NOD mouse which closely mimics the human disease (see Ta ble 1-2). A major strength of this model has been the ability to study a large number of congenic pa rtner gene KO strains, e.g., NODscid , NOD. Ifn -/-, NOD. IL2-/-, NOD. IL4-/-, NOD. IL10-/-, NOD. Igµ-/-, and NOD. Stat6-/-, permitting investigations into the role of individual genes in the development and onset of SjS-like disease in th is model (56-59). Taken together, these studies suggested that the dis ease could be divided into th ree distinct consecutive but continuous phases (59-62). In phase 1 (ini tiation of glandular pa thology), a number of

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8 aberrant genetic, physiological and biochemical activities associated with retarded salivary gland organogenesis a nd increased acinar ce ll apoptosis occur sequentially prior to and independent of detectab le autoimmunity (62). In phase 2 (onset of autoimmunity believed to result from the acinar cell apoptos is), leukocytes expressing pro-inflammatory cytokines infiltrate the exocrine glands, establishing lymphocytic foci, first of T cell clusters followed by recruitment of B lymphocytes (63, 64). In phase 3 (onset of clinical disease), loss of salivary and lacrimal gla nd secretory functions o ccur, most likely the result of (auto)-antibodies reactive with the M3Rs (37, 56, 65, 66). These three phases are portrayed in Figure 1-1. Although the onset of SjSlike disease in NOD mice is independent of the production of ANAs (anti-nucl ear antibodies), Scofield et al. (67) have recently shown that immunization of BALB/c mice with Ro antigen induces a SjSlike disease, pointing to the im portance of individual antibodies in the onset of exocrine gland dysfunction. Interestingly, the recently derived IQI/Jic mouse line appears to mimic the disease profile of the NOD mouse (68). A critical observation derived from studies with NOD mice is the important role of B lymphocytes in the development and onset of disease. First, NOD. Igµ-/mice lacking mature B cells fail to develop glandular dys function despite exhibi ting peripheral T cell activation and T cell in filtrations of the salivary and lacrimal glands (64). Second, NOD. IL4-/mice lacking the ability to produce the cytokine IL-4 also fail to develop glandular dysfunction despite exhibiting both T and B cell ac tivation in the periphery, T and B cell infiltration of the salivary and lacrimal glands , and the production of ANAs (58). Third, NOD. IL4-/mice fail to produce IgG1 subcla ss antibodies reactive with the muscarinic acetylcholine receptors, a defici ency that appears to be circumvented

PAGE 23

9 following an adoptive transfer of T cells isolated from NOD. Igµ-/mice or injections of recombinant IL-4 cytokine protein (58). Considering these obse rvations, it is not surprising that passive transfer of serum IgG from human SjS patients or from diseased NOD mice, but not healthy human subjects or pre-diseased animals, into NOD. Igµ-/mice results in a temporary loss of saliva secreti on (66). Furthermore, serum IgG fractions from human SjS patients, but not healthy human subjects, can competitively inhibit the binding of the muscarinic receptor agonist, [3H]-quinuclidinyl benzilate, to salivary gland membranes (66). The disease profile observed in NOD. IL4-/mice raises several questions pertinent to the role of the B lymphocyte in development and onset of SjS-like disease. First, do B lymphocytes act as antigen-presenting cells (APCs) for initiation of the SjS-like autoimmune response, as has been proposed for initiation of type 1 diabetes in NOD mice? Second, do the B lymphocyte populati ons in NOD mice respond abnormally to the presence of IL-4 resulting in circumvention of normal homeostatic mechanisms that would prevent over-proliferation, survival and escape from negative selective pressures? Third, is there an autoimmune B cell populati on present in NOD mice that is activated by IL-4 to produce a specific subclass of IgG antibody that can inhibit normal acinar cell function? Whereas sorting out the possibility that the B cell popula tion can act as APCs might be difficult due to the fact that the autoimmunity is generally characterized as a hypersensitivity type 2 (or antibody-mediated ) immunological response , recent studies indicate that IL-4 may be important in bot h survival of M3R (auto)-reactive B cells and in their IgG isotype switching to promot e production of IgG1 anti-M3R autoantibody (58). This latter concept is supported by observations from studies using BAFF-

PAGE 24

10 transgenic mice in which there is an over-p roduction of BAFF. These BAFF-transgenic mice exhibit enhanced B cell proliferation and survival, as well as altered differentiation patterns, and develop an autoimmune condition resembling SLE (69). Interestingly, by 4 months of age, these mice develop a secondary pathology reminiscent of SjS characterized by severe sialad enitis, decreased saliva produc tion, and destruction of the submaxillary glands. Infiltrates within the salivary glands of BAFF-transgenic mice appear to be a marginal zone (MZ) B cell population, a population known to be increased in the spleens of BAFF-transge nic mice and thought to partic ipate in the maintenance of germinal centers in the target tissue and subsequent antibody production in SjS (70). A role for aberrant lymphocyte survival is also supported by studies using the MRL/ lpr mouse model (71). MRL/ lpr mice carry a mutation in the lpr gene that causes a defect in the Fas protein involve d in the Fas/FasL apoptotic pa thway. This defect leads to impairment of normal lymphocyte apoptosis, resulting in an abnorma l proliferation and survival of lymphocytes, especia lly B cells (72). While the MRL/ lpr mouse was developed for the study of SLE, it manifests an autoantibody pattern found in part in SjS, including anti-dsDNA, anti-ssDNA, ANA and rheuma toid factor (73). In addition, nearly 30% of mice develop anti-52 KDa SS-A/Ro antibodies, 6% develop anti-60 KDa SSA/Ro antibodies, and 6% develop anti-SS-B/ La antibodies (74), how ever other studies have not been able to detect similar frequency of these antibodies in the MRL/ lpr mouse. MRL/ lpr mice also develop sialadenitis of the submandibular, parotid and lingual glands and dacryoadenitis of the lacrimal glands (75) . The infiltrates are generally comprised of CD4+ T cells, with lesser numbers of CD8+ T cells and B cells. In addition, there are scattered macrophages and dendritic cells (76). However, the infilt rates appear diffuse

PAGE 25

11 and not as compact, tightly packed fo ci found in NOD mice. Although lymphocyte infiltrations may cause destruction of exocri ne gland tissues, perh aps by iNOS / nitric oxide (NO) (77), tumor necrosis factor (TNF)and/or various cyt okines (78), anti-M3R antibodies have not been observed in MRL/ lpr mice, thus loss of secretory function is not detected (79). To put these observations in pe rspective, it is important to note the fact that, in the SjS-like disease process of NOD mice, a high rate of acinar cell apoptosis occurs in the submandibular glands around 2 mo nths of age, or approximately 2-4 weeks prior to onset of detectable le ukocytic infiltration of the salivary glands , and this process is associated with an up-regulation of Fa s/Fas-ligand expression by acinar cells (62). Genetic Predisposition of SjSlike Autoimmune Exocrinopathy Genetic manipulations of a variety of mi ce can either result in the appearance of various disease traits observed in SjS patie nts or delay / prevent development and onset of pre-clinical and clinical disease. This is obvious in such mouse lines as MRL/ lpr , NFS/ sld , the Baff -KI transgenic, the Id3 -KO transgenic, and Aromatase -KO transgenic. A remaining question, however, is whether we can identify those genetic regions of the mouse genome that predispose mice to de velop SjS-like disease. The NOD mouse provides an excellent model to investigate th is issue. A large collection of congenic NOD mice are available defining th e diabetes su sceptibility ( Idds) loci that predispose these mice to autoimmune type 1 diabetes (T 1D). Unlike the genetic predisposition for T1D in both humans and NOD mice which is dependent on specific genes mapping to the major histocompatibility complex (MHC), th e genetic predisposition for SjS-like disease in NOD mice appears independent of, or only weakly dependent on, MHC-associated genes, thus mimicking SjS in humans. The first indication involve d the studies of the congenic strain, NOD.B10H2b, in which the NOD MHC I-Ag7 Idd1 T1D susceptibility

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12 locus is replaced by MHC I-Ab (53). These mice, while fail ing to exhibit insulitis and development of diabetes, continue to show a complete SjS-like syndrome including salivary and lacrimal gla nd dysfunction. Thus, NOD.B10H2b mice were advanced as the first naturally-occurri ng model for primary SjS (53). Replacing other diabetes suscepti bility loci in the NOD mouse (e.g., Idd10, Idd9 , Idd13 , and so forth), while lowering the incide nce of insulitis and diabetes, proved to have little effect on its SjS-like disease. However, when both the Idd3 and Idd5 loci were replaced with the corresponding genetic in tervals derived from C57BL/6 mice, the severity of the biological markers of epithe lial cell pathology was reduc ed and the loss of secretory function reversed (31, 32). In a reverse a pproach, introducing both the Idd3 and Idd5 genetic regions derived from NOD mice into the SjS non-susceptible C57BL/6 mouse resulted in the appearance of SjS-lik e disease, confirming the contributions of these two genetic loci to developmen t and onset of disease (54, 55). The Role of B Lymphocytes in the Development of SjS-like Autoimmune Exocrinopathy in Mice Development of Germinal Center (GC)-lik e Lymphocytic Foci in the Exocrine Glands Considerable attention has been focused on attempts to define the organization of the immune cell infiltrates, referred to as ly mphocytic foci, which a ppear in salivary and lacrimal glands with the onset of disease. We and others have reported that these lymphocyte infiltrations within the submandibular and lacrim al glands of NOD mice are composed predominantly of CD4+ T cells wi th lesser numbers of CD8+ T cells and B cells, a fact generally accepted universally (63). Subsequent ly, we have found that these findings appear to be misleading due to the proc edures used to isolat e the infiltrating cells from the exocrine tissues for flow cytometric analyses. We now be lieve that vigorous

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13 digestion and subsequent purif ication steps may account for the lost of many fragile cells, thereby leading to possible erroneous conclu sions. Recently, we have revisited this question using immunofluorescent staining of paraffin-embedd ed salivary and lacrimal tissues that not only maintain the native stru cture of the glands but retain the cellular organizations. Results strongly indicate that lymphocytic foci are dynamic entities whose cellular compositions and organizations change dramatically as they mature. Thus, to state that the major cell type is a CD4+ T ce ll appears to over-simp lify the real situation. As presented in Figure 1-2, Immunofl uorescent staining of paraffin-embedded sections of glandular tissues freshly explante d from NOD mice of di fferent ages depicts a rapidly changing cellular composition of the lym phocytic foci. During the early stages of lymphocyte infiltration into the salivary glands, small densely packed foci appear that are mostly composed of CD3+ T cells virtually free of detectable B cells. As the foci mature, an increasing number of B220+ B cells are detected which histologically appear to surround T cell cores. Over time, the number of T cells decrease relative to the B cells and become more dispersed throughout the B cell areas. An important observation from studies using NOD mice, first reported by van Blokland and colleagues (76), is the fact that dendriti c cells (DCs) and possibly macrophages appear to be the first cells to migrate into the glands, resulting in the activation of epithelial cells to express adhesion molecule s and produce chemokines that attract T cells, and subsequently additi onal macrophages and B cells. Most BM8+ macrophages are found to occupy areas around th e periphery of the focus and T cells regions (76).

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14 We have recently shown by immunohistoch emistry that F4/80 macrophage are also scattered throughout the foci ( unpublished data). However, car eful examination of these well-defined lymphoepithelial sialadenitislike foci does not appear to mimic ectopic germinal centers. It is still uncertain if th ese ectopic lymphocytic foci have any biological significance in terms of the clinical manifestation of SjS, but their absence generally results in loss of pr ogression of disease into the c linical phase. It has been reported that the lymphocytic infiltrates ar e made up of clonally expanded T cells with the TCR repertoire of V 8.1,2, V 6, and V 4 (80). However, it is also unknown if there is localized or systemic activations that re sult in effector molecules for the clinical development of SjS. Consisten tly, histological analys is of salivary and lacrimal glands in human SjS patients revealed the presence of leukocytic infiltrations which consists mostly of T cells and significantly less B cells. However, our findings have clearly demonstrated that the infiltrates are mo stly B220+ B cells (75%) and a smaller percentage of CD3+ T cells when the disease appears to be most progressive; however, to fully understand the nature of the organization of these lymphocytic foci, additional work needs to clarify what subpopulations of B220+ B cells and T cells are present in the infiltrates. Development and Characteri stics of B Cells in SjS The life-history of B lymphocytes is now a well-studied and well-documented area on which many reviews have been written (8184). While it is recognized that T cells play an important role in regulati ng B cell activation, ma turation and antibody production, considerable attention has r ecently been focused on the role of B lymphocytes in the development and onset of SjS in humans and SjS-like disease in mice. This is due, in part, to reports documenting a number of apparent innate developmental

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15 abnormalities in B cells for both patients and mouse models. However, identifying the stage(s) at which this abnormal B cell behavi or is induced and/or how it is maintained remains unknown and probably represents a multi-factorial process, since multiple environmental factors dictate the fate and behavior of B cells.. The Role of Marginal zone (MZ) and B-1 B Cells in SjS As previously discussed, mice over-expr essing BAFF develop SLE-like disease with significant increases in the number of MZ B cells (69). When these mice are aged to about 16-18 months, they exhibit a SjS-like di sease with a subset of B cells in the salivary glands that resemble MZ B cells (70). In GraveÂ’s di sease, MZ cells are found to infiltrate the thyroid glan d (85), and NOD mice have been reported to also have infiltrations in their thyroid gl ands (86). While it is not kno wn if these MZ B cells play any role in the pathogenesis of autoimmunity, they may provide insight into how chronic stimulation of auto-reactive B lymphocytes within a tissue eventually transform to lymphomas. Hyperproliferation of MZ B cells is hypothesized to be the cause and source of transformed B cells that often arise in Sj S patients and are capable of developing into non-Hodgkin B cell lymphomas (87). Dependent on location, MZ B cells can be induced to transform into splenic MZ lymphomas, nodal MZ lymphomas, and/or extra-nodal MALT lymphomas (87). Therefore, this particular subset of B cells appears to be an important connection between autoimmunity and tumorigenesis in SjS patients. For B-1 cells, a vast majority is found in the peritoneal and pleural cavities of the mouse, thus only 5% of this B cell population is pr esent in the spleen and virtually none are present in the peripheral lymph nodes (88). B1 cells can be further separated into B-1a cells that express membrane CD5 molecules or B-1b cells that are CD5 negative. The development and survival of B-1 cells are critically dependent on the strength of B cell

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16 receptor signaling. Numerous studies in mi ce involving the genetic disruption (either by deletion or over-expression) of molecules that contribute to the signal transduction pathways of the BCR greatly effects the development of B-1 cell populations. For example, genetic alterations that enhan ce BCR signaling, e.g., deletion of CD22 or CD72, and over-expression of CD19, lead to significant increases in the number of B-1 cells. In contrast, genetic manipulations that lower BCR signaling, e.g., deletion of CD19, CD21/35 or Vav-1, reduce the number of B-1 cel ls (89). Therefore, these observations indicate the importance of maintaining homeostasis of BCR signaling to ensure appropriate development, survival or ev en expansion of this B cell population. B-1 cells manifest many unique characteris tics important to proper B cell function, including longevity of survival with high potential for self-renewal, refractory to activation by ligand binding to BCRs, lack of somatic mutation, a nd restriction of N insertions that result in lim ited immunoglobulin gene repertoires (90). Since B-1 cells arise from fetal precursors, they express limited Ig gene repertoires, mostly restricted to “natural” IgM, where the te rm “natural” indicates the production of IgM subclass of antibody in the absence of stimulation by exoge nous antigens (91). Therefore, B-1 cells produce antibodies that are predominantly au toreactive, but highl y polyreactive with numerous naturally or evolutionarily conserved pathogen-associated carbohydrate antigens, such as phosphoryc holine (PC) (92), phosphatidyl choline (PtC) (93) and lippolysaccharide (LPS) (94). The specificiti es of these B-1 produced IgM antibodies suggest that B-1 cells are cri tical for protection against b acterial infection (95), acute septic peritonitis (96), vascular diseases such as atherosclerosis (97), and viral infections (in particular, influenza) (98).

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17 A correlation between the func tions of B-1 cells and systemic rheumatic autoimmune diseases has drawn considerable attention. Several reports have demonstrated that in both humans and mice there is a significant correlation between th e frequency of B-1 cells and the eventual development of auto immune diseases (99, 100). Interestingly, dramatic increases in the number of B-1 cel ls are found in human patients with SjS (101) and RA (102). While B-1 cells are present in mi nor labial glands of SjS patients (103), it is not certain whether B-1 cells can initiate au toimmunity or provide protection against it. In the latter case, it is important to note the similarity between B-1 cells and anergic B cells, including failure of BCR-antigen stimulation to induce Ca++ influx (104), the expression of low levels of CD5 (105), and the expre ssion of high levels of the transcription factor NFATc (106, 107). Theref ore, it has been speculated that B-1 cells may induce tolerance of potentially autore active B cells rendering them anergic or nonpathogenic. On the other hand, B-1 cells are often auto -reactive, producing au toantibodies with low affinity compared to conventional B cel ls, thereby possibly involved in initiating autoimmunity (90). A smaller number of this B-1 cell population, however, can migrate to and reside in the germinal centers where they will receive signal from helper T cells, the consequence of which is an induction of class switching and somatic hypermutation (108). This newly emerged set of B-1 cells can then give rise to high affinity autoantibodies that are potentially pathogenic. Expression of Restricted Repertoire of the B Cell Receptor in SjS Disease An interesting hallmark feature of B cells in SjS is the relatively restricted B cell receptor (BCR) repertoires found on peripheral B cells and exocrine gland-infiltrating B cells, as well as the MALT-associated B cell lymphomas that develop in a subset of SjS

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18 patients. In a recent study by Kaschner et al . (109) involving three patients, a significant over-representation of specific VLs was observed: for V, four genes (2A2, 2B2, 2C and 7A) represented 56% of all functional Vs. In the productive V repertoires, three genes (L12, 012/02, and B2) represented 43% of all VJs. VA27, a gene frequently found on autoantibodies, rheumatoid factor and lympho mas in SjS patients, was identified at an increased frequency of 29% in the parotid gl and compared to only 8% in the peripheral blood. In addition, significant enrichment of VA19 and V2E, specifically the clonal expansion of VA27-J5 and VA19-J2 has also been reported in the parotid glands of SjS patients (110). Since B cells take advant age of receptor editing to escape apoptosis and prevent recognition of se lf-antigen, and because ther e is a marked decrease in receptor editing observed in primary SjS pa tients, this restricted BCR V-region usage may be a result of a defect in receptor editing. However, also influencing this restricted V-region usage may be the reported depleti on of memory B cells from the peripheral blood with a concomitant elevated level of antigen-activated B cells in the parotid glands (109), an observation implying a defect in selection. This peripheral memory B cell population, characterized as CD19+CD27+, was found to have a mutational frequency of 8.6% in SjS patients in the im munoglobulin VH transcripts co mpared to 4.3% in normal healthy controls, as well as an elevated mutational freque ncy in Cµ transcripts for multiple Ig heavy chain isotypes (111). No t addressed by these data is whether the restricted use of BCR repertoi res is due to general innate selection mechanisms for B cell survival or an antigen-driven expansion and survival of a restricted receptor-bearing B cell population.

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19 Signal Transduction of the B Cell Receptor The BCR complex is made up of membranebound immunoglobulin (Ig) associated with CD79a and CD79b, two heterodimers of Ig and Ig . Following interaction of the BCR with a ligand, protein tyrosine kinases (PTK s) including Syk and Btk of the Tec family and Lyn, Fyn, Blk, and Lck of the Src family, are activated. In addition, the BCR is finetuned by crosstalk between PT Ks, protein tyrosine phosphata ses and adapter proteins such as B cell linker (BLNK) and BAM32 (B lymphocyte adapter molecule of 32 kDa) (112). Activated Src-family PTKs phosphorylate the immunoreceptor tyrosine-based activation motifs (ITAM) in the cytoplasmic tails of the Ig /Ig heterodimers, resulting in recruitment of Syk and Btk through their tandem Src homolo gy 2 (Src 2) domains. Activated Syk, through BLNK, can activate PLC 2 (phospholipase C 2) by tyrosine phosphorylation. PLC 2 can hydrolyze PIP2 (phosphatid ylinositol 4, 5-bi sphosphate), a membrane phospholipids, to produce DAG (diacylglycerol) and IP3 (inositol 1, 4, 5trisphosphate). IP3 then triggers the release of intracellular Ca2+. Together, DAG and Ca2+ activate PKC (protein kinase C). Activat ed PKC is required for the activation of mitogen-activated protein kinase s (MAPKs), such as extrace llular signal-regulated kinase (ERK), c-JUN NH2-terminal ki nase (JNK), p38 MAPK, and even tually the transcription factors, nuclear factorB (NFB) and nuclear factor of activated T cells (NFAT). Signals from activated Syk can also phosphorylate I -B which results in the release of transcription factor NFB into the nucleus. Furthermore, activated Syk, through BCR ligation, can trigger the activa tion of the Ras pathway. Ac tivated Syk phosphorylates and activates Ras. Activated Ras will in turn activate RAF1 by tyrosi ne phosphorylation, and ultimately activate effector molecules similar to the PKC/Ca+ pathway (112).

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20 Intimate Interaction of the B Ce ll Receptor and B cell Co-Receptors Activation of BCR-associated signaling path ways is critical to the generation of humoral immunity as these signaling pa thways determine B cell proliferation, differentiation, selection, survival and eventu ally function. B cell function, however, is also regulated by B cell surface co-receptor mo lecules. These co-receptors modulate the intensity, quality, and duration of the BCR signal transduction pathways. These coreceptor molecules, which include CD19, CD21, and CD22 and complement component C3d, modify the intrinsic intracellular signa l transduction threshold by adjusting the strength of the signals n eeded to initiate BCR-mediat ed activation. CD19, CD21 and CD22, are functionally linked with Lyn, Vav, and SHP1 in a common signal transduction pathway that is initiated by BCR binding to its ligand (113). CD19 is a 95 KDa transmembrane glycoprotei n of the Ig superfamily expressed by all B cells from the late pro-B cell to the plasma cell (114). Th e cytoplasmic domain contains nine highly conserved tyrosine residues that recognize active SH2 (Src Homology 2) domain motifs of regulatory mol ecules. The CD19 tyrosine residue is phosphorylated constitutively in splenic B cells at low level and complexed constitutively with Lyn and Vav. When BCR binds its liga nd, a local increase in the number of activated Lyn molecules permits phosphoryla tion of the Immunoreceptor Tyrosine-based Activation Motifs (ITAMs) of Ig /Ig heterodimers. Lyn also phosphorylates the tyrosine residue at position 513, plus a numbe r of other tyrosine residues on CD19. Phosphorylation of CD19 at ty rosine position 391 recruits Vav to bind at this site, and due to the close proximity with Lyn, Vav is also phosphorylated by Lyn. This process functions to activate MAPK and induce a prolonged Ca2+ influx, which initiates additional downstream signaling pathways, incl uding cytoskeletal re organization (113).

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21 CD19 also interacts with phosphatidylinosit ol-3 kinase (PI3K) by providing a docking site for the p85 subunit of PI3K when the tyrosine residues are phosphorylated by Lyn. Activated PI3K facilitates Ca2+ mobilization and activation of the serine/threonine kinase AKT pathway (112). Once Lyn has performed th is function, it loses its affinity for the SH2 domain. Despite all, the ligand for CD19 remains undefined. However, CD19 can form a non-covalent quaternary complex on the surf ace that includes CD21, CD81 and CD225. CD81 is a member of tetra-spans family that is involved in regulation of cell growth, mobility and signaling. CD81 is physically associated with CD225, whose precise function is unknown but thought to be involved in anti-proliferative activity regulated by interferon and B cell growth ( 113). CD21 is the receptor fo r C3d, a cleavage product of C3 that forms covalent bonds with foreign an tigen and immune complexes. It is also a receptor for Epstein Bar Virus (EBV). The ab ility of C3d-antigen complexes to crosslink the CD19/CD21/BCR complex provides a possi ble link between innate and adaptive immunity (115). Functioning as positive regulators of B cell activation, signals generated by the co-ligation of CD19/CD21 and BCR ar e additive. Co-ligation of BCR and CD19/CD21 by a C3d-antigen complex lowers the threshold for B cell activation, thus lowering the amount of antigen required to activ ate B cells (116). Currently, there are at least two models for the CD19/CD21 comp lex in its function as a regulator of transmembrane signals. The “co-stimulato ry” model postulates that transmembrane signaling results from cross-linking between the BCR and CD19/CD21 by the simultaneous binding of C3d-antigen comple xes. The “response regulator” model postulates that the BCR independently tran smits the transmembrane signal, whereas

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22 CD19 merely regulates intrinsic levels of Lyn and Vav phosphorylation and activation. In this model, an association between CD 21 and C3d-antigen complexes promotes the intrinsic functions of CD19 (113). In opposition to CD19/21, CD22 plays an im portant inhibitory role in B cell activation. The cytoplasmic domain of CD22 c ontains six tyrosines located in the three Immunoreceptor Tyrosine-based Inactivation Mo tifs (ITIMs) and two ITAMs, suggesting the potential for both negative and positive sign aling. Phosphorylated CD22 recruits the phosphotyrosine phosphatases, SHP1 and SH IP, to limit BCR signaling (117), thus, CD19/CD21 and CD22 are reciprocally regula ted by each other. Only limited numbers of Lyn and Vav proteins are available in this system and must be shared by BCRs, CD19/CD21 and CD22. The phosphorylations of CD19 by Lyn and Vav also results in the phosphorylation of CD22 that, in turn, recruits SHP-1 and SHIP, bringing these molecules in close proximity to CD19/C D21 and BCRs. SHP-1 and SHIP, when bound to CD22, dephosphorylates CD19 and BCRs thereby down-regulating CD19 and BCR function. In addition, SHIP can convert PI P3 to PI3,4-P2 which down-regulates the PI3K-dependent pathway (118). The Role of Autoantibodies in the Development of SjS In recent years, the nature of numer ous (auto)-antigens in connective tissue disorders have been identifie d using various molecular appr oaches. Many autoantigens have proven to be intracellu lar enzymes and regulatory factors required for cellular function, e.g., gene replica tion, transcription, RNA proce ssing and protein synthesis (119). Antibody against autoantigen such as anti-Ro IgG antibody can cross the placenta and cause neonatal lupus (120). However, li mited data are available indicating that antibodies directed against these molecules have any direct eff ect in eliciting a

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23 pathological consequence, for example, lo ss of fluid secretion by exocrine glands. Nevertheless, antibodies targ eting nuclear proteins (ANAs ) in SjS, especially the ribonuclear proteins Ro/SS-A a nd La/SS-B (121), have long been used as a diagnostic marker of disease in both humans and anim al models. Different immuno-fluorescence patterns of ANAs have, over time, led to the identification of other nuclear proteins targeted by autoantibodies, such as Sm, dsDN A, the nuclear mitotic apparatus (NuMA), proteasomes, mitotic chromosomal autoan tigens (MCAs), and poly-(ADP-ribose) polymerase (33). Autoantibodies detected in SjS patients and various animal models are not limited to targeting nuclear proteins. One well-s tudied autoantigen is the intracellular cytoskeletal protein -fodrin, targeted by proteolytic enzy mes in the salivary glands of both SjS patients and NFS/ sld mice (122). In addition, as NFS/ sld mice age, they develop autoantibodies against ssDNA, IgG1 and IgG2a subclasses, rheumatoid factor and type II collagen (123). Other exampl es of intracellular autoantigens are tissue kallikrein-1 (Kik-1) and kallik rein-13 (Kik-13) as defined by detectable autoantibodies in sera of IQI/Jic mice affected with SjS (12 week and older). Ho wever, only Kik-13 was shown to induce a prolif erative response by splenic T cells (124). IQI/Jic is an inbred strain that originated, like the NOD strai n, from the ICR mouse colony in Japan and exhibits spontaneous SjS-like disease that mi mics that of NOD mice (68). The fact that Kik-13 is highly expressed in the salivary gl and ductal cells may explain target tissue specificity of SjS as it is consistent with periductal infiltrati on of immune cells. Furthermore, since only reduced forms of Kik13 are recognized by se ra of IQI/Jic mice, one might conclude that cryptic epitopes ar e involved, not unlike the case proposed for

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24 the potential development of autoantibody resp onses to PSP in NOD mice (60). Lastly, both SjS patients and NOD mice form autoantibodi es reactive with islet cell autoantigen69 (ICA-69) expressed in pancreatic islets, the brain and both salivary and lacrimal glands (125). Disruption of the ICA69 lo cus in the NOD mouse prev ents lacrimal gland disease and greatly reduces salivary gla nd, suggesting that immunoreactivity against ICA-69 might play a role in disease progression (125). An intriguing question in autoimmune diseases like SjS pertains to how intracellular components, i.e ., self-proteins, become recognized and presented as dominant neo-antigens by immune cells. The recent reviews and journal articles by Rosen et al . (126-128) advance cellular a poptosis as an initial even t. In this work, Rosen and colleagues describe how molecules w ithin the subcelluar compartment are redistributed in apoptotic cells. Small memb rane blebs could be shown to contain 52kDa Ro and other molecules, such as calreticu lin, normally present within the ER lumen. Nuclear antigens also ex hibit redistribution during apoptosis, showing an increase in localization of 60-kDa Ro/SSA, La/SS-B, the snRNPs, Ku and PARP as a rim around the condensing chromatin (128, 129). Such clustering of potential autoantigens occurs during apoptosis, but not during necrosis (130, 131). Although the degree of acinar cell apoptosis in the salivary and lacrimal glands is not well-established or universally accepted, proteolysis, phosphorylation, glutathiolation, tr ansglutamination, citrullination, and/or formation of novel protei n-protein or prot ein-nucleic acid complexes probably play a role in the alteration of molecular st ructures permitting exposure of neoor cryptic epitopes to the immune system (130, 132, 133).

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25 The importance of T lymphocytes in the ac tivation, proliferation and differentiation of antigen-reactive B cells in autoimmune-prone mice is nicely demonstrated in the Id3 gene KO mouse (134). The Id proteins bind basic helix-loop-helix tr anscription factors and function as dominant negative inhibitors of gene expression. Id3 is an immediate early-response gene regulating growth and is involved directly in TCR-mediated T cell selection during T cell development in the thymus. Id3-deficient mice with various genetic backgrounds develop a SjS-like diseas e, including the synthesis of anti-Ro/SS-A and anti-La/SS-B antibodies at approximately one year of age (135). In the absence of T cells, these Id3-deficient mice failed to exhibi t development of a SjS-like disease. One fascinating observation in this mouse model is the appearance of secretory dysfunction as early as 6 weeks of age, a time point prior to other visible di sease symptoms. This raises the possibility that organogenesis of the sa livary glands may be impaired in this Id3 -gene KO mouse, resulting in aberrant antigen pr esentation and autoreac tive T cell activation, as Id3 is known to be a Smad4-dependent TGFresponsive gene whose pathway is important for salivary gland development (136). We have speculated that delayed organogenesis of the salivary glands in NOD mi ce is critical for subsequent development of SjS-like disease in the NOD mouse, as well (57). Anti-Muscarinic Acetylcholine Type-3 Recep tor Autoantibodies – The Effectors of Glandular Dysfunction Although the loss of saliva and tear flow in SjS was initially believed to be a consequence of acinar cell apop tosis elicited by cytotoxic T lymphocytes, an interesting paradigm shift has occurred based on studies showing the requirement for B lymphocytes and immunoglobulin. First, B cell deficient NOD.Ig µnull mice fail to develop secretory dysfunction, and second, IgG from SjS patie nts can induce a reversible stimulation or

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26 inhibition of salivary function when infused into NODscid mice (66). Accumulating evidence suggests that disturbances in lymphocyte homeostasis, including ectopic germinal center formation in the target ti ssue and/or aberrations of cellular signaling regulated by B cell activating factor (BAFF), are present in SjS (70, 111, 137). As shown in both transgenic mice over-expressing BAFF an d patients with SjS, B cell hyperactivity may lead to excessive immunoglobulin produ ction and prolonged B cell expansion that eventually lead to the monoc lonal expansion of B cells and transformation to B-cell lymphomas in a subset of patients (46, 47). Additionally, intrinsic defects in the B cell compartment associated with SjS may play a role in the generation of SjS autoantibodies with diversified prevalence and specificity, as evidenced by the wide array of autoantigens targeted. Thus, identification of autoantibodies that di rectly cause dryness in SjS patients is essential for understandi ng the pathogenesis and onset of clinical disease. Of the many autoantibodies identified in SjS patients to date, the association between anti-M3R autoantibodies and secretor y dysfunction seems most intuitive since the M3R is the major receptor mediating secret ion in the salivary and lacrimal glands in response to parasympathetic stimuli. Studies strongly indicate that serum or purified IgG from SjS patients down-regulate carbachol-e voked bladder muscle contraction by 50 %, while anti-idiotypic antibodies neutralize this inhibition of ch olinergic neurotransmission (36, 39, 138). Furthermore, anti-idiotypi c antibodies were able to neutralize autoantibodies that inhibit cholinergic neur otransmission (138). Similarly, studies using the human salivary gland ductal cells, HSG, show ed that pretreatment of the cells with

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27 SjS IgG for 12 or 24 hours reduced the magnitude of subsequent carbachol-induced intracellular calcium release (38). In a recent study, Cha et al.(37) reported that M3R desensitization occurs in mice with anti-M3R autoantibodies, as reveal ed in a comparison of carbachol-evoked responses in NOD mice >20 weeks of age ve rsus either age-matched C57BL/6 or antibody-negative 8-10 week old NOD mice. These observations, therefore, would be consistent with the hypothesis that chronic stimulation by anti-M3R antibody induces an inhibitory affect on M3Rs. Importantly, NOD mice with overt SjS-like disease initially responded well to pilocarpine-induced stimula tion; however, this stimulation was downregulated following chronic in jections. We currently interpret these results as an augmented desensitization of M3Rs in the presence of anti-M3R autoantibodies. Extrapolating these data to a clinical setting, chro nic intake of pilocarpine might enhance saliva secretion initially, yet may induce even tually a more rapid desensitization in human patients positive for anti-M3R autoan tibodies. The effect of prolong usage of pilocarpine on desensitization of M3Rs might need further investigation to elucidate the mechanism and other potential clinical consequence. Furthermore, mixed response profiles were s een when bladder strips isolated from normal, healthy C57BL/ 6 mice were incubated with sera from a number of different SjS patients. While a few sera enhanced smoot h muscle contraction in comparison with either normal sera or Krebs physiological buffe r, other sera inhibite d the contractions. This could represent effects of different antibody titers, lengt h of incubation time, and/or different titers of a pathogenic subset of anti-M3R autoantibodies. In light of recent studies discussed below, SjS-like disease ma y be dependent on anti-M3R autoantibodies

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28 of a specific isotype (56). If the effects are dependent on a specific IgG subclass, then it will be necessary to determine if its inhibitory activity on acinar cell secretion is dependent on the constant region of an imm unoglobulin subclass or relies on a variable region whose specificity is shap ed by the constant region.

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29Table 1-1. Mouse strains used in the study of Sjögren’s syndrome Mouse strains Characteristics Disease Manifestations / Phenotypes NZB/W [(NZB x NZW)F1 hybrid] Naturally occurring mouse model by crossing NZB and NZW (139) Lacrimal gland involvement A greater percentage of B cells compared with MRL/lpr mouse MRL/lpr Mutation in lpr that encodes Fas protein(140) Diffuse lymphocytic infiltration Autoantibodies (against ssDNA, RNPs, IgG) No loss of secretion No detection of anti-M3R Abs NOD/LtJ / NODcongenics NOD/LtJ NOD.B10H2b NOD-scid NOD.Igµ-/NOD.IL4-/Spontaneous insulitis and diabetes(141) NOD with H-2b from C57BL/10 (142) Homogygous mutation in scid locus (no functional lymphocytes) (143) No functional B-lymphocytes (66) Cytokine IL-4 gene knockout (56, 58) SjS-like disease phenotype; Loss of secretion, anti-M3R Abs, focal lymphocytic infiltration Disease phenotype w/ diabetes Disease phenotype w/o diabetes No disease phenotype but abnormal organogenesis Disease phenotype w/ normal flow Disease phenotype w/ normal flow

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30Table 1-1. Continued Mouse strains Characteristics Disease Manifestations / Phenotypes NOD.IFN -/NOD.IFN R-/NODscid. IFN -/NOD.Q NOD.P IFN gamma KO IFN gamma receptor KO No functional lymphocytes in IFN gamma KO (57) NOD with H2q from C3H.Q NOD with H2p from C3H.NB (144) Neither disease phenotype nor abnormal organogenesis observed Neither disease phenotype nor abnormal organogenesis observed Severity of sialadenitis greater than that of NOD Severity of sialadenitis lesser than that of NOD C57BL/6.NODAec1Aec2 C57BL/6 carrying Aec1 (Idd3) genetic region on Chr 3 and Aec2 (Idd5) genetic region on Chr 1 (55) SjS-like disease phenotype in a C57BL/6 genetic background IQI/Jic Inbred strain originating from ICR (68) Anti-kallikrein 1 and -13 Abs NFS/sld Autosomal recessive gene with sublingual gland differentiation arrest (145) Anti-alpha fodrin Abs Id3 gene KO Id3; basic helix-loop-helix transcription factor, a dominant negative inhibitor of gene expression (135) Impaired TCR-mediated T cell selection Loss of secretion Anti-Ro and anti-La Abs

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31Table 1-1. Continued Aromatase gene KO Estrogen deficiency due to absence of enzyme catalyzing the conversion of testosterone to estradiol (146) B cell hyperplasia in the BM and spleen Anti-alpha fodrin Abs Alymphoplasia (aly/aly) Homozygous mutation in aly (alymphoplasia) (147) Conserved CDR3 in TCR of infiltrating T cells in the lacrimal glands, salivary glands and kidneys GVHD (graft-vs-host disease) GVHD induced by the injection of DBA/2 spleen cells into nonirradiated (C57BL/6 x DBA/2) F1 mice (148) Lymphocytic infiltration w/ majority of T cells BAFF transgenic Transgenic for B-cell survival factor (69) Loss of secretion by 15-17 m onths of age. Lymphocytic infiltration w/ majority of B cells BALB/c Immunized with short peptides o f 60 kDa Ro antigen (67) Loss of secretion, anti-Ro, lymphocytic infiltration

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32 Table 1-2. Comparison of general symptoms of Sjögren's syndrome patients and NOD mice Characteristic Sjögren's Syndromed NOD micee Dacryoadentitis (Yes) a Yes b Sialadenitis Yes Yes b Decreased tear & saliva flow rates Yes Variable b Altered proteins in tears & saliva Yes Yes b Pro-inflammatory cytokine production Yes Yes Autoantibodies Anti-Ro/SS-A, Anti-La/SS-B Yes Probably not c Anti-DNA (ANAs) Yes Yes Anti-fodrin Yes Yes Anti-adrenergic receptor Yes Yes Anti-type-3 muscarinic ACh receptor Yes Yes Keratoconjunctivitis sicca Yes (?) Ocular epithelium dessication (Rose-Bengal Dye) Yes Yes b Break-up time testing Decreased (?) Lysozyme & Lactoferrin activity Decreased Decreased b Stomatitis sicca Yes Yes Buccal epithelium dessicati on Yes (?) Serine protease activity against PSP (?) Yes Amylase & EGF activity Decreased Decreased b a Biopsies of lacrimal gla nds not often performed b Data presented under Research & Methods Section c Possibly detected as part of ANAs

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33 Phase I Phase II Phase IIIDelayed organogenesis Aberrant gene & protein expression Acinar cell apoptosis Autoantibody production Loss of secretory function Leukocyte infiltration of exocrine glands Wk: 0 4 8 12 Wk: 0 4 8 12 16 20 >24 16 20 >24SMX expression of parotid secretory protein (PSP) PSP-proteolysis ( marker of SjS ) Detection of anti-M3R antibody Figure 1-1. Model for the progression of SjS-like autoimmune exocrinopathy

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34 Figure 1-2. Dynamic cellular composition of ly mphocytic infiltration in submandibular glands of NOD mouse. Green: CD3.FIT C. Red: B220-Texas Red. A) 8-10 weeks of age. 400X magnificati on. B) 12-16 wks of age. 200X magnification. C) 20-24 weeks of age. 100X magnification.

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35 CHAPTER 2 EFFECT OF COBRA VENON FACTOR (CVF) ON COMPLEMENT AND B LYMPHOCYTES IN THE NOD.B10.H2b MICE Introduction Sjögren's syndrome (SjS) is a human auto immune disease char acterized by loss of exocrine function as a result of a chronic im mune attack directed primarily against the salivary and lacrimal glands l eading to xerostomia (dry mouth) and xerophthalmia (dry eyes) (1, 3, 11, 18). Although the underlying cause of SjS rema ins elusive, a number of studies using the NOD mouse model of SjS have led us to conclude that autoimmune exocrinopathy progresses in thr ee consecutive phases (61, 14 9). In phase 1, a number of genetically predetermined physiological and biochemical activities associated with retarded salivary gland organogenesis occur prior to and independent of initiation of autoimmunity (59). In phase 2, leukocytes infiltrate the exocrine glands with a concomitant increase in the expression of pro-inflammatory cy tokines. In phase 3, secretory dysfunction of the saliv ary and lacrimal glands occu rs, most likely, the result of production of autoantibodies (39, 56, 65, 66, 150, 151). Interruption within any one of these three phases can prevent onset of clinical SjS-like disease. SjS is considered a lymphoproliferative B ce ll disorder, thus, the clinical phase of disease fails to develop in the absence of e ither B cells or autoantibodies. Although B cell development is stringently regulated, B ly mphocytes in SjS are hyper-proliferative, capable of evading apoptosis and overly sensitive to activation and maturation, suggesting escape from tolerance-induci ng mechanisms. One mechanism controlling

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36 activation, survival and proliferation of B cells is through a cross-linking between B cell receptors and their co-receptors CD1 9, CD21 and CD22. While the ligand for CD19 remains unknown, CD19 can form a non-covale nt quaternary complex that includes CD21, CD81 and CD225 (113). CD21, in tur n, is the receptor for C3d, a cleavage product of C3 that forms covalent bonds w ith foreign antigen or immune complexes. Signals generated by the co-ligation of CD19/CD21 and BCR by C3d fragment act as positive regulators of B cell activation. Co -ligation of BCR and CD19/CD21 by a C3d; Ag complex lowers the threshold for B cell activation, possibly cont ributing to the hyperproliferative and hyper-active properties of autoreactive B cells (116). A number of studies using anti-complement treatment have shown that complement plays an important role in several autoimmune diseases, includi ng rheumatoid arthritis, multiple sclerosis, myasthenia gravis and systemic lupus erythe matosus (152). However, little attention has focused on the role of complement, especially complement component C3, in SjS. In the present study, we have examined whether C3 plays a role in the pathophysiological aspects of SjS-like disease of NOD.B10H2b mice. Materials and Methods Animals NOD.B10H2b and BALB/cJ mice were bred and maintained under specific pathogen-free conditions within the mouse facility of the Department of Pathology, Immunology and Laboratory Medici ne at the University of Florida, Gainesville, FL. Breeder pairs of both strains were purchased from the Jackson Labor atories (Bar Harbor, ME). All mice received water and food ad libitum.

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37 Complement Depletion by Cobra Venom Factor Cobra venom factor (Naja naja kaouthi a) was purchased from Calbiochem (#233552, stated to be >95% pure by SDS-PAGE) (La Jolla, CA). Star ting at 10 weeks of age, mice received intraperitoneal injections of either 200 µl CVF (one unit diluted in sterile PBS) or 200 µl of ster ile PBS. The injections were carried out twice a week over the course of the study (14 weeks). Bloods we re collected from the tail veins once each week, the sera prepared and tested for levels of complement by ELISA. In brief, goat IgG anti-mouse C3 antibody (#55463, Cappel, ICN Pharmaceuticals Inc., Aurora, OH) diluted 1:250 was used as the capture antibody. A peroxidase-conjugated goat IgG anti-mouse C3 antibody (#55557, Cappel-ICN Pharmaceutical s Inc.) diluted 1:500 was used as the detection antibody. ELISAs were run in tripli cate for each serum sample diluted 1:12,500 in PBS. For a standard, mouse compleme nt provided by Cappel, ICN Pharmaceuticals Inc. was used and diluted over the range from 1:100 to 1:1,562,500. Color was developed with TMB (Sigma, St. Louis, MO) for appr oximately 10 min and stopped by adding 50 µl of 2N H2SO4; OD readings were determined at 450 nm. Measurement of Salivary Rates To measure stimulated flow rates of saliva, individual mice were weighed and given an intraperitoneal (i .p.) injection of a cocktail containing isoproterenol (0.2 mg/100 g body weight) and pilocarpine (0.05 mg/100 g body weight) dissolved in PBS. Saliva was collected for 10 min from the oral cavity of individual mice using a micropipette starting 1 min afte r the injection of the secret agogue. The total volumes of saliva sample were measured.

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38 Histology and Immunofluorescent St aining for B and T Lymphocytes Submandibular glands were surgically rem oved at time of euthanasia (24 weeks of age), placed in 10% phosphate-buffered forma lin for 24 h, then embedded in paraffin and sectioned at 5 µm thickness. Paraffin-em bedded slides were de-paraffinized by immersing in xylene, followed by dehydrating in ethanol. Tissue s ections were stained with H&E dye (Gainesville Service Tec h., Gainesville, FL) and observed at 100× magnification. For immunohistochemical staining, the tissu e sections were washed in PBS for 5 min, then incubated 1 h with blocking solu tion containing normal rabbit serum diluted 1:50 in PBS. Each section was incubated with rat anti-mouse B220 (BD BiosciencesPharmagen, San Diego, CA) diluted 1:10 a nd goat anti-mouse CD3 (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:50 fo r 1 h at 25 °C. The slides were washed three times with PBS for 5 min per wash followed by a 1 h incubation with Texas Redconjugated rabbit anti-rat IgG (Biomeda, Fo ster City, CA) diluted 1:25 and FITCconjugated rabbit anti-goat IgG (Sigma Chem icals, St. Louis, MO) diluted 1:100 at 25 °C. The slides were washed thoroughly with PBS, treated with Vectashield DAPImounting medium (Vector Laboratory, Bur lingame, CA) and overlayed with glass coverslips. Stained sections were visualized at 200× magnification. Flow Cytometry Splenic leukocytes were prepared for fl ow cytometric analyses as detailed elsewhere. Aliquots of each ce ll preparation containing 1 × 105 cells were incubated 45 min with either R-PE-conjugated rat anti-mouse CD19 monoclonal antibody (#557399), FITC-conjugated rat anti-m ouse CD19 monoclonal antibody (#557398), FITC-conjugated rat anti-mouse CD21 monoc lonal antibody (#553818) or PE-conjugated

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39 mouse anti-mouse CD22.2 monoclonal antibody (#5533384) (BD BiosciencesPharmingen, San Diego, CA), washed in FACS buffer, then analyzed for fluorescence staining on a FACScan (BD Biosciences, San Jose, CA). Detection of Anti-Nuclear Au toantibodies in the Sera ANA in the sera of mice were detected us ing the Sigma ANA sc reening kit (Sigma Chemicals, St. Louis). HEp-2-fi xed substrate slides were ov erlaid with the appropriate mouse serum diluted 1:50. Slides were inc ubated for 3 h at room temperature in a humidified chamber. After three washes for 5 min with PBS, the substrate slides were covered with FITC-conjugated goat anti-mouse IgG (Sigma, St. Louis) diluted 1:50 for 1 h at room temperature. After three wa shes, nuclear fluorescence was detected by fluorescence microscopy at 100× magnification. Results Effects of CVF Treatment on the SjSlike Disease Profile in NOD.B10-H2b Mice SjS-like disease in NOD.B10H2b mice, similar to SjS in humans, is characterized by high levels of B cell survival and pro liferation, plus hyper-gammaglobulinemia with production of autoantibodies(53). One mechanis m postulated to regulat e B cell survival involves the cross-linking of the BCR with the CD19/CD21 complex via complement component C3d. In a series of stud ies using either the NOD or NOD.B10H2b mouse models of SjS, we have shown that the crit ical time point to delay or prevent onset of clinical disease is between 12 and 16 weeks of age, a time when B cell hyper-activity and appearance of anti-acinar cell autoantibodies are occurring. Based on these two independent observations, we examined the eff ects of depleting systemically complement component C3 from NOD.B10H2b mice by routine injections of CVF starting at 10 weeks of age. CVF resembles C3 functionally and binds to factor Bb, a proteolytic

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40 product of factor B × factor D. CVF-Bb, however, is resistant to the complement regulator proteins; thus, the pe rsistence of CVF-Bb consumes plasma C3 resulting in the depletion or inactivation of C3(153). Taking advantage of this property, CVF was injected intraperitoneally into NOD.B10H2b mice twice weekly to block the production of C3d. For comparative controls , ageand sex-matched NOD.B10H2b mice were injected with PBS. Beginning 2 days prior to the first inject ions and at weekly intervals thereafter, the individual mice were bled, their sera prepared and pooled and the C3 levels in the pooled sera determined by ELISA. As presented in figure 2-1, NOD.B10H2b mice treated with CVF showed a rapid, but temporar y decrease in the levels of detectable C3. In contrast, NOD.B10H2b mice treated with PBS maintained constant levels of serum C3. An early marker predictive of subsequent onset of SjS-like disease in NOD and NOD.B10H2b mice is the activation of a serine protease capable of proteolytic digestion of parotid secretory protein ( PSP), a zinc-dependent anti-bact erial protein secreted into saliva(60). Measurements of serine proteas e activity in the salivas of PBS and CVFtreated NOD.B10H2b mice revealed that all NOD.B10H2b mice at 7 weeks of age failed to show detectable serine protease activity, but exhibited positive serine protease activity by 24 weeks of age (data not presented). These data indicate that treatment with CVF did not alter this acinar cel l-associated pathophysiological manifestation. The unique characteristic of NOD and NOD.B10H2b mice as models for SjS is the gradual decrease in salivary flow rates starting around 12–14 weeks of age concomitant with the appearance of leukoc yte infiltrates with in the exocrine glands. To measure changes in salivary flow rates, each mouse wa s weighed and injected with isopreterenol

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41 and pilocarpine. Saliva flow rates were meas ured 1 day prior to treatment and every 2 weeks thereafter. NOD.B10H2b mice treated with PBS showed a 20% decrease in saliva secretion over the first 12 weeks of treatment , as depicted in figure 2-2. In contrast, the NOD.B10H2b mice treated with CVF showed nearly a 70% average increase in their salivary flow rates during the same period, consis tent with their increasing age and size. At 24 weeks of age or 14 week s after initiating treatment, the mice were euthanized and their submandibular glands explanted for histological analysis. As presented in figure 2-3A, NOD.B10H2b mice treated with PBS showed multiple areas of leukocyte infiltration, having an average of 13 foci pe r histological section. In contrast, NOD.B10H2b mice treated with CVF showed fewer ar eas of leukocyte infiltration, having an average of only two foci per section (Figur e 2-3C). In addition, the foci in the CVFtreated mice appeared to be smaller. Furt hermore, when immunostained using anti-B220 and anti-CD3 antibodies to detect B and T cells, respectively, the number of B cells within the foci of the CVF-treated animals was significantly reduced (Figure 2-3B and 23D). Several clinical manifestations of SjS are mediated by autoantibodies, thus, the detection of such autoantibodies are important in the diagnosis of disease. The presence of ANA in sera of human patie nts, as well as NOD and NOD.B10H2b mice, indicates development of the clinical phase. Thus, at time of euthanization, blood was collected from each animal and the sera prepared and pooled within each group. The two-pooled sera were assayed for the presence of AN A using HEp-2 cells. As expected, NOD.B10H2b mice treated with PBS were strongly posi tive for ANA in their serum (Figure 2-4C), while NOD.B10H2b mice treated with CVF proved to be nearly negative (Figure 2-4D).

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42 Effects of CVF Treatment on B cell Sub-Populations NOD.B10H2b mice injected with CVF or PBS were euthanized at 24 weeks of age, their splenocytes collect ed and examined for different sub-populations of B cells based on expressions of the BCR's co-recep tors CD19, CD21 and CD 22. Mice injected with CVF had a two-fold decrease in the num ber of B cells within unfractionated total spleen cell preparations that expressed CD 19 molecules on their surface when compared to normal BALB/c mice or NOD.B10H2b mice treated with PBS (Figure 2-5C versus Figure 2-5A and B, respectively; Table 2-1). In addition to the altered distribution of CD19-positive B cells in animals treated with CVF, there was also a concomitant reduction in the MFI of CD19 expression on the surface of the B cells, as shown in Figure 2-5D. This shift in MF I was observed for both the CD19hi and CD19low B cells populations. Similar to the altered expression of CD19 on B cells from CVF-treated mice, expression of CD21, a receptor for the C3d mol ecule, was also reduced as compared to the CD21 expression on B cells from PBS-trea ted mice. As presented in Table 2-1, the numbers of CD19-positive B cells expressing high levels of CD21 nearly disappeared (0.1% for CVF-treated mice versus 41.0 % for the PBS-treated control mice). Interestingly, expression of CD22 on CD19-positive B cells, a negative regulator of B cell responses, proved similar for splenic B ce lls isolated from either PBSor CVFtreated mice (Table 2-1). Discussion In the present study, we have investigated the possible involvement of complement component C3 in the development of SjS-like disease of NOD.B10H2b mice by temporally reducing C3 during the early stag e of disease by treatment with CVF. The

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43 NOD.B10H2b mouse is a model of primary SjS exhibiting many of the immunological manifestations observed in SjS patients(53 ), including leukocyte infiltration of the exocrine glands, hyper-reactive B cells, hyper-gammaglobulinemia and production of autoantibodies thought to be the effectors of clinical disease. Recent studies of both humans and animal models have suggested that B cells and pat hogenic autoantibodies play major roles in SjS and SLE due in part to a breakdown in B lymphocyte selftolerance, possibly from a cross-linking of BCRs with co-receptors CD19 and CD21(114, 154). One mechanism involved in survival, ac tivation and prolifera tion of B cells is through such cross-linking involvi ng C3d. We reasoned, therefore, that inactivation of C3 at the time of disease onset (12 ± 2 weeks of age) might prevent this over-reactivity of B cells and reduce the severity of the SjS-like disease in NOD.B10H2b mice. Results of the present study indica te that CVF-treatment of NOD.B10H2b mice starting at 10 weeks of age, while having little or no effect on the aberrant physiological activities (e.g., activ ation of unique serine pr oteases), reduced the severity of lymphocyte infiltration into the salivary glands, decreased the production of ANAs and prevented the onset of xerostomia sicca. Inte restingly, this reduction in disease severity correlated with significant reductions in both the numbers of splenic B cells expressing CD19 and a concomitant reduction in the m ean fluorescent intensity (MFI) of CD19 expression in the absence of any major changes in CD22 expr ession levels. Stronger cross-linking of the CD19/CD21 complex to the BCR is known to be mediated by complement-tagged antigen (155, 156), the effect s of which is a greatly lo wered signaling threshold, a prolonged BCR signaling, sustained tyrosine phosphorylation of proteins and enhanced responses of B cells to antigen.

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44 Based on the present findings, we would postu late that the importance of C3 in SjSlike disease of NOD.B10H2b mice involves C3d crosslinking BCRs with the coreceptors CD19/CD21 molecules or CD21/CD 35 through CR1/CR2 to provide powerful secondary signals within B lymphocytes. Although we were able to find direct correlations between C3-d epletion, loss of CD19hi/CD21hi B cell sub-populations and reduced autoimmunity in CVF-treated NOD.B10H2b mice that could suggest one mode of action might be the loss of C3d crosslinking of the BCR a nd its co-receptors, a number of other explanations ar e also possible. First, however , it may be possible to rule out any involvement of membrane–attack co mplex formation since the NOD mouse is a C5-deficient animal (157). Nevertheless, the importance of C3 in both innate and adaptive inflammatory responses cannot be underestimated. C3a is a critical mediator of inflammatory responses, especially in recr uiting monocytes/macrophages to the site of cell injury. Furthermore, C3 products can bind to or form comple xes with antigens to facilitate inflammatory and immunological resp onses, in part through binding to specific complement receptors, such as CR1/CR2 pres ent on FDCs. Such localization of antigen on FDCs in secondary lymphoid tissues prom otes germinal center formation, B cell retention, survival and activati on within germinal centers, as well as subse quent antibody formation (158-160). Thus, reduced germinal cen ter formation in the salivary glands of CVF-treated animals could be due to reduced levels of functional C3 affecting FDC secretion of chemokines that would normally recruit B lymphocytes to the germinal centers. The role of complement in either developm ent or severity of SjS disease in humans has not been easily defined, resulting in conflicting reports appearing over the past two

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45 decades. Molina et al. (161) reported that complement levels in SjS patients with neutrophilic inflammatory vasc ular disease were decreased , while Thomsen et al.(162) reported increased levels in primary SjS patient s. Both of these reports, however, differed from an earlier study by Fishbach et al. (163) indicating that SjS patients exhibited normal levels of complement. Recently, renewed interest in this area has surfaced with reports that hypocomplementaemia (specificall y, reduced levels of C3 and/or C4) is closely associated with B cell lymphoma deve lopment in SjS and increased pathogenicity (164). Considered together, levels of complement may vary during progression and activity of SjS; however, the role of comp lement components, like C3, appears to be essential for prolonged hyper-ac tivity of B cells. Understanding the mode of action for C3 and its active components could be useful in designing highly sp ecific intervention therapies in treating SjS patients, assuming i ndividuals predisposed to develop SjS can be identified early enough in the disease state.

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46 Figure 2-1.C3 levels in the plasma of NOD.B10H2b mice following treatment with cobra venom factor (CVF) or PBS. Starting at 10 weeks of age and continuing for 14 weeks, mice received i.p. injections tw ice weekly of either CVF or sterile PBS. Bloods were collected, the sera pr epared and the levels of C3 were determined using ELISA. The star ( ) indicates time of the first injections.

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47 Figure 2-2. Salivary flow rates of NOD.B10H2b mice treated with CVF or PBS. NOD.B10H2b mice treated either with PB S (open bars) (n=8) or CVF (striped bars) (n=8) were given an i.p. injection of isoproterenol and pilocarpine on the day prior to (day 1) and 7 weeks after (day 83) treatment. Saliva was collected for 10 min from the oral cavity of individual mice using a micropipette starting 1 min after the inj ection of the secretagogue. The volume of each saliva sample was measured and standardized against the weight of the individual mice. Data are the mean s of eight animals per group ± S.D. Difference in salivary flow rate wa s determined by Student-Newman-Keuls test. p<0.05 was considered significant.

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48 Figure 2-3. Histological examination of the exocrine glands of NOD.B10H2b mice treated with CVF or PBS. Submandibul ar glands, removed from each mouse at 24 weeks of age and fixed in 10% formalin, were stained with Mayer's H&E dye. Stained sections from glands of NOD.B10H2b mice treated with PBS (A) or CVF (B) were observed at 40× magnification for glandular structure and leukocyte infiltrations. Lymphocyte foci are indicated with arrows. The distribution of the T and B ce lls within the foci were determined by staining first with rat anti-mouse B220 or goat anti-mouse CD3 antibodies, followed by an incubation in Texas Redconjugated rabbit anti-rat IgGor FITC-conjugated rabbit anti-goat IgG antibodies, respectively. The slides were treated with Vectashield DAPImounting medium and overlayed with glass coverslips. Stained sections cont aining a focus from a PBS (C) or CVF (D) treated animal were visualized at 200× magnification. T cells stain green, B cells stain red and cell nuclei stai n blue. All indi vidual figures are representative.

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49 Figure 2-4. Detection of anti-nucle ar autoantibodies (ANAs) in NOD.B10H2b mice treated with CVF or PBS. ANAs in a positive control serum (A), a negative BALB/c serum (B) or the sera of NOD.B10H2b mice treated with PBS (C) or CVF (D) mice were detect ed using the ANA screening kit supplied by Sigma Chemicals. HEp-2-fixed subs trate slides were incuba ted with the appropriate mouse serum for 3 h, followed by deve lopment with FITC-conjugated goat anti-mouse IgG secondary antibody for 1 hour. Nuclear fluorescence was detected by fluorescence microscopy at 100× magnification. All individual figures are representative.

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50 Figure 2-5. Reduced expression of CD19 on splenic B lymphocytes of NOD.B10H2b mice treated with CVF. Spleens were freshly explanted from normal, untreated BALB/c (A) as well as NOD.B10H2b mice at 24 weeks of age treated with either PBS (B) or CVF (C ), prepared as single-cell suspensions and incubated with R-PE-conjugate d rat anti-mouse CD19 monoclonal antibody. Each cell preparation was analyzed by flow cytometry for fluorescence staining to identify CD19hi or CD19lo expressing B cells. The fluorescent intensities of CD19 in splenic B cells of NOD.B10H2b mice treated with CVF (dark solid line) vs. PBS (dotted line) and BALB/c (light solid line) are shown in the histogram of panel D. All individual plots are representative.

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51 Table 2.1. Changes in the splenic CD19-positive B cell phenotypes. Each group included eight female mice.

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52 CHAPTER 3 EFFECT OF KNOCKING OUT COMPLEMENT COMPONENT 3 ON COMPLEMENT AND B LYMPHOCYTES IN THE DEVELOPMENT OF SJS-LIKE AUTOIMMUNE DISEASE Introduction Sjgren’s syndrome is a chronic human auto immune disease in which the clinical symptom is highly dependent on the activity of B lymphocytes (1-10) One mechanism that controls survival, activa tion and proliferation of B cells is through cross-linking of B cell receptors and their co-receptors, especi ally CD19 and CD21. Cross-linking of BCRs and co-receptors that re sult in the hyperproliferation of B cells involves C3d (165). To investigate this issue in greater detail, I turned to the NOD.B10H2b mouse, our model of primary SjS exhibiting many of the immunol ogical manifestations observed in SjS patients (53), including hyper-reactivity of B cells, hypergammaglobulinemia, and production of numerous autoantibodies . We have shown that NOD.B10H2b mice exhibited reduced apoptosis, hyper-prolifera tion and over-activation of B lymphocytes starting around 10 weeks of age. The result is the production of both organ-specific and organ-nonspecific autoantibodies. In chapter 2, I reasoned that inactivat ion of complement component C3 might prevent this over-reactivity of B cells and subsequently reduce th e severity of the SjS-like disease in NOD.B10H2b mice by preventing production of C3d, thereby reducing the cross-linking of BCRs and their CD19/CD 21 co-receptors. Treatment of NOD.B10H2b mice with cobra venom factor (CVF), known to deplete C3 from circulation, while not preventing the aberrant physiological activities of pre-clinical diseas e (e.g., activation of

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53 unique serine protease), reduced the severity of lymphocyte infiltration into the salivary glands, the production of autoantibodies, and th e degree of salivary gland dysfunction. Interestingly, this reduction in clinical di sease severity correlated with significant reductions in the co-expressions of CD19/CD 21 on the B cell subpopulations. No major changes were noted in the CD22 expre ssion levels of CD19-positive B cell subpopulations (166). These initial findings suggested a direct correlation between C3 depletion, loss of CD19hi/CD21hi B cell subpopulations and reduced autoimmunity in CVF-treated NOD.B10H2b mice (166). However, the usage of CVF to deplete C3 might manifest other unexpected immunologica l consequences. The acti on of CVF on complement appeared to last approximately 2 weeks, due to the fact that the immune system reacts to the CVF by making antibodies against it, renderi ng CVF ineffective (153). It is unknown whether immune response that results in an tibodies against CVF has any consequences on the on-going autoimmune process. St udy has shown that autoimmunity can be prevented if the immunological response is deviated by exposing the immune system to stimulating antigen prior to the initiation of autoimmunity (167). In addition to the known immunological effect, the purity of CVF resolved by SDS-PAGE is estimated to be 95% as described in the manufacture product sheet. The effect of the undetermined portion of CVF (~5%) remains unknown. To eliminate any unforeseeable consequences of CVF, it is critical to examine th e role of C3 by genetically knocking out C3 gene in a newly described mouse referred to as C57BL6.NODAec1Aec2 or double congenic (DC/DC).

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54 In this study I have reassesse d the critical role of C3 in the pathogenesis of SjS by examining SjS-like disease in a C3 gene KO model predisposed to this autoimmune disease. In this chapter, the effect mani fested by C3 on the severity of autoimmune exocrinopathy and its impact on the developm ent of B lymphocytes is presented. Materials and Methods Generation of C57BL/6.NODAec1Aec2.C3-/Mouse Previously, we have generated a mo use strain designated as C57BL/6.NODAec1Aec2 or DC/DC that contained both the Idd3 and Idd5 chromosomal intervals from the NOD in the non-autoimmune C57BL/6 mice genetic background. This animal manifested all the phenotypes of human Sj ögren’s syndrome. To generate the C3 KO mice, C57BL/6.NODAec1Aec2 female mice were bred with B6.129S4C3tm1Crr/J male purchased from The Jackson Laboratory (Bar Ha rbor, ME). F1 generation offspring were interbred. F2 generation offspring were screened by genotyping for homozygosity for Aec1 locus, Aec2 locus, and C3 gene knockout. Additional mit markers were used during the genotyping process to select for non-crossover by the C57BL/6 background at the Aec1 and Aec2 loci of the NOD mice. All the animals used in this experiment were bred and maintained at the Animal Care Services at University of Florida. Proteolysis of Parotid Secretory Protein (PSP) Detection of PSP proteolysis was carried out by incubating whole saliva specimens with a synthesized oligopeptide correspondi ng to amino acids 20 through 34 of the published sequence for mouse PSP. This oli gopeptide contains the proteolytic site (NLNL) for a serine kinase present in salivary glands during development and onset of SjS-like disease in the NOD mouse (unpublished data). Eight µ l of saliva collected from individual mice were mixed with 42 µ l of the PSP oligopeptide (2.5mg/ml) and incubated

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55 at 42oC for 12 hrs. Following incubation, 50 µ l Tris-HCl buffer (50 mM, pH 8.0) was added and the mixture centri fuged through Micro-spin filt er tubes at 14,000 rpm for 10 min. The filtrates were analyzed by HP LC (Dionex Systems, Sunnyvale, CA) for proteolytic products. Contro l samples consisted of 50 µ l of the PSP oligopeptide. Detection of Cleaved Caspase3 in the Submandibular Glands Submandibular glands were surgically re moved at time of euthanasia (4-7 and 2427 weeks of age), placed in 10% phosphate-bu ffered formalin for 24 h, then embedded in paraffin and sectioned at 5 µm thickness. Pa raffin-embedded slides were de-paraffinized by immersing in xylene, followed by dehydrating in ethanol. The tissue sections were washed in PBS for 5 min, and then incubated 15 min at RT in Sniper blocking solution (Biocare Medical Cat# BT967H, Concord, CA). Each section was incubated with Rabbit anti-Cleaved Caspase-3 diluted at 1:4 00 (BioCare Medical #CP229B, Concord, CA) overnight at RT. The slides were washed three times with PBS for 5 min per washed followed by 30 min incubation at RT with Mach-2 goat anti-rabbit HRP polymer secondary antibody (BioCare Medical #RHRP5 20, Concord, CA). The slides were washed thoroughly with PBS, and then staine d for Cardassian DAB chromagen (Biocare Medical Cat# DBC859L10, Concord, CA). Rinsed in deionized water, and counterstained for methyl green (DakoCytomation Cat# S1962, Clostrup, Denmark). Stained sections were visualized at 200X magnification. For each whole submandibular gland of the animals, number of cleaved Caspase-3 positive cells was identified and counted Salivary Protein Concentration and Salivary Amylase Activity Total protein content was determined usi ng the Bradford method. Amylase activity in saliva was determined using the InfinityTM Liquid Amylase Kit (Thermo Trace

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56 Electron Corp. Waltham, MA) in which starch was the substrate. Saliva samples were diluted 250-fold with deionized water and added to 1 ml of the InfinityTM Amylase Liquid Stable Reagent. Following 1 min and 2 min incubators at 37oC, absorbance was measured at a wavelength of 405 nm. Amylas e activity was calculated according to the manufacture’s instructions using the formula: Amylase activity (U/L) = A/2 x 5140 x 400 (sample dilution). Histological Examination of Subm andibular and Lacrimal Glands The animals were euthanized at 5, 9, 13, 17, and 25 weeks of age. Submandibular and lacrimal glands were surgically rem oved from each mouse and placed in 10% phosphate buffered formalin for 24 hrs. Fi xed tissues were embedded in paraffin and sectioned at 5 µ m thickness. Paraffin-embedded s lides were de-paraffinized by immersing in xylene, followed by dehydrati ng in ethanol. The tissue sections were stained with H&E dye (Gainesville Service Te ch, Gainesville, FL). Stained sections were observed at 100X and 200X magnificati ons for glandular structure and leukocyte infiltration. Detection of Anti-Nuclear Autoantibody in Sera ANA in the sera of mice were detected using ANA screening kit (Immunoconcepts, USA). HEp-2 fixed substrate slides were overlaid with the appr opriate mouse serum diluted 1:40. Slides were incubated for 30 min at room temperature in a humidified chamber. After three washes for five minut es with PBS, the substrate slides were covered with FITC-conjugated goat anti-mous e IgG (Immunoconcepts) diluted 1:50 for 30 min at room temperature. After three wa shes, nuclear fluorescence was detected by fluorescence microscopy at 200X magnification.

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57 Detection of Immunoglobulin Specific Musc arinic Type III Receptor Autoantibody Anti-M3R antibodies in sera of C57BL/6.NODAec1Aec2 mice were determined as described in detail elsewhere (44). In brie f, Flp-In CHO cells transfected with mM3R were collected from growing cultures, wa shed once with phosphate-buffered saline (PBS), and resuspended in FACS buffer (PBS, 0.5% BSA, 0.07%NaN3). Aliquots of cells at a density of 1 x 106 cells/0.1 ml were incubated 2 hrs at 4°C with 10 l sera from individual mice or pooled from appropriate gr oups. Cells were washed once with FACS buffer, resuspended in 50 l of FACS buffer and incubated for 30 min at 4oC with either FITC-conjugated goat anti-mouse IgG1, IgG2b, IgG2c, IgG3, IgM (Southern Biotechnology Associates, Inc., Birmingham AL). After a final wash with FACS buffer, the cells were resuspended in FACS buffe r and analyzed using a FACScan cytometer equipped with Cell Quest software (Becton Di ckinson, Mountain View, CA). Control reactions included cells incubated with s econdary antibody alone or an appropriate isotype control. An increase in fluorescen ce intensity compared to secondary antibody alone was considered a positive reaction. Flow Cytometry for Subpopulations of B cells Spleens were freshly explanted from euth anized mice and gently minced through a steel sieve. Following a singl e wash with PBS, the red blood cells were lysed by a 7 min exposure to 0.84% NH4Cl. The resulting cell suspensions were washed two times in PBS, counted and resuspended in FACS buffer (PBS supplemented to 2% ABS and 0.01%NaN3) to 1x108cells/ml. Aliquots of each cell preparation containing 1 x 105 cells were incubated 45 min with either R-PE -conjugated rat anti-mouse CD19 monoclonal antibody (#557399), FITC-conjugated rat anti-mouse CD19 m onoclonal antibody

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58 (#557398), FITC-conjugated rat anti-mous e CD21 monoclonal antibody (#553818), PEconjugated mouse anti-mouse CD22.2 monoclonal antibody (#5533384), or PEconjugated goat anti-mouse CD23 (#X) (BD Bi osciences – Pharmingen, San Diego, CA), washed in FACS buffer, then analyzed for fluorescence staining on a FACScan (BD Biosciences, San Jose, CA). Measurement of Stimulated Saliva Secretion To measure stimulated flow rates of saliva, individual mice were weighed and given an intraperitoneal (ip) injection of a cocktail containi ng isopreterenol (0.2 mg / 100 gm body weight) and pilocarpine (0.05 mg / 100 gm body weight) dissolved in PBS. Saliva was collected for 10 min from the oral cavity of individual mice using a micropipette starting one min after the injection of the secretagogue. The volume of saliva sample was measured. The saliva samples were then frozen at -80°C until analyzed. Statistical Analysis Differences in cleaved Caspase-3, stimula ted saliva volume, amylase activity, or salivary protein concentration were analy zed with the Student-Newman-Keuls test. p<0.05 was considered significant. Results Profiling of Phase I of SjS-like Auto immune Exocrinopathy of C57BL/6.NODAec1Aec2.C3-/Mice The salivary glands include the submandibular gland, sublingual gl and and the parotid gland as well as numerous minor salivary gla nds. It is innervat ed by both sympathetic and parasympathetic nerves. The parasympathetic nerves utilize the muscarinic type III receptor (M3R)to regulate the fluid secretion in the salivary gland while the sympathetic

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59 nerves use the -adrenergic receptor to modulate the prot ein secretion (17). In SjS, the pathway which regulates fluid secretion is t hought to be dysfunctiona l due to the activity of the M3R autoantibody binding to its receptor ( 10). In addition, acinar cells made up of mucous cells (release mucins) and serous cells (secrete kallikrein proteins, enzymes such as amylase, peroxidases, lysozyme, lactoferri n, cystatins, and histatins) are sporadically destroyed (59). Therefore, Ph ase I of this autoimmune res ponse is often characterized by the loss of fluid secretion resu lting in an elevated concentr ation of salivary proteins. Because of the sporadic cellular destructi on, this phase is mainly accompanied by an increase in activity of Caspase-3 and expr ession of cysteine pr otease that cleaves PSP. For the past few decades, tremendous amounts of research devoted to delineate the progression of SjS using animal models. It proposed that SjS-lik e autoimmune disease progresses through three different but slightly overlapped phases. At Phase I from 4-8 weeks of age, the animals undergo delay in organogenesis allowing the delay expression of acinar cell antigens, and consequently, the lack of clonal deletion of autoreactive lymphocytes escaping into the periphery. In addition, the constant remodeling of the cellular physiology results in e xpression of aberrant or inco rrect proteins or enzymes, leading the loss of cellular homeostasis at Ph ase I of the disease. Furthermore, the cellular dynamic of th e animal is accompanied by the in itiation of acinar cell apoptosis resulting in the loss of glandular mass (59). As presented in chapter 2, depletion of C3 did not change the intrinsic properties of the acinar cells of the animal which are main ly regulated by the genetic elements of the animal. It further supported the observation that depletion of C3 did not change the ability of the cysteine protease to cleave pa rotid secretory protein (PSP). Consistently,

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60 this study has shown that knocking out C3 also did not affect the activity of cysteine protease in degrading PSP in saliva samples (Figure 3-1). As expected, PSP cleavage occurred as early as 5 weeks of age in the disease control animals, C57BL/6.NODAec1.Aec2 , predisposed to SjS-like disease. concurrent with the aberrant expression of PSP cleavage pro duct at Phase I, apoptosis can also be observed at this phase. Apoptosis is the mechanism in which cells are programmed to undergo cell deat h in response to detrimenta l trigger. Caspases are a family of proteases known to mediate program med cell death. Apoptosis of exocrine cells has been suggesting to be critical duri ng the early phase of the development of autoimmune exocrinopathy (60). The contro lled cell death is thought to be involved in exposing and releasing of cryp tic antigens that the immune system has never recognized previously. As the result of recognizing newly exposed antige ns, when sufficient level of antigens are being presented, the immune system reacts by possibly recruiting inflammatory cells to the site of injury such as the exocrine glands in SjS. One of the ways to identify apoptosis is by using e xpression of cleaved caspase-3 to detect endogenous levels of the large fragment (1 7/19 kDa) of activated caspase-3. As anticipated, C57BL/6 animals serving as disease-free norma l control mice showed little positivity for cleaved caspase-3 in the submandibular glands from 4-7 to 24-27 weeks of age, while C57BL/6.NODAec1.Aec2 mice with SjS exhibited a significant increase of approximately five-fold in the number of cleaved caspase-3 positive cells in the submandibular glands at 4-7 weeks of ag e and decreased to a normal level at 24-27 weeks of age. Inte restingly, C57BL/6.NODAec1Aec2.C3-/mice displayed a similar level of apoptosis or a similar number of cleaved caspase-3 positive cells to C57BL/6

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61 disease-free control mice, and a five-f old decrease compared to C57BL/6.NODAec1.Aec2 at 4-7 weeks of age. However, at 24-27 weeks of age, C57BL/6.NODAec1Aec2.C3-/mice showed no significant increase in cleaved caspase-3 compared to C57BL/6 and C57BL/6.NOD-Aec1Aec2 mice (Figures 3-2a, 3-2b). Profiling of Phase II of SjS-like Auto immune Exocrinopathy of C57BL/6.NODAec1.Aec2.C3-/Mice In response to the pathophysiological abnor mality that occurred during Phase I of SjS-like autoimmune exocrinopathy, the anim al initiated an immunological reaction to changes which signify Phase II of the dis ease. The immunological response includes leukocyte infiltrations in submandibular and la crimal glands that may play an important role of glandular destruction by occupyi ng the space that once resided by acinar and ductal cells of the glands. In addition, the Phase II of SjS is characterized by presence of autoantibodies produced by autoreactive B ly mphocytes that escaped developmental selection (61). Many studies have now indicat ed that the clinical manifestation of SjS which are involved in the d ecline of salivary flow rates, is possibly mediated by autoantibody against M3R (56, 65, 150). Other autoantibodies such as the presence of ANAs, anti-Ro and anti-La, found in sera of human patients and SjS animal models, indicate the advancement of SjS to a clinical phase invol ving secretory dysfunction (121). To examine the leukocytic infiltrations in the exocrine glands, mice were euthanized at 5, 9, 13, 17, and 25-27 weeks of age. The submandibular and lacrimal glands were explanted for histological examination by routin e H&E staining. Interestingly, meticulous examination of the submandibular and lacrimal glands revealed the complete absence of lymphocytic infiltrations in the C57BL/6.NODAec1Aec2.C3-/mice. As expected, there was a tremendous num ber of infiltrates in the exocrine glands

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62 including the submandibular and lacrimal glands of the diseased control mice in C57BL/6.NODAec1Aec2 while the C57BL/6 normal control mice exhibited no lymphocytic infiltrations (Figure 3-3). Concurrently, sera of the mice were collect ed prior to their euthanization. The expression level of ANAs was performed using collected mice sera. Sera were incubated on the Hep2 cells and visualized by staini ng with goat anti-mouse whole IgG-FITC conjugated secondary antibodies. Expression of ANAs can be detected as early as 13 weeks of age in males and females of C57BL/6.NODAec1Aec2 mice with higher frequency and intensity at ol der ages suggesting the adva ncement of the autoimmune disease process. Interestingly, ANAs were not seen at a 1:40 d ilution in C57BL/6.NODAec1Aec2.C3-/mice even at 27 weeks of age in both male and female animals. C57BL/6 serving as normal control showed the absence of ANAs level when aged to 27 weeks old (Figure 3-4). One of the most critical autoantibodies be lieved to be involved in the shutdown of salivary flow rate is anti-M3R. Previous studies using the NOD.IL4-/and NOD.B10. H2b.IL4-/animals indicated that IgG1 specifi c anti-M3R is the most important effector autoantibody that is required for the secretory dysfunction of submandibular glands. In this study, to determine the pres ence of anti-M3R, sera were incubated with CHO cells expressing M3R on their surface. Se ra that are positive for M3R will bind to the cells and fluorescent at different intensity depending on the antibody titers using isotypic fluorochrome conjugated secondary an tibodies by flow cytometry. As shown in Figure 3-5, pooled sera from C57BL/6.NODAec1Aec2 mice were exhibited positivity for IgG1-M3R, IgG2b-M3R, and IgG2c-M3R autoantibodies, while C57BL/6.NOD-

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63 Aec1Aec2.C3-/mice pooled sera failed to produce any IgG1-M3R and IgG2b-M3R autoantibodies, or showed a decreasing level of IgG2c-M3R compared to the wild type mice. Pooled sera from all the mice were positive for IgM-M3R, and were negative or express at the same level for IgD-M3R a nd IgA-M3R respectively (Figure 3-6). Profiling of Phase III of SjS-like Auto immune Exocrinopathy of C57BL/6.NODAec1Aec2.C3-/Mice Phase III of SjS-like autoimmune exoc rinopathy is characterized by glandular secretory dysfunction exemplified by the loss of amylase activity due to the destruction of the acinar cells and temporal decrease in stimulated salivary flow rates concomitant with the appearance of leukoc yte infiltrates within the e xocrine glands occurred during Phase II of the disease. To measure change s in secreted saliva volume, each mouse was weighed and injected with a secretogue contai ning both isopreterenol and pilocarpine. Stimulated saliva secretion was collected and measured. Analysis was performed separately for male and female animals due to the gender dichotom y existing between the two sexes in terms of the severity of the auto immune process. SjS-like diseased controls male and female C57BL/6-NOD. Aec1Aec2 mice displayed a 30% loss of stimulated saliva volume being secreted from 4 to 25 weeks of age. Interestingly, male and female C57BL/6-NOD. Aec1Aec2.C3-/mice exhibited no loss of stimulated saliva volume over time similar to the non-diseased C57BL/6 control mice. C57BL/6-NOD. Aec1Aec2.C3-/male and female mice actually showed a sign ificant increase in secreted saliva volume from 4 to 25 weeks of age (Figures 3-7 and 3-8). Alpha-amylase activity was measured by th e ability to degrade the substrate, etylidenepNP-G7 using whole saliva samp les collected from C57BL/6, C57BL/6.NODAec1Aec2 , and C57BL/6.NODAec1Aec2.C3-/mice at 4, 8, 12, 16, and 24-27 weeks of

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64 age. As shown Figure 3-9, C57BL/6 mice serv ing as normal control exhibited no loss in -amylase activity from 4 to 24-27 weeks of age, while the SjS-like diseased control animals, C57BL/6.NODAec1Aec2 , exhibited significant decrease in -amylase activity over the same period of time, indicating the loss of acinar cell function in the salivary gland. Interestingly, C57BL/6.NODAec1Aec2.C3-/mice with no functional C3 gene showed a slight increase in -amylase activity from 4 to 24-27 weeks of age. No loss of -amylase activity of C57BL/6.NODAec1Aec2.C3-/mice could be directly contributed by down-regulation of cellular apoptosis, or possibly is the result of regeneration of acinar cells in the absence of C3. Normal -amylase activity over time found in C57BL/6.NODAec1Aec2.C3-/mice is also accompanied by constant concentration of salivary proteins while the disease control anim al exhibited an elevat ed level of salivary protein concentration over time (Figure 3-10). Characterization of the changing dynamics in the subpopulation of B lymphocytes: Marginal Zone (MZ) and Follicular (FO) B Cells The clinical manifestation of SjS is t hought be highly dependen t on the activity of autoantibodies produced by B lymphocytes. Mo st evidence supports th e notion that these autoantibodies must be produced by autore active B lymphocytes(90). However, it remains speculative which subpopulation of B ce lls contributes more significantly to the autoimmune process. Adding to this complex ity, sex hormones play a critical role in the changing dynamic of different B cell populations (168). Therefore, it is essential that analysis of these B cell populations must be determined based on the difference in male and female sexes of the animals. Splenocyt es collected from male and female of C57BL/6, C57BL/6.NODAec1Aec2 , and C57BL/6.NODAec1Aec2.C3-/mice were analyzed by flow cytometry. Separation of MZ and FO B cells is based on the

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65 fluorescent intensity of CD21 and CD23 ma rkers. MZ B cells are CD23+CD21hi while FO B cells are CD23+CD21int. Figure 3-11 presents a representative finding for male C57BL/6, C57BL/6.NODAec1Aec2 , and C57BL/6.NODAec1Aec2.C3-/experimental models, and similar analysis were done for female from each of the three experimental mouse models (data not shown). Data for both sexes are compared in Table 3.1. As presented in Table 3.1, comparing th e male among the three different mouse models, C57BL/6, C57BL/6.NODAec1Aec2 , and C57BL/6.NODAec1Aec2.C3-/-, of the CD19+ splenic cells, the percen t of MZ B cells found in the spleens of all three models appeared to be very similar, whil e both male C57BL/6 and C57BL/6.NODAec1Aec2 mice have similar percentage of splenic FO B cells at 61.70% and 64.63% respectively. However, there is a reduction of approximately 9% of FO B cells found in the spleen of male C57BL/6.NODAec1Aec2.C3-/mice compare to C57BL/6.NODAec1Aec2 male mice. In the three female models, the numbe r of MZ and FO B cells found to be very interesting. Of the female mouse CD19+ sp lenic B cells, the per cent of splenic MZ B cells appeared to be at 7.33, but in crease up to 13.0 % in the C57BL/6.NODAec1Aec2 female mouse. However, only 7.3% of CD19+ MZ B cells are found in C57BL/6.NODAec1Aec2.C3-/mice which is similar to female C 57BL/6 mice, possibly indicating the decrease in hyperproliferation of the MZ B ce lls in C3KO female mice and returning it to a normal number. Interestingly, the increase in the percentage of female splenic CD19+ MZ B cells in the C57BL/6.NODAec1Aec2 mice is accompanied by the decrease in the percentage of splenic CD19+ FO B cells which found to be at 57.94%, while there are 65.60% and 66.70% of splenic CD19+ FO B cells are found in C57BL/6 and

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66 C57BL/6.NODAec1Aec2.C3-/mice respectively. Therefor e, changing dynamic in the populations of MZ and FO B cells is appeared to regulated and driven by both C3 and the sex differences among the three animal models. Discussion In the present studies, I have exte nded our understanding on the role of complement during the development of auto immune exocrinopathy by using an animal model in which C3 gene was knocked out in the anim al model predispos ed to SjS-like disease, referred to as C57BL/6-NOD. Aec1Aec2 . Results from the current study have clearly indicated the critical requirement of C3 for the full initiation of the SjS. By eliminating C3 gene using speed congenic breeding in the C57BL/6.NODAec1Aec2 animals, the clinical manifestations of SjSlike disease were diminished. These included significant reduction of cellular apoptosis, the absence of ly mphocytic foci within the exocrine glands, lack of ANAs production, and elimination of IgG1-M3R autoantibody with decreasing level of ot her IgG isotypic M3R autoanti bodies. Most importantly, C57BL/6-NOD. Aec1Aec2.C3-/mice restored normal saliva secretion in both female and male animals. In addition, a lterations of splenic B cell subpo pulations were identified. In the three male models, C57BL/6, C57BL/6.NODAec1Aec2 , and C57BL/6.NODAec1Aec2.C3-/, of the CD19+ splenic cells, the pe rcent of MZ B cells is similar among the three models while splenic CD 19+ FO B cells of the C57BL/6.NODAec1Aec2.C3-/mouse showed decline of close to 9% compared to C57BL/6 and C57BL/6.NODAec1Aec2 mice. However, in the three female models , the number of splenic CD19+ MZ and FO B cells appeared to be sim ilar between the C57BL/6 and C57BL/6.NODAec1Aec2.C3-/mice while there is an increase of 8% in the percent of MZ B cells and decrease of 9% in the percent of FO B cells found in C57BL/6.NODAec1Aec2 mice

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67 compare to C57BL/6 and C57BL/6.NODAec1Aec2.C3-/-. The trends in the alterations in various B cells populations among the three st rains of mice were very interesting. However, because of the small number of e xperimental animals in each strain, the final output results performed by St udent-Newman-Keuls test appeared to be statistically insignificant The previous study has suggested an importa nt role of C3 during the development of SjS in the NOD.B10. H2b, the animal model of primary SjS (166). Data presented in chapter 2 indicated that when the level of serum C3 was temporally reduced by intraperitoneally injec ting 10-weeks old NOD.B10. H2b mice with CVF, the mice showed reduced severity of lymphocytic infiltrati ons in the submandibular glands, decreased production of ANAs and retention of normal sa livary flow rates. Therefore, the SjS phenotypes were greatly improved when C3 was depleted in serum at 10 weeks old animals. However, the side effect of CVF on the biological and im munological reactions on administered animals has never been dete rmined and its role on autoimmune process is unknown. Furthermore, the im purity of the C3 preparation raised questions about its mechanism of action. Therefore, in this st udy, we focused our attention in investigating the role C3 has on the pathogenesis of SjS by using genetic knockout out of the C3 gene in a mouse model that is predisposed to SjS-like autoimmune disease. SjS is one of the connective tissue autoimmune diseases in which the membrane attack complex (MAC) is not involved in the cellular destruction in animal models and human patients (169). In this context, NOD mice an d NOD congenic strains are consistent with that conclusion because they ar e deficient in C5 but still manifest the full phenotypes of SjS. Saliva and salivary glands of SjS patient s were found to contain or

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68 express elevated levels of complement regulato ry proteins compared to healthy subjects. These complement regulatory proteins in clude protectin (CD59), decay accelerating factor (CD55), membrane cofactor protei n (CD46) and clusterin (SP-40, 40), which function to prevent complement mediated dest ructions on the exocrine glands in patients (169). Therefore, it is possible to eliminate the possibility of complement mediated lysis of acinar tissues in the glands. In this st udy, the complete absence of leukocyte infiltrate in the exocrine glands in the C57BL/6-NOD. Aec1Aec2.C3-/mice was quite striking because of the fact that C5 is still active in these animals. One of the cleavage product of C5, C5a, is the most potent inflammatory mediat or of the complement system (114). It is clear that C5 is not required for the initiati on of infiltrations into the injured tissues. Additionally, C5a directly regulates the functi ons of PMNs by increasing their adherence to vessels walls and their migra tion to site of tissue destruct ion (114). Interestingly, the presence of PMNs has never been detected in salivary glands of human patients and animals, but was found to be circulating in se ra of patients with pS jS (170, 171). These studies suggest the irrelevant role of PMNs in the development of SjS in term of occupying the exocrine gland as part of leuko cytic focus to carry out their destructive function, but they reinforced the biological requirement an d importance of C3 in SjS autoimmune process. Based on our previous study, the importance of C3 in adaptive immunity of SjS must be addressed. SjS appear s to be a lymphoproliferative B cell disorder in which the clinical phase of disease fa ils to develop in the abse nce of either B cells or autoantibodies. B lymphocytes in SjS are believ ed to be hyper-prolif erative, capable of evading apoptosis, and overly sensitive to activation and maturation. C3 plays an

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69 important role in the adaptive immunity by controlling activ ation, survival and proliferation of B cells by cross-linking B cell receptors and their co-receptors CD19, CD21 and CD22. Cross-linking of BCRs and B cell co-receptors by a cleavage product of C3, C3b, can modulate the strength, intensity and duration of the signal generated by BCR (113). Signals generated by the co-ligation of CD19/CD21 and BCR by the C3d fragment act as positive regul ators of B cell activation, resu lting in the lowering of the threshold for B cell activation, possibly cont ributing to the hyper-pro liferative and hyperactive properties of autoreactive B cells (165). Activation of B lymphocytes requires s econdary signals that mediated by C3d cross-linking BCRs with the co-receptors CD19/CD21 molecules. In addition, C3 products can bind to or form complexes with antigens to facilitate inflammatory and immunological responses, in part through binding to specific complement receptors, such as CR1/CR2 present on FDCs. Such localiz ation of antigen on FDCs in secondary lymphoid tissues promotes germinal center formation, B cell retention, survival and activation within germinal centers, as well as subsequent antibody formation (158, 159). Therefore, elimination of C3 could prevent formation of functional germinal center in secondary lymphoid tissues of the animals and formation of ectopic germinal centers-like foci, often found in the exocrine glands of human and SjS-like animal models. The role of complement is well establis hed in the etiology of other autoimmune diseases such as SLE and rheumatoid arthriti s. However its role in the pathogenesis of SjS has remained elusive. Early studies ha ve shown a correlation in the deposition of immune of complex in the form of IgA or IgM with the clinical manifestations of SjS (172). As mentioned previously, Cuida et al. (169) has demonstr ated the presence of

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70 complement regulatory proteins in saliva and salivary glands of SjS patients serving to prevent activation of complement on the tissues . Recent reports have demonstrated that hypocomplementaemia (specifically, reduced levels of C3 and/or C4) is closely associated with B cell lymphoma development in SjS and increased pathogenicity (164). Since all of the above studies using SjS patients were examin ed only at Phase III or the clinical phase of the disease in which comp lement activation may not be relevant or required to the development of Sj S at this stage. Each patien t behaved slightly differently, which may explain the inconsistency in the inte rpretation of the data collected. With our animal models, we were able to show that complement is required at Phase II of the disease, not for the complement mediated ly sis destruction but instead for activation, proliferation, and survival of B cells. The low level of C3 f ound in sera of patients in the study previous mentioned may be due to lack of circulating C3 in the periphery, but it is probably the result of B cells taken up a ll the circulating C3 molecules for their hyperactivity and hyperproliferation. This study reinforces the importance of C3 in the pathogenesis of SjS. Its implication in translational research may pr ove to be very effective and beneficial. Further data is required to dete rmine if C3d has any preferences of binding to self or nonself antigens, and if reversal of disease can occur once C3 is infused back into the C3KO mice.

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71 Figure 3-1.Proteolysis of paroti d secretory protein. A. PSP pe ptide alone. B. 24 wks old C57B/6 (n=2). C. 24 wks old C57BL/6-NOD. Aec1.Aec2 (n=2). D. 24 wks old C57BL/6-NOD. Aec1.Aec2.C3-/(n=5). All indi vidual figures are representative.

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72 Figure 3-2.Examination of apoptosis by the presence of cleaved Capase-3 in the submandibular glands. (a) Immunohist ochemistry staining for Caspase-3. A. 4-7 weeks old C57BL/6 (n=4). B. 24-27 weeks C57BL/6. (n=4). C. 4-7 weeks old DC.DC (C57BL/6-NOD. Aec1Aec2 ). (n=4). D. 24-27 weeks old DC.DC. (n=4). E. 4-7 weeks old DC.DC.C3-/(C57BL/6NOD. Aec1Aec2.C3-/-). (n=2). F. 24-27 weeks old DC.DC.C3-/-. (n=2) All individual figures are re presentative. (b) Number of apoptotic cells or Caspase-3 positive cells in the submandibular glands determined by counting total number of cells stained positiv e for cleaved Caspase-3 per whole histological gland from 4-7 weeks old to 24-27 weeks old mice. Statistical analysis was performed by St udent-Newman-Keuls test. p<0.05 was considered significant.

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73 Figure 3-3. Histological examination of the exoc rine glands. Submandibular and lacrimal glands were removed from each mouse approximately 24-27 weeks of age and fixed in 10% formalin, were stained w ith MayerÂ’s H&E dye. Stained section of C57BL/6, lacrimal glands (A) subma ndibular glands (B). Stained section of C57BL/6-NOD. Aec1Aec2 , lacrimal glands (C) submandibular glands (D). Stainined section of C57BL/6-NOD. Aec1Aec2.C3-/-, lacrimal glands (E) submandibular glands (F). All indi vidual figures are representative

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74 Figure 3-4. Detection of antinuclear autoantibodies (ANAs) us ing Hep2 cells as subtrate. 24 weeks old C57BL/6 serum as nega tive control (A). 24 weeks old C57BL/6-NOD. Aec1Aec2 serum as postive control (B). 24 weeks old C57BL/6-NOD. Aec1Aec2.C3-/serum (C). All individual figures are representative

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75 Figure 3-5. Detection of anti-muscarinic acetylcholine type-3 receptor antibodies. Pooled sera collected from 27 wks old C57BL/6 (n=5), 30 wks old C57BL/6NOD. Aec1Aec2 (n=5), and 24 wks old C57BL/6-NOD. Aec1Aec2.C3-/(n=5) mice. Sera were incubated 2 hrs at 4° C with either mouse-M3R transfected Flp-In CHO cells or respective isotype controls. Cells were washed with FACS buffer, resuspended in 50 l of FACS buffer and incubated for 30 min at 4oC with either FITC-conjugated goat anti-mouse IgG1, anti-mouse IgG2b, anti-mouse IgG2c or anti-mouse IgG3 an tibodies. After a final wash with FACS buffer, the cells were resuspended in FACS buffer and analyzed using a FACScan cytometer.

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76 Figure 3-6. Detection of anti-muscarinic acetylcholine type-3 receptor antibodies. Pooled sera collected from 27 wks old C57BL/6 (n=5), 30 wks old C57BL/6NOD. Aec1Aec2 (n=5), and 24 wks old C57BL/6-NOD. Aec1Aec2.C3-/(n=5) mice. Sera were incubated 2 hrs at 4° C with either mouse-M3R transfected Flp-In CHO cells or respective isotype controls. Cells were washed with FACS buffer, resuspended in 50 l of FACS buffer and incubated for 30 min at 4oC with either FITC-conjugated goat an ti-mouse IgM, anti-mouse IgD, or anti-mouse IgA antibodies. After a fina l wash with FACS buffer, the cells were resuspended in FACS buffer and analyzed using a FACScan cytometer.

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77 Figure 3-7. Stimulated saliva flow of female animals. C57BL/6 (4 weeks, n=3, 25 weeks, n=3), DC.DC (4 weeks, n=5, 25 weeks, n=5), and DC.DC.C3-/(4 weeks, n=9, 25 weeks, n=6) were given an i.p. inj ection of isoproterenol and pilocarpine and collected for 10 min from the oral cavity of individual mice using a micropipette starting 1 min after the inj ection of the secretagogue. The volume of saliva samples was measured. Sta tistical analysis was performed by Student-Newman-Keuls test. **: p<0.01 , ***: p<0.001 .

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78 Figure 3-8. Stimulated saliva flow of male an imals. C57BL/6 (4 weeks, n=4, 25 weeks, n=4), DC.DC (4 weeks, n=8, 25 weeks, n=10), and DC.DC.C3-/(4 weeks, n=6, 25 weeks, n=9) were given an i.p. injection of isoproterenol and pilocarpine and collected for 10 min from the oral cavity of individual mice using a micropipette starting 1 min after the injection of the secretagogue. The volume of saliva samples was measured. Statistical analysis was performed by Student-Newman-Keuls test. *: p<0.05 , ***: p<0.001 .

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79 Figure 3-9. Amylase activity in saliva. Saliva of C57BL/6 {4 wks old (n=7), 24-27 wks old (n=7)}, DC/DC {4 wks old (n =13), 24-27 wks old (n=11)}, and DC/DC.C3-/{4 wks old (n=10), 24-27 wks old (n=14)}were collected. Amylase activity in saliva was determined using the InfinityTM Liquid Amylase Kit. Saliva samples were diluted and added to InfinityTM Amylase Liquid Stable Reagent. Absorbance was measured at a wavelength of 405 nm after incubating at 37oC. Statistical analysis was performed by StudentNewman-Keuls test. *: p<0.05

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80 Figure 3-10.Salivary protein concentration. Sa liva of C57BL/6 {8 wks old (n=7), 24-27 wks old (n=7)}, DC/DC {8 wks old (n=8), 24-27 wks old (n=8)}, and DC/DC.C3-/{8 wks old (n=8), 24-27 wks old (n=8)} were collected. Total salivary protein content was determ ined using the Bradford method. Statistical analysis was performe d by Student-Newman-Keuls test.

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81 Figure 3-11.An representation of an approach to delineate marginal zone (MZ) and follicular (FO) B lymphocytes in male animals based on CD21 and CD23 markers. A. Male C57BL/6. B. Male C57BL/6-NOD. Aec1Aec. C. C57BL/6-NOD. Aec1Aec2.C3-/

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82 Table 3-1.Changes in CD19+ splenic B cell populations

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83 CHAPTER 4 INVOLVEMENT OF STAT6 IN THE IL4 SIGNALING PATHWAY DURING THE CLINICAL PHASE OF SJOGREN-LIKE SYNDROME Introduction Although the underlying cause of Sjgren’s syndrome (SjS) remains elusive, a number of studies using the NOD mouse mode l and numerous congenic strains have led us to propose the concept that this auto immune exocrinopathy progresses in three consecutive phases (61, 149). In phase 1, a number of aberrant genetic, physiological and biochemical activities asso ciated with retarded saliva ry gland organogenesis occur sequentially prior to and independent of initia tion of an autoimmune attack. In phase 2, leukocytes infiltrate the exocrine glands with a concomitant increase in the expression of inflammatory cytokines. In phase 3, secret ory dysfunction of the salivary and lacrimal glands occurs, most likely the result of production of anti-muscarinic a cetylcholine type-3 receptor (M3R) autoantibodies (4, 65). An in terruption within any one of these three phases prevents onset of c linical SjS-like disease. SjS is an autoimmune disease in whic h B cells and autoantibodies play an important role in the glandul ar dysfunction (66). Hyperpro liferation and hyperactivity of autoreactive B lymphocytes result in severe hypergammaglobulinemia that is often found in patients with SjS and NOD mice (53, 87). In addition, SjS patients, as well as NOD mice and some of its congenic partners, deve lop specific autoantibod ies against nuclear antigens, intracellular components, membrane pr oteins and secreted products of exocrine tissues (27, 30, 32, 121, 173). Approximately 40-70% of SjS patients’ sera contain

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84 autoantibodies that are reactiv e to SS-A/Ro and SS-B/La antigens. These two specific anti-nuclear autoantibodies have been used as diagnostic markers of SjS disease (18). However, recent reports have focused consid erable attention on an ti-M3R. Preliminary studies suggest that anti-M3R autoantibodies may be pr esent in 80-100% of sera from both SjS patients and NOD mice ( 44, 65) and may be an important effector of glandular dysfunction by blocking normal signal transduc tion pathways possibly leading to the internalization of the antibody -receptor complex or desensitiz ing acinar cells to normal neural stimulations (38, 39, 138). This c oncept is supported, in part, by study showing that the IgG fractions of se ra obtained from SjS patients or NOD mice with disease can suppress stimulated salivary flow rates when infused into healthy, normal mice (66). The possibility that anti-M3 R autoantibodies may be involved in development of xerostomia and xerophthalmia in SjS has led us to examine this issue in greater detail. Preliminary studies utilizing a number of congenic partner strains of NOD and NOD.B10H2b with non-functional cytokine genes revealed that the IL-4 gene knockout (KO) mice, NOD.IL4-/and NOD.B10H2b.IL4-/-, failed to develop salivary gland dysfunction despite severe leuko cytic infiltration of the sa livary glands, accompanied by detectable increases in the expr ession of pro-inflammatory cy tokines. Interestingly, both NOD.IL4-/and NOD.B10H2b.IL4-/mice failed to produce M3R autoantibodies of the IgG1 isotype (56, 58). IL-4 is a pleiotropi c cytokine involved in cell proliferation, activation and differentiati on. Function of this Th2 cytokine is known to be involved in two different pathways, the IL4-IRS (Insulin Receptor Substrate) pa thway responsible for the cellular proliferation and activation wh ile the IL4-STAT6 (Signal Transducers and Activators of Transcription) pathway involved in isotype switching to IgG1 and IgE by

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85 specifically stimulating germline 1 and immunoglobulin gene transcription (174). Even though IL-4 acting thr ough the IRS pathway might be critical in activation and proliferation that results in the survival and expansion of autoimmune T and/or B cells, recent studies have pointed to the requirem ent of IgG1 isotypic autoantibodies against M3R in the secretory dysfunction in NOD mice (56, 58). In support of this concept, adoptive transfer of T cells capa ble of producing IL4 into NOD.B10H2b.IL4-/mice incapable of producing IgG1/IgE resulted in shutdown of secretory function due to of IgG1-M3R autoantibody (58). In the present study, I have investigated th e biological roles of IL-4-STAT6 pathway; specifically ex amining the role isotypi c switching plays in the development and onset of clinical SjS-like disease. Material and Methods Animals The following animals were used in this study: NOD.B10. H2b, C.129S2Stat6 tm1Gru/J, NOD.B10H2b.CStat6+/+, NOD.B10H2b.CStat6+/, NOD.B10H2b.CStat6-/and Balb/C. All the mice were bred a nd maintained under specific pathogen-free conditions within the mouse facility of the Departme nt of Pathology, Immunology & Laboratory Medicine at Univer sity of Florida, Gainesvill e, FL. Breeder pairs of NOD.B10H2b and C.129S2Stat6tm1Gru/J mice were purchased from the Jackson Laboratories (Bar Harbor, ME). All mi ce received water and food ad libitum. Histology Submandibular glands were surgically removed from each mouse at time of euthanasia (24 weeks of age), and placed in 10% phosphate buffered formalin for 24 hrs. Fixed tissues were embedded in paraffin and sectioned at 5 µ m thickness. Paraffinembedded slides were de-paraffinized by im mersing in xylene, followed by dehydrating

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86 in ethanol. The tissue sections were stai ned with H&E dye (Gaine sville Service Tech, Gainesville, FL). Stained sections were observed at 100X magnification for glandular structure and leukocyte infiltration. Immunofluorescent Staining for B and T Lymphocytes Paraffin-embedded tissues of the subma ndibular glands were sectioned and mounted onto microscope slides. Slides we re de-paraffinized by immersing in xylene, then dehydrated in ethanol. Following a 5 mi nute wash with PBS at 25°C, the sections were incubated 1 hr with blocking solution containing normal rabbit serum diluted 1:50 in PBS. Each section was incubated w ith rat anti-mouse B220 (BD BiosciencesPharmagen, San Diego, CA) diluted 1:10 a nd goat anti-mouse CD3 (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:50 for 1 hr at 25°C. The slides were washed 3X with PBS for 5 min per wash followe d by a 1 hr incubation with Texas Redconjugated rabbit anti-rat IgG (Biomeda, Fo ster City, CA) diluted 1:25 and FITCconjugated rabbit anti-goat IgG (Sigma Chemical s, St. Louis, MO) diluted 1:100 at 25°C. The slides were washed thoroughly with PB S, treated with Vectashield DAPI-mounting medium (Vector Laboratory, Burlingame, CA ) and overlayed with glass coverslips. Stained sections were visua lized at 200X magnification. Flow Cytometry Spleens were freshly explanted from euth anized mice and gently minced through a steel sieve. Following a singl e wash with PBS, the red blood cells were lysed by a 7 min exposure to 0.84% NH4Cl. The resulting cell suspensions were washed two times in PBS, counted and resuspended in FACS buffer (PBS supplemented to 2% ABS and 0.01%NaN3) to 1 x 108 cells/ml. Aliquots of each ce ll preparation containing 1 x 105 cells were incubated 45 min with eith er R-PE-conjugated rat anti-mouse CD19

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87 monoclonal antibody (#557399), FITC-conjuga ted rat anti-mouse CD19 monoclonal antibody (#557398), -conjugated rat antimouse CD21 monoclonal antibody (#553818) orRPE-conjugated mouse anti-mous e CD22.2 monoclonal antibody (#5533384) (BD Biosciences – Pharmingen, San Diego, CA), wash ed in FACS buffer, then analyzed for fluorescence staining on a FACScan (B D Biosciences, San Jose, CA). Proteolysis of Parotid Secretory Protein Detection of PSP proteolysis was carried out by incubating whole saliva specimens with a synthesized oligopeptide correspondi ng to amino acids 20 through 34 of the published sequence for mouse PSP. This oli gopeptide contains the proteolytic site (NLNL) for a serine kinase present in salivary glands during development and onset of SjS-like disease in the NOD mouse (unpublished data). Eight µ l of saliva collected from individual mice were mixed with 42 µ l of the PSP oligopeptide (2.5mg/ml) and incubated at 42oC for 12 hrs. Following incubation, 50 µ l Tris-HCl buffer (50 mM, pH 8.0) was added and the mixture centri fuged through Micro-spin filt er tubes at 14,000 rpm for 10 min. The filtrates were analyzed by HP LC (Dionex Systems, Sunnyvale, CA) for proteolytic products. Contro l samples consisted of 50 µ l of the PSP oligopeptide. Detection of Anti-Nuclear Au toantibodies in the Sera ANA in the sera of mice were detected using ANA screening kit (Immunoconcepts, USA). HEp-2 fixed substrate slides were overlaid with the appr opriate mouse serum diluted 1:40. Slides were incubated for 30 min at room temperature in a humidified chamber. After three washes for five minut es with PBS, the substrate slides were covered with FITC-conjugated goat anti-mous e IgG (Immunoconcepts) diluted 1:50 for 30 min at room temperature. After three wa shes, nuclear fluorescence was detected by fluorescence microscopy at 400X magnification.

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88 Measurement of Salivary Flow Rates To measure stimulated flow rates of saliva, individual mice were weighed and given an intraperitoneal (ip) injection of a cocktail containi ng isopreterenol (0.2 mg / 100 gm body weight) and pilocarpine (0.05 mg / 100 gm body weight) dissolved in PBS. Saliva was collected for 10 min from the oral cavity of individual mice using a micropipette starting one min after the injection of the secretagogue. The volume of saliva sample was measured. The saliva samples were then frozen at -80°C until analyzed. Profile of Immunoglobulin in Serum Determination of all the immunoglobulin is otypes in sera samples was determined using the Beadlyte Mouse Immunoglobulin Is otyping Kit (Cat. # 48-300) purchased from Upstate (Lake Placid, NY). Please refer to manufacture protocol for complete instruction. Immunoglobulin Specific M3R Autoantibodies Detection Using Immunoflourescence Detection of anti-M3R anti bodies in sera of NOD.B10H2b.CStat6+/+ and NOD.B10H2b.CStat6-/was determined using immunoflo urescence. Flp-In CHO cells were transfected with mouse M3R gene, and se lected for the antibiotic resistance. M3R transfected Flp-in CHO and nontransfected Flp-In CHO were plated on multi-well slides with 50% confluence. Sera diluted at 1/50 in PBS were incubated with the cells for 1 hour in a humidified chamber at room temp erature (RT). After incubation, cells were washed with PBS for 5 times, 5 min eac h. Isotypic FITC conjugated secondary antibodies, IgG1, IgG2a, IgG2b, IgG3, IgM, IgG (Serotec, Oxford UK) diluted at 1/100 were added, and incubated for 30 minutes at RT. Cells were washed 5 x 5 min, and

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89 visualized with similar exposure time using Zeiss (Thornwood, NY) Axiovert 200 M microscope. Statistical Analysis Differences in level of immunoglobulin isot ypes and stimulated salivary flow rates were analyzed with the St udent-Newman-Keuls test. p<0.05 was considered significant. Results Generation of NOD.B10H2b.CStat6+/+, NOD.B10H2b.CStat6+/-, NOD.B10H2b.CStat6-/As presented in Chapter 1, the NOD.B10H2b mouse represents a good mouse model of primary SjS. To genetically knock out the STAT6 gene in this model, we backcrossed NOD.B10H2bmouse with C.129S2Stat6 tm1Gru/J mouse. The advantage of this backcross breeding is that we could identify all three different genotypes of STAT6 mice, NOD.B10H2b.CStat6+/+, NOD.B10H2b.CStat6+/-and NOD.B10H2b.CStat6-/-. As presented in Figure 4-1, Dr. Gao (175) in our laboratory initia lly interbred NOD.B10H2b with C.129S2Stat6 tm1Gru/J to generate F1 offspring. Female F1 mice were backcrossed with male NOD.B10H2b mice. The first backcro ss generation was screened by PCR for heterozygocity of the STAT6 gene and MHC H-2b locus by microsatellite marker genotyping. Any female mouse that found to exhibit both MHC H-2b locus and heterozygous for STAT6 gene were selected, and b ackcrossed with male NOD.B10H2b mice to generate second backcross generation. The second backcross generation that was female and positive for MHC H-2b locus with heterozygous for STAT6 gene were selected and bred with NOD.B10H2b to generate the third backcross generation. At third backcrossed generation, Dr. Gao sele cted mice that exhibited both MHC H-2b locus

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90 and heterozygous for STAT6 gene, and interbred them, thereby generated NOD.B10H2b.CStat6+/+, NOD.B10H2b.CStat6+/-and NOD.B10H2b.CStat6-/mice. Animals at the third backcross contained 93.75% of NOD.B10H2b background, therefore the remaining 6.25% belonged to the Balb/c b ackground and could possibly interfere with the development of autoimmunity. Due to the contamination contributed by the Balb/c background, there were a number of mice that we re either showed or failed to show PSP cleavage in the saliva when analyzed by HPLC. Interestingly, when she separated out the PSP positive and PSP negative groups, there were significant reduction in salivary flow rates of the animals from 4 weeks old to 24 weeks old in the PSP positive group. However, she observed no change in salivar y flow rates over time in PSP negative group. Her data suggested that we need to furt her examine this inte resting observation. To further examine the role of STAT6 in this disease, third backcross generation animals were backcrossed to the sixt h generation to reach 99.2% of NOD.B10H2b background. Interbred sixth backcrossed mice, we obtained NOD.B10H2b.CStat6+/+, NOD.B10H2b.CStat6+/-and NOD.B10H2b.CStat6-/mice. All the experiment presented in this study used these mice. Detail schematic generation of three mouse models mentioned previously is presented in Figure 4-1. Pathophysiological Examination of NOD.B10H2b.CStat6+/+, NOD.B10H2b.CStat6+/-and NOD.B10H2b.CStat6-/Mice Previous study has shown that when C.129S2Stat6 tm1Gru/J mice were backcrossed with NOD.B10. H2b mice to the third generation, the re sulting offspring exhibited all the pathophysiological abnormalities phenotypes th at are commonly found in animal models predisposed to SjS-like autoimmune disease ( 175). Early preliminary results from that study indicated that these pathophysiological phenotypes encompassed the expression of

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91 a serine protease that cleaves PSP into smaller peptides, decrease in -amylase activity, and increase in salivary protein concentrati on. For the current study, we have continued the same backcross to the sixth generation, and interbred them to generate three different strains of mice which are NOD.B10H2b.CStat6+/+, NOD.B10H2b.CStat6+/-and NOD.B10H2b.CStat6-/mice. Full analysis of the sixt h backcross generation revealed similar pathophysiological pheno types found in the third gene ration including decrease in amylase activity and increase in salivary prot ein concentration from 4 weeks to 30 weeks of age (data not shown). As exp ected male and female of NOD.B10H2b.CStat6+/+, NOD.B10H2b.CStat6+/-and NOD.B10H2b.CStat6-/mice exhibited complete cleavage product of PSP at early as 12 weeks of age (Fi gure 4-2). Therefore, these data suggested that STAT6 may not have any significant im pact on the pathophysiological consequence during the autoimmune process of SjS. Leukocytic Infiltrations in the Submandibular and Lacrimal Glands in Both the NOD.B10H2b.CStat6+/+ and NOD.B10H2b.CStat6-/Mice Lymphocyte infiltration in the submandibular and lacrimal glands is one of the hallmarks for detection of the immunological phase of SjS in human and animal models. A lymphocytic focus is defined as a clus ter of leukocytes containing at least 50 lymphocytes. A focus score is defined as the number of foci in a 4 mm2 area (18). There is considerable debate whether the focus score in human SjS patients correlates with severity of the disease. However, the corr elation of focus score and severity of the disease appear more predictable in NOD mice because so far the loss of secretion function occurs concomitantly with leukocytic infiltration in the exocrine glands. Submandibular and lacrim al glands of NOD.B10H2b.CStat6+/+and NOD.B10H2b.CStat6-/mice were explanted from euthanized an imals at 4, 8, 12, 16 and 24 weeks of age.

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92 Tissues were embedded in pa raffin and sectioned at 5 µ m thickness. H&E staining of the histological sections indicat ed that formation of lym phocytic foci initiated at approximately 12 weeks of age in wildt ype and knockout animals. Lymphocytic infiltration was observed to be more severe in terms of the number and size of foci at 16 weeks of age in both wildtype and knockout mice (Figure 4-3). Within histological sections in both the submandibular and lacr imals glands of wildtype and knockout mice, lymphocytic foci appeared to make up of bot h CD3+ T cells and B220+ B cells localized in a well-defined organization (Figure 4.3). These observations suggest that T cells enter the glands first and possibly signal the infl ux of B220+ B cells into the glands. Detection of Anti-Nuclear Antigens in the Sera of NOD.B10H2b.CStat6+/+and NOD.B10H2b.CStat6-/Mice As previously reported, many of the clin ical manifestation of SjS are possibly mediated by the activity of autoantibodies pr oduced by autoreactive B lymphocytes (3). The presence of ANA including anti-Ro/SSA, anti-La/SSB and many others in sera of human patients and occasionally seen in so me SjS animal models may indicate the progression of SjS to a clinical phase invol ving secretory dysfuncti on. To detect ANAs, sera from NOD.B10H2b.CStat6+/+and NOD.B10H2b.CStat6-/mice were incubated with the Hep2 cells. Positive reaction was visualized by staining with goat anti-mouse whole IgG-FITC conjugated secondary antibodies. Both STAT6 wildtype and knockout mice appeared to be strongly positive for ANA (Figure 4-4). A ho mogeneous staining pattern of nuclei was observed for sera collected from both STAT6 wildtype and knockout mice, and this pattern was similar to that observed with sera from parental NOD.B10H2b mice.

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93 Determination of Immunoglobulin Subc lass Levels in Sera of NOD.B10H2b.CStat6+/+and NOD.B10H2b.CStat6-/Mice STAT6 is required for C and C 1 germline transcripti on by upregulation of activation-induced cytidine deaminase (AID ) in response to IL4 stimulation (176). Therefore, STAT6-deficient mice fail to produce significant amounts of IgG1 and IgE (177, 178). Furthermore, SjS in human patien ts and animals models is characterized over-production of immunoglobulins or hypergammaglobulinemia. To further investigate the role STAT6 on the various immunoglobulin isotypes in STAT6 wildtype and knockout mice, the levels of the individu al immunoglobulin isotype subclasses were determined. As shown in Figure 4-5, using the Luminex system to measure the amount of immunoglobulin in both STAT6 wildtype and knockout mice, there was approximately a 90% reduction in the levels of IgG1 and IgE of the knockout compared to the wildtype. The amount of IgE in the knockout was virtua lly undetectable. Levels of IgM and IgA were reduced about two fold in the STAT6 knockout mice. Small difference was seen in IgG2a level, however no signifi cant changes in the levels of IgG2b and IgG3 isotypes in the STAT6 wildtype and knockout mice were observed . These data imply an overall trend of reduction in the level of immunoglobulins found in the STAT6 knockout compared to the wildtype. Therefore NOD.B10H2b.CStat6-/mice appeared to exhibit the most drastic reductions in the levels of IgG1 and IgE with smaller decreases in IgM and IgA amounts. These data suggested that the hypergammagl obulinemia phenotype has been corrected in the STAT6 knockout mice reducing signif icant levels of IgG1 and IgE. Therefore, the total level of im munoglobulins appeared drastically reduced , suggesting a normal level of immunoglobulins.

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94 Detection of Specific Immunoglobulin against the Muscarinic Type 3 Receptor Autoantibody The polyclonal and monoclonal proliferati on of autoreactive B lymphocytes leads to the production of organ-specific and orga n-nonspecific autoanti bodies that corresponds to the progression of the disease developm ent (87). The presence of autoantibodies against the intracellula r ribonuclear proteins such as an ti-Ro/SSA and anti-La/SSB in SjS patients although not in NOD mice is important as a diagnostic disease biomarker and patientsÂ’ classification ( 18). Autoantibodies to these antige ns can be of either IgG or IgM subclass (172). However, the IgA auto antibody titers to Ro/La antigens are well correlated with sicca syndrome in primary SjS, especially with titers of rheumatoid factor present in patients with primary SjS (172). Di fferent levels of IgM rheumatoid factor are not related to disease duration, but the levels are highly significant in patient developing extra-glandular manifestations (121). Other autoantibodie s that are found to positively correlate with SjS patients and documented to be also found in the NOD animal include anti-foldrin, anti-carbonic anhydrase , anti-nuclear anti gen, and especially antimuscarinic type III receptor that, SjS-like autoimmune exocrinopathy mouse model (61). Especially critical is autoantibody against M3R since antibody may be involved as an effector of glandular dysfunction due to the f act that IgG fractions of sera obtained from SjS patients or old NOD mice containing an ti-M3R antibody suppr esses stimulated salivary flow rates when infused into norma l mice (66). Therefore, the isotype or subclass specific for a certain pathogenic autoantibodies may results in the clinical manifestation of the disease. Coincide ntally, anti-Ro/SSA but not anti-La/SSB autoantibodies found in human patients were mostly existed in the IgG1 isotype.

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95 However, the role of IgG1 specific anti-M 3R autoantibody SjS has never been precisely investigated. Utilizing the NOD.B10H2b.CStat6 knockout and its inability to produce IgG1 and IgE subclass antibody, the question of whet her IgG1 specific an ti-M3R autoantibody causes glandular dysfunction was examined. As previously described, we constructed Flp-in CHO cells transfected with a vect or to over-express the mouse M3R gene. Transfected Flp-in CHO cells express M3R protein on their su rface, acting as a target to detect serum antibody against M3R ( 44). As shown in Figure 4-6, both STAT6 wildtype and knockout mice were positive for IgG-M3R, IgM-M3R, and IgG2b-M3R autoantibodies. However, they were bot h negative for IgG2a-M3R and IgG3-M3R autoantibodies (data not shown). Interesti ngly, STAT6 wildtype mice were positive for IgG1-M3R autoantibody, but STAT6 knockout mi ce were completely negative for IgG1M3R autoantibody. Measurement of Salivary Flow Rates Glandular dysfunction in NOD.B10H2b mice models for SjS is characterized by the temporal loss of secretory functions c oncomitant with the appearance of leukocyte infiltrates within the exocrine glands. To measure changes in salivary flow rates, each mouse was weighed and injected with a s ecretogue containing isopreterenol and pilocarpine. Stimulated saliva secretion was collected and measured as discussed in Methods section. Both STAT6 wildt ype and heterozygote animals showed approximately 40% reduction in their salivar y flow rates when comparing 24 weeks old with 4 weeks old animals, implying an onset of dry mouth phenotype over time. When examining the STAT6 knockout mice in both male and female animals, there was no statistically significant reduction in salivary fl ow rates, although there was some loss of

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96 saliva volumes (Figures 4-7 and 48). Consider altogether, NOD.B10H2b mice lacking the STAT6 gene appear to maintain nearly normal secretory function in the salivary glands. Discussion In the present studies, I have investigat ed the role of STAT6 in the IL4 signal transduction pathway in the development of SjS-like disease in the NOD mouse using our recently constructed STAT6 -gene KO mouse referred to as NOD.B10H2b.CStat6-/-. The NOD.B10H2b.CStat6-/mouse was generated to further examine the role of STAT6 in isotype switch in SjS-like autoimmune exocrinopathy. NOD.B10H2b.CStat6-/mice exhibited a number of char acteristics observed in dise ase-prone NOD/Lt and NOD.B10H2b mice, and as identified in the NOD.B10H2b.IL4-/mouse (56, 58). These included (a) the presence of leukocytic infiltration of th e submandibular and lacrimal glands, (b) the production of anti-nuclear an tibodies and autoantibodies r eactive with tissues of the salivary glands, and (c) the activation of a PSP-specific se rine protease. Most importantly, NOD.B10H2b.STAT6-/mice behave like NOD.B10H2b .IL4-/mice by not exhibiting the temporal loss of stimulated salivary flow rates and failed to produce an IgG1-M3R specific autoantibody. Although earlier studies using the NOD.IL4-/and NOD.B10H2b.IL4-/mice indicated that secretory functi on of the salivary gland was restored to normal level when the IL4 gene was genetically knocked out, desp ite exhibiting the pathophysiological abnormalities phenotypes and lymphocytic inf iltrations in the exocrine glands concomitantly with production of ANAs (56, 58) . These earlier data suggested that IL4 plays a much more important role at the clin ical phase, having little or no affect on the pathology associated with preclinical disease of Phase I or Phase II of the SjS-like

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97 disease. IL4, signaling via either STAT 6 or IRS pathway, appears to modulate the secretory function of exocrine glands. Interestingly, IL4 gene knockout mice failed to produce IgG1 isotypic antibody against M3 R autoantibodies while producing normal level of M3R with other isotypes, IgG2a, IgG2b, IgG3, IgM, and IgA, pointing to a critical role in IgG1 isotype switching (58) . As previously discussed, IL4 mediates its function by two separate pathways. IL4 -IRS pathway responsib le for the cellular proliferation and activation while the IL4STAT6 account for the isotype switching to IgG1 and IgE by specifically stimulating germline 1 and immunoglobulin gene transcription. As indicated, NOD.B10H2b.STAT6-/mice failed to make IgG1 antibody against M3R. Therefore, the role of IgG1-M3R antibody appear ed to be very critical for the secretory dysfunction. The mechanisms that lead to secretory dysfunction by the salivary and lacrimal glands are still under intensive investigati on. There are several hypothesis presented by several groups trying to explain the m echanics of how antibody reactive against muscarinic type III receptor could result in th e shutdown of fluid secretion. First, in a normal physiological system, fluid secretion by the exocrine glands is initiated by binding of acetylcholine to M3R. Antibodi es binding to M3R could mask the binding site of neurotransmitter, resulting in the down regulation of the cascade (39). Second, the complex of antibody and M3R could initiate in ternalization of the complex resulting in degradation of both antibody and receptor (38). Last, recent studies by Cha et al. have shown that chronic binding of antibodies to M3 R could result in rece ptor desensitization, therefore shutting down the whol e cascade of downstream events resulting in termination of fluid secretion (37).

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98 There is controversy implying whether cl inical symptom of SjS could be the mediated only by the activity of autoanti bodies with specific isotype. There are numerous autoantibodies found in SjS patie nts and NOD mice such as autoantibodies against the intracellular ribonuc lear proteins specifically anti-Ro/SSA and anti-La/SSB (61). Autoantibodies to these antigens can be existed in the IgG and IgM isotypes (172). However, the IgA autoantibody titers to Ro or La antigens have been shown to correlate well with sicca syndrome in primary SjS (172) . Levels of anti-Ro and anti-La have a strong association with the ti ter of rheumatoid factor on ly found patients with primary SjS (121). Different levels of IgM rheumatoid factor were not related to the disease duration, but the levels were highly signifi cant in patient devel oping extra-glandular manifestations (121). Other autoantibodies that are found to positively correlate with SjS patients such as anti-foldrin, anti-carboni c anhydrase, and anti-nuclear antigen, are also found in our NOD animal model of SjSlike autoimmune exocrinopathy (61). However, it remains questionable if a specific isotype to any of these autoantibodies play significant role in the pathogenesi s of SjS, or if this is simply one of the mere correlations documented in SjS patients. Many reports investigating spontaneous lupus prone mice have indicated that IgG2a and IgG3 are potential pathogenic au toantibodies that are regulated by type 1 cytokine such as IFN(179, 180). However, the predominant autoantibodies and immunoglobulins deposited in the kidneys in th e surrogate peptide or mercuric induced models of lupus are IgG1 or IgE isotype wh ich interestingly, would be driven by IL4STAT6 (181). Therefore, autoimmune connect ive tissue diseases such as SLE, the mode of autoimmune regulation is hi ghly dependent on the initiati on factors. Consistent with

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99 observation found in NOD.B10H2b.CStat6-/mice, knocking out either IL4 gene or STAT6 gene in lupus mice models such as BXSB, (MRL-lpr/lrp x B6) F2 and NZM.2410 mice has no effect on production of autoantibodies and the degree of lymphocytic infiltration in secondary lymphoid organs or kidneys (182, 183). Thus, the presence of general ANA production and leukocyte infilt rations may not have any apparent consequence in the clinical aspect of the autoimmunity, instead merely establish the environment and response necessary. Howeve r, isotypic antibody may possess the ability to initiate adverse effect on the disease. Ou r previous data and newly generated data in this study have clearly demonstrated the n eed for an IgG1 antibody against M3R in the pathogenesis of SjS. It may possibly serve as an effector molecu le that involve in shutdown down of secretory function in the exocrine glands. Perhaps one of the more unusual observation in this aspect is the fact that Fab fragments isolated from IgG fraction of Sj S human patients sera or NOD mice, when infused to NOD.Igµ-/mice can also cause temporary shutdown of salivary flow rates. Infusion of the remaining fraction of IgG into the NOD.Igµ-/mice failed to dysregulate the exocrine glands (66). Therefore, the Fab portion seems very criti cal in this phase of disease, while the Fc portion appears to ha ve no biological significant to the secretory physiology. Thus, a dichotomy exists, so the secretory mechanism pertaining to regulation of fluid secretion in animal models of SjS is proving to be ve ry intriguing. It is also possible that since IgG1 fraction is the most abundant immunoglobulins in both human and mouse, antibodies to self and nonself usually found to be exist in IgG1 isotypic form. Therefore, elimination STAT6 could result in the diminished

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100 concentration of IgG1. Cons equently, there is insufficien t quantity to initiate any biological and physiologi cal responses.

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101 Figure 4-1.Genera tion of NOD.B10. H2b.C. STAT6-/mouse.

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102 Figure 4-2.Detection of proteoly tic activity agai nst PSP in saliva . Saliva was collected and assayed for protease activity on an oligopeptide containing the NLNL enzymatic site of PSP. Using HPLC, the whole synthetic oligopeptide eludes at 13.9 min ( A ) while the two degradation produc ts of the olig opeptide appear at 9.2 and 12.8 minutes. Saliva from Balb/c ( B ) is not capable of enzymatically digesting the oligope ptide, while saliva from NOD ( C ) and NOD.B10. H2b ( D ) mice showed complete digestion of the oligopeptide. In addition, both NOD.B10. H2b.STAT6+/+ ( E ) and NOD.B10. H2b.STAT6-/mice exhibited enzymatic activity of protea se that completely digested the oligopeptide. All individual figures are representative.

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103 Figure 4-3. Histological examination of the exoc rine glands. Submandibular and lacrimal glands were removed from each mouse approximately 24-27 weeks of age and fixed in 10% formalin. Fixed hist ological sections were cut at 5 µ m thickness with two layers 50 µ m apart and stained with Mayer’s H&E dye and immunoflourescence for dist ribution of B and T cells in lymphocytic foci (Red: B220+ B cells, green: CD3+ T cells, blue: DAPI). The number of focus were counted on the whole hist ological glands and average for two layers. H&E staining (A) and i mmunoflourescent staining (B) of submandibular glands of NOD.B10. H2b.STAT6+/+ mice (n=23). H&E staining (C) and immunoflour escent staining (D) of su bmandibular glands of NOD.B10. H2b.STAT6-/mice (n=18). H&E staining (E) and immunoflourescent staining of l acrimal glands of NOD.B10. H2b.STAT6+/+ mice (n=23). H&E staining (G) and i mmunoflourescent staining of lacrimal glands of NOD.B10. H2b.STAT6-/mice (n=18). A C E G B D F H

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104 Figure 4-4.Detection of anti -nuclear autoantibodies (ANAs). Serum samples obtained from groups of mice at various ages we re pooled, diluted 1:40 and incubated with HEp-2 fixed substrate slides fo r 30 min at room temperature in a humidified chamber. The substrate s lides were then incubated with FITCconjugated goat anti-mouse IgG diluted 1:50 for 30 min at room temperature and viewed by fluorescence micros copy at 400X magnification. A. NOD.B10H2b.STAT6+/+ 24 wks old female mice (n=10). B. NOD.B10H2b.STAT6-/24 wks old female mice (n=10). All individual figures are representative.

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105 0 100 200 300 400 500 60 0 IgA IgG1 IgG2a IgG2b IgG3 IgE IgMSTAT6+/+ STAT6-/g /ml Figure 4-5.Amount of immunoglobulin isotype s present in sera of female NOD.B10H2b.STAT6+/+ (n=5) and female NOD.B10H2b.STAT6-/(n=5). Statistical analysis was performed by Student-Newman-Keuls test.

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106 Figure 4-6. Detection of M3R isotypic au toantibodies by immunoflourescence. Sera were incubated with M3R-transfecte d Flp-In CHO cells. Cells were visualized using isotypic FITC-conguga ted secondary antibodies at 100X magnification.

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107 Figure 4-7.Salivary flow rates of female animals. Groups of female NOD.B10H2b.STAT6+/+ with NOD.B10H2b.STAT6+/{4 wks old (n=7), 24 wks old (n=12)}, and NOD.B10H2b.STAT6-/{4 wks old (n=5), and 24 wks old (n=8)} were injected with isopreter enol/pilocarpine to stimulate saliva secretion. Saliva was collected from each mouse for 10 min starting 1 minute after injection of the secretagogue. The volume of each sample was measured. Statistical analysis was pe rformed by Student-Newman-Keuls test

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108 Figure 4-8.Salivary flow rates of male animals. Groups of male NOD.B10H2b.STAT6+/+ with NOD.B10H2b.STAT6+/{4 wks old (n=8), 24 wks old (n=17)}, and NOD.B10H2b.STAT6-/{4 wks old (n=5), and 24 wks old (n=6)}mice were injected with isopreterenol/pilocarpine to stimulate saliva secretion. Saliva was collected from each mouse for 10 min starting 1 minute after injection of the secretagogue. The volume of each sample was measured. Statistical analysis was pe rformed by Student-Newman-Keuls test

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109 CHAPTER 5 SJGREN’S SYNDROME-LIKE DISEASE OF C57BL/6.NODAEC1AEC2 MICE: GENDER DIFFERENCES IN KERATOC ONJUNCTIVITIS SICCA DEFINED BY A CROSS-OVER IN THE CHROMOSOME 3 AEC1 LOCUS Introduction Sjgren’s syndrome (SjS) is a chronic, systemic human autoimmune disease in which an immunological attack against the salivary and lacrimal glands results, respectively, in severe dry mouth (stomatitis sicca) and dry eye (keratoconjunctivitis sicca) disease(s) (1, 4). Despite considerable efforts to define the genetic, environmental and immunological bases of SjS, the unde rlying etiology of this disease remains relatively obscure, especia lly concerning the genetic contribution. While there is evidence emerging to suggest possible invo lvement of both majo r histocompatibility complex (MHC)-associated and non-MHC-associat ed factors, studies show only a weak tendency toward familial aggregations. Ne vertheless, in different human populations, specific alleles of the MHC class II HLA-DQ an d HLA-DR genes have been reported to have an increased frequency in SjS patients (5 2). However, studies from different ethnic groups have yielded inconsiste nt results suggesting only weak MHC class II a ssociations; thus, these associations may reflect, instead, the presence of anti-nuclear autoantibodies, such as anti-SS-A/Ro and anti-SS-B/La, us ed as biomarkers of disease (18). For a number of years, a variety of mouse models exhibiting various aspects of SjS have been studied extensively in an attempt to identify the nature of this autoimmune disease. One of the more intensively studi ed models of SjS is the NOD mouse (61). Through the use of various congenic partners and gene knock-out strains of NOD, the

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110 development and onset of SjS-like disease in these mice has been divided into three distinct consecutive phases (61, 149). In Phase 1, a number of aberrant genetic, physiological and biochemical activities asso ciated with retarded salivary gland organogenesis and increased acinar cell apop tosis occur sequentially prior to and independent of detectable autoimmunity. In Phase 2, believed to result from acinar cell apoptosis, leukocytes expressi ng pro-inflammatory cytokine s infiltrate the exocrine glands, establishing lymphocytic foci, first of T cell clusters followed by recruitment of B lymphocytes. In Phase 3, onset of clinical disease as depicted by loss of salivary and lacrimal gland secretory function, most likely occurs as the result of (auto)-antibodies reactive with the muscarinic acetyl choline type-3 receptors (M3Rs). A genetic predisposition for the developmen t and onset of this SjS-like disease in NOD mice has also been identified. Firs t, SjS-like disease in NOD mice appears independent of, or only weakly associated w ith, MHC-associated genes, thus mimicking SjS in humans. The first indication invol ved studies of the congenic strain, NOD.B10H2b, in which the NOD MHC I-Ag7 Idd1 diabetes susceptibility locus was replaced by MHC I-Ab (53). These mice, while expressing a different MHC class II region, continue to show a complete SjS-like disease includi ng salivary and lacrimal gland dysfunction. Second, replacing other Idd loci in the NOD mouse (e.g., Idd10, Idd9 , Idd13 , and so forth), resulted in the identification of Idd3 and Idd5 loci as critical genetic regions for the development of this disease (54). In a reverse approach, introducing the Idd3 and/or Idd5 genetic regions derived from NOD mice into the SjS non-susceptible C57BL/6 mouse resulted in the appearance of SjS-lik e disease, confirming the contributions of these two genetic loci to development and ons et of disease (55). Furthermore, the pre-

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111 clinical, non-immune aspects manifested in Phase I of the disease are associated with the Idd5 locus of chromosome 1, while the immunol ogical aspects of the disease manifested in Phases 2 and 3 of the di sease are associated with Idd3 of chromosome 3. Recapitulation of the full disease profile was shown to require genes within both these genetic loci. This SjS-susceptible C57B L/6 congenic mouse currently carries our designation C57BL/6.NODAec1Aec2 where Aec1 corresponds to Idd3 and Aec2 corresponds to Idd5 . Unfortunately, Aec1 ( Idd3 ) represents a 48.5 cM centromeric region of chromosome 3 which precludes easy analysis of candidate gene(s) essential for the onset of autoimmunity in response to the salivary a nd lacrimal gland injuries associated with Phase 1 of the disease. Thus, we have initia ted studies to define smaller genetic regions that contain those genes regulating the events of Phase 2. To this end, we have now narrowed the Aec1 region to a centromeric stretch less than 20 cM. This region contains several potential candidate genes, incl uding genes shown to have polymorphisms associated with SjS in humans. Materials & Methods Animals The recombinant inbred (RI) C57BL/6.NODAec1R1Aec2 mice were bred and maintained under specific pathogen-free condition s in the animal facility of Animal Care Services, University of Florida, Gainesville, FL. C57BL/6.NODAec1R1Aec2 mice were generated by crossing C57BL/6.NODAec1Aec2 mice with C57BL/6J mice purchased from the Jackson Laboratory (Bar Harbor, ME). The F1 heterozygotes were screened for the presence of cross-overs within the Aec1 or Aec2 genetic regions by microsatellite marker genotyping. Primer sequences for the microsatellite markers were based on

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112 sequences available from Jack son Laboratory and purchased fr om IDT (Coralville IA). Any mouse indicating a cross-over in one of the Aec regions was bred with a C57BL/6J mouse to produce Aec -cross-over heterozygous male and fe male offspring that were then used to produce F2 generations. Mice of the F2 generations were sc reened for a male and female homozygous for the cross-over chro mosome. One recombinant pair having a cross-over within Aec1 at 19.2 cM on chromosome 3, referred to as C57BL/6.NODAec1R1Aec2 , is being carried as a single line of descent and is currently at the fourth generation. Studies described he rein were approved by the Un iversity of Florida IACUC. Histology Male and female C57BL/6.NODAec1R1Aec2 mice were euthanized at various ages as indicated in the text. Submandibul ar and lacrimal glands were surgically removed from each mouse and placed in 10% phosphate buffered formalin for 24 hrs. Fixed tissues were embedded in paraffin and sectioned at 5 µ m thickness. Paraffinembedded sections were de-paraffinized by immersing in xylene, followed by dehydrating in ethanol. The tissue sections were stained with H&E dye (Histology Tech Services, Gainesville, FL). Stained sectio ns were observed at 100X magnifications for glandular structure a nd leukocyte infiltration. Measurement of Saliva and Tear Flow Rates To measure stimulated flow rates of saliva, individual mice were weighed and given an intraperitoneal (ip) injection of a solution contai ning isopreterenol (0.2 mg/100 gm body weight) and pilocarpine (0.05 mg/ 100 gm body weight) dissolved in PBS. Saliva was collected from the oral cav ity of individual mice for 10 min using a micropipette starting one min after the injection of the secretagogue. The volume of saliva sample was measured and then stored at -80°C until analyzed.

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113 To measure stimulated flow rates of tear s, individual mice were injected with pilocarpine hydrochloride (4.5 mg/kg in saline ) and allowed to rest comfortably for 10 minutes. Tear volumes were determined usi ng a phenol red thread, a modification of the Shirmer test. In brief, the bent end of a small piece of Zone-Quick*, Phenol Red Thread (FCI Ophthalmics, Pembrooke, MA) was placed ca refully at the intercanthus of each eye of a resting mouse lightly anes thetized using inhalation anesthesia isoflurane. The thread was held in place with forceps for 20 sec, re moved from the eye, and the length of the red area measured using the scale provided. The red portions of the th reads containing the tear samples from both left and right eyes of an individual mouse were cut into small fragments (~ 2-3 mm in length) and pooled in a microcentrifuge tube. A volume of saline solution equivalent to the total length of th read was added and the samples stored at 80 C until analyzed. Saliva Protein Concentration and Salivary Amylase Activity Saliva and tear samples were analyzed fo r total protein using the Bradford assay using BSA as the standard. Amylase activity in saliva was determined using the Infinity TM Liquid Amylase Kit (Thermo Trace Electron Corp., Waltham, MA) in which ethylidenepNP-G7 was the substrate. Saliv a samples were diluted 250-fold with deionized water and added to 1 ml of the Infinity TM Amylase Liquid Stable Reagent. Following 1 min and 2 min incubations at 37oC, absorbance was measured at a wavelength of 405 nm. Amylase activity was calculated according to the manufactureÂ’s instructions using the formul a: Amylase activity (U/L) = A/2 x 5140 x Sample dilution.

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114 Detection of Caspase-3 Activity Caspase-3 activity was determined using the Caspase-3/CPP32 Fluorometric Assay Kit (#K105-200) purchased from BioVision, In c. (Mountain View, CA). For each assay, 50 µg of tissue cell lysate was used. Each samp le was run either in duplicate or triplicate. Proteolysis of Parotid Secretory Protein (PSP) Detection of PSP (splunc2) proteolysis wa s carried out by incubating whole saliva specimens with a synthesized oligopeptid e corresponding to amino acids 20 through 34 of the published sequence for mouse PSP. This oligopeptide contains the proteolytic site (NLNL) for a serine kinase present in salivary glands during development and onset of SjS-like disease in the NOD mouse (unpublished data). Eight µ l of saliva collected from individual mice were mixed with 42 µ l of the PSP oligopeptide (2.5mg/ml) and incubated at 42oC for 12 hrs. Following incubation, 50 µ l Tris-HCl buffer (50 mM, pH 8.0) were added and the mixture centri fuged through Micro-spin filt er tubes at 14,000 rpm for 10 min. The filtrates were analyzed by HP LC (Dionex Systems, Sunnyvale, CA) for proteolytic products. Contro l samples consisted of 50 µ l of the PSP oligopeptide. Detection of Anti-Nuclear Autoan tibodies (ANAs) in the Sera ANAs in the sera of mice were de tected using an ANA screening kit (ImmunoConcepts, Sacramento, CA). HEp-2 fi xed substrate slides were overlaid with the appropriate mouse serum diluted 1:40 and incubated for 30 min at room temperature in a humidified chamber. After three 5-minute washes with PBS, the substrate slides were covered with FITC-conjugated goat anti -mouse IgG diluted 1:50 and incubated for 30 min at room temperature. After three wa shes, nuclear fluorescence was detected by fluorescence microscopy at 400X magnification. Detection of Immunoglobulin-Spe cific Anti-M3R Autoantibodies

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115 Detection of anti-M3R antibodies in sera of mice as well as humans was determined as described in detail elsewhere ( 44). In brief, Flp-In CHO cells transfected with mM3R were collected from growing cu ltures, washed once with phosphate-buffered saline (PBS), and resuspended in FACS buffer (PBS, 0.5% BSA, 0.07%NaN3). Aliquots of cells at a density of 1 x 106 cells/0.1 ml were incubated 2 hrs at 4°C with 10 l sera from individual mice or pooled from appropr iate groups. Cells were washed once with FACS buffer, resuspended in 50 l of FACS buffer and incubated for 30 min at 4oC with either FITC-conjugated goat anti-mouse IgG or one of the following subclasses, IgG1, IgG2b, IgG2c, IgG3, IgM (Southern Biotec hnology Associates, Inc., Birmingham AL). After a final wash with FACS buffer, the cells were resuspended in FACS buffer and analyzed using a FACScan cytometer e quipped with CellQuest software (Becton Dickinson, Mountain View, CA). Control r eactions included cel ls incubated with secondary antibody alone or an appropriate isotype control. An increase in fluorescence intensity compared to sec ondary antibody alone was cons idered a positive reaction. Statistical Analyses For this study, we have standardized both saliva and tear coll ections based on the body weight of the individual mice in an atte mpt to better control comparisons. Although this has not been common practice while working with mice of the NOD genetic background, we have incorporated this fo r mice of the C57BL/6 genetic background for two reasons. First, the disease tends to o ccur in the C57BL/6 ge netic background strains at an earlier age, necessitati ng collections of saliva and te ars when the mice are as young as 4-5 weeks of age and are less than half the size of adult mice. Second, there are greater size differences between male and female mice of these C57BL/6 genetic

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116 background strains during the early time course of the dis ease. Statisti cal significance between saliva and tear collections was de termined using Student-Newman-Keuls test. Values of p<0.05 were considered significant. Results Genetic Profile of Recombinan t Inbred Line C57BL/6.NODAec1R1Aec2 Mice In earlier studies that examined the SjS-like disease profiles of C57BL/6.NOD c3 mice carrying the NOD-derived Idd3 locus, C57BL/6.NOD c1t mice carrying the NODderived Idd5 locus, and C57BL/6.NODAec1Aec2 mice carrying both the NOD-derived Idd3 and Idd5 loci, we observed several interest ing aspects concerning the development and onset of the autoimmune response. First, C57BL/6.NOD c1t mice, by 20 weeks of age, exhibited the various ab errant physiological and bi ochemical disease markers, including acinar cell apoptosi s, seen in the NOD mouse stra in, but showed no signs of a developing autoimmune attack against the salivary or lacrimal glands. Second, C57BL/6.NOD c3 mice failed altogether to develop signs of a SjS-like disease. Third, the presence of both NOD-derived Idd3 and Idd5 loci in the C57BL/6.NODAec1Aec2 mice recapitulated the SjS-like disease seen in NOD mice, indicating a need for the aberrant physiological and bioc hemical changes in the salivary and lacrimal glands for the subsequent onset of an immunological re sponse resulting in stomatitis sicca and keratoconjunctivitis sicca. In analyzing the boundaries of the Aec1 ( Idd3 ), Aec2 ( Idd5 ) and Aec1R1 genetic regions using microsatellit e markers, we identified Aec2 as lying between 32.8 and 101.2 cMs on chromosome 1, as defined by the mi crosatellite markers D1mit18 (B6)/D1mit302 (NOD) and D1mit360 (NOD)/D1mit117 (B 6), respectively (Figure 5-1). Aec2 , then, appears to be a 68.4 cM region rather than a 46.1 cM region orig inally reported for Idd5

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117 (184). In addition, this region may contai n a small region at 70.0 cM derived from C57BL/6. On the other hand, Aec1 appears to cover the gene tic region of chromosome 3 from the centromere to 48.5 cM, as defined by D3mit164 (NOD) and D3-Tshb (NOD)/D3mit286 (B6), respectively, wh ile the cross-over in C57BL/6.NODAec1R1Aec2 has been identified as occurring at or near position19.2 cM, as defined by D3mit151 (NOD)/D3mit21 (B6) (Figure 1). Thus, by maintaining the Aec2 locus, but splitting the Aec1 locus, it seemed possibl e to better identify gene s involved in regulating the onset of the autoimmune respons e in this mouse model of SjS. Phase 1 Profile of SjS-Like Disease in C57BL/6.NODAec1R1Aec2 Mice Since C57BL/6.NODAec1R1Aec2 mice were selected to retain the Aec2 locus of C57BL/6.NODAec1Aec2 mice, it would be expected th at the aberrant physiological changes associated with the pre-immune phase of SjS-like disease would remain intact. Two markers of these physiologi cal changes are an elevated le vel of caspase-3 activity or cysteine protease activity in the salivary gland indicating cell apoptosis (60) and appearance of an activated serine protease in the submandibular gland that degrades parotid secretory protein (PSP) , a member of the BPI-PLUNC family of innate antibacterial proteins (185) . As anticipated, caspase-3 activ ity in the submandibular gland tissue of RI C57BL/6.NODAec1R1Aec2 mice increased between 4 and 14 wks of age, then decreased as the mice aged (data for female mice shown in Table 5-1). Interestingly, caspase-3 activity in the l acrimal gland tissue of female C57BL/6.NODAec1R1Aec2 mice remained relatively low over this same time frame, but increased with age. In addition, serine proteas e activity in salivary gland tissue was detected starting as early as 10 wks of age (Figure 5-2). Thus, the C57BL/6.NODAec1R1Aec2 mice exhibit

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118 the salivary and lacrimal gland pathology thought to be necessary to induce phase 2 of the immune response. Phase 2 Profile of SjS-Like Disease in C57BL/6.NODAec1R1Aec2 Mice Although the number of lymphocytic foci present in the salivary and lacrimal glands often does not correlate directly with disease or severity of disease, both SjS patients and NOD-derived mouse strains exhi biting SjS-like disease typically have lymphocytic infiltrates in thei r salivary glands whose hist ological appearance is termed lymphoepithelial sialadenitis (LES). To determine if RI C57BL/6.NODAec1R1Aec2 mice develop LES, the submandibular and extr a-orbital lacrimal glands from both male and female mice euthanized at 5, 10, 19 and 22 weeks of age were freshly explanted, fixed in formalin, embedded in paraffin, sectio ned and stained. As presented in Figures 3A and 3B, histological examination revealed multiple foci of leukocy tic infiltrates in the submandibular glands of males started at a pproximately 10 weeks of age and females at 19 weeks of age. In keeping with the earlier observations that female mice tend to have a more severe sialadenitis than male mice, C57BL/6.NODAec1R1Aec2 females appeared to have larger infiltrates in their submandibular glands than males at 22 weeks of age. In contrast, C57BL/6.NODAec1R1Aec2 males displayed high leve ls of dacryoadentitis (Figure 5-3), whereas females surprisingl y exhibited none (Figure 5-4). With the appearance of T and B lymphocytes within the salivary and lacrimal glands, there is a subsequent production of an increasing number of detectable serum autoantibodies (10). The presence of ANAs, in particular anti-SS-A/ Ro and anti-SS-B/La in the sera of human patients, is one parame ter in the diagnosis of clinical SjS. To identify ANAs in the sera of RI C57BL/6.NODAec1R1Aec2 mice, both male and female

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119 mice were serially bled between 5 and 22 w eeks of age, the sera were collected and pooled according to age and gender, and test ed on Hep-2 cells. The presence of ANAs was visualized by staining with FITC-conj ugated goat anti-mouse whole IgG. ANAs were detected as early as 5 wks of age in ma les (1 of 5), but not until 10 wks of age in females. All mice of both sexes were positiv e for ANAs from 10 to 22 wks of age. The pattern of ANAs showed mostly homogeneous nuclear staining similar to that in NOD and NOD.B10. H2b mice, but unlike its parental strain C57BL/6-NODAec1Aec2 which commonly showed speckled staining of the nucleus (Figure 5-5). Phase 3 Profile of SjS-Like Disease in C57BL/6.NODAec1R1Aec2 Mice Phase 3 of SjS-like disease in the different NOD-derived mouse lines is characterized by the loss of stimulated sa liva and tear volumes, possibly due to the synthesis of anti-M3R autoanti bodies (44, 65). Secondary to this secretory dysfunction is the concomitant initial increase in saliva a nd tear protein concentr ations followed by the decrease in specific protein activities, especi ally alpha-amylase in the saliva (63). To determine if the RI C57BL/6.NODAec1R1Aec2 mice develop salivary and lacrimal gland dysfunction, temporal cha nges in saliva and tear flow rates were determined for both male and female mice at 5, 10, 16, 17, 19 a nd 22 weeks of age, as described in the Materials and Methods. As shown in Fi gure 5.6A, both female and male RI mice exhibited losses of approximately 35-40% in sa livary flow rates as the mice aged from 5 to 22 weeks of age. These da ta are consistent with the de creases of saliva fluid volumes observed with NOD, NOD.B10H2b and C57BL/6.NODAec1Aec2 mice. In contrast, while male RI C57BL/6.NODAec1R1Aec2 mice showed a temporal loss of tear flow rates, female RI C57BL/6.NODAec1R1Aec2 mice displayed no loss of lacrimal gland

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120 secretory function (Figure 5-6B). These data correlated with the hist ological findings of lymphocyte infiltrations of the lacrimal glands (see Figures 5-3 and 5-4). As expected, protein concentrations of saliva and tear specimens from males and saliva specimens from female RI mice were increased as th e volumes decreased (data not presented). To detect antibodies reactive with the M3R in C57BL/6.NODAec1R1Aec2 mice, flow cytometric analyses were carried out us ing serum samples prepared from individual male and female mice of different ages. Serum samples, diluted 1:50 were incubated with either non-transfected or mM3R-transfect ed Flp-In CHO cells. The cells were then washed, re-incubated with FITC-conjugate d goat anti-mouse Ig M or IgG subclassspecific antibody, and analyzed by flow cytometr y. As presented in Figure 5-7, all sera exhibited positive IgM binding with mM3R-transfected Flp-In CHO cells, but not with non-transfected cells. Sera from C57BL/6.NODAec1R1Aec2 mice older than 18 wks of age showed various patterns of positive IgG subclass staining, while sera from young mice (5 wks of age) failed to show positive IgG staining on the transfected cells. Furthermore, neither non-transfected nor mM 3R-transfected Flp-In CHO cells treated with secondary antibody alone showed any positive staining (data not presented). Lastly, alpha-amylase, an enzyme whose function in digestio n is to break down starch, appears to be the most abundant protein produced by the acinar cells of the salivary glands (186). As such, the loss of am ylase activity is directly correlated with the loss of acinar cell function in the salivary gland. Whole sa liva samples, collected from C57BL/6.NODAec1R1Aec2 male and female mice at 5, 10, 16, 17, 19 and 22 weeks of age, were used to measure alpha-amylase activ ity by the ability to degrade the substrate, ethylidenepNP-G7. As indicate d in Figure 5-8, mice exhibited an increase in enzymatic

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121 activity (608.78 ± 123.4 vs. 927.44 ± 323.85 U/ml) as they aged from 5-10 to 16-17 weeks accompanied by an increase in size. However, by 19-22 weeks of age, the enzymatic activity declined to 603.23 ± 248.36 U/ml indicating the onset of glandular tissue dysfunction. Discussion In the present study, we examined the SjSlike disease profile in a newly generated RI mouse, designated C57BL/6.NODAec1R1Aec2 , in which the genetic region on chromosome 3 of C57BL/6.NODAec1Aec2 mice known to regulate the onset and development of the autoimmune attack agains t the submandibular and lacrimal glands has been reduced to a centromeric segment of less than 20 cM. Results of the disease profiling showed that both male and female C57BL/6.NODAec1R1Aec2 mice exhibited the expected pre-immune abnormalitie s observed in NOD mice (from which the Aec1 and Aec2 regions are derived) that indicate an impending immune phase of the disease. These abnormalities include up-regulated cystei ne kinase activity indicative of cell apoptosis and aberrant expression of PSP. De spite elimination of almost 30 cM of the Aec1 region, both male and female C57BL/6.NODAec1R1Aec2 mice exhibited a full autoimmune response that led to clinical SjS-like disease of the salivary gland, while male, but not female, C57BL/6.NODAec1R1Aec2 mice exhibited clinic al disease of the lacrimal glands. This diseas e profile included activation of serine kinase activity, appearance of lymphocytes in the salivary and lacrimal glands, production of ANAs, synthesis of anti-M3R antibodi es, temporal loss of saliva an d lacrimal flow rates with concomitant increases in salivary and lacrimal protein concentrations, and decreases in amylase activity. Unexpectedly, female C57BL/6.NODAec1R1Aec2 mice showed no signs of autoimmunity in the lacrimal glands and retained full lacrimal gland function.

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122 Why this dichotomy in dacryoadenitis and xerophthalmia occurs between males and females is unknown, but suggests an important gene or set of gene s lying within the deleted region of the Aec1 locus that imparts gender diffe rences. Finding a cross-over that identifies this region regulating this difference be tween males and females is currently in progress. Mapping of the cross-over re gion in the RI C57BL/6.NODAec1R1Aec2 mice revealed the cross-over apparent ly occurred at the site of the IL2 gene encoding a cytokine associated with T1D in NOD mice. The IL2 gene, in addition to being located at 19.2 cM of chromosome 3, is found within 0.35 cM of the Idd3 locus. IL-2 is associated with a wide spectrum of effect s on the cells of the immune system, including CD4+ T cells, CD8+ T cells, B cells and NK cells. IL -2 can induce proliferation and expansion of antigen specific CD4+ and CD8+ T cell clones, as well as augmenting the functions of other cytokines in the immune response (187). Recently, IL-2 has been implicated as an important cytokine in th e development of regulat ory T cells during the autoimmune process (188), as well as promot e proliferation of B lymphocytes (189). Interestingly, IL-2 plays a role in enha ncing antibody production wh ich could contribute to the hypergammaglobulinemia and excessi ve production of auto reactive antibodies observed in SjS patients. Mice deficient in IL-2 can develop autoimmune hemolytic anemia and ulcerative colitis (190). At the same time, transgenic animals have been shown not to experience autoimmunity, but instead develop lymphoc yte infiltrations in peripheral tissues (191). In human studies, elevated IL-2 se rum levels are correlated with progression of some autoimmune diseases such as scleroderma and rheumatoid arthritis (192, 193). Thus, the wide range of functions associated with IL-2 suggests that either

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123 the cytokine, its receptor or its regulatory factors could play critical roles in the SjS-like disease phenotype, both in human patients and in animal models. As a result, identification of the origin of this ge netic region at 19.2 cM is imperative. Although this Aec1R1 region contains a large number of genes that could play a role in the pathogenesis of SjSlike disease, there are several genes of particular interest at this early stage of an alysis. The first is the Il7 gene located at position 6.6 cM. IL-7 is produced by reticular stromal cells in the bone marrow and regulates development of B lymphocytes, especially at the stage of proB cell differentiation to pre-B cell (194). In addition, IL-7 is important in B cell lymphopoies is as well as B cell maturation at later stages of development via th e activation of three critical signaling pathways: JAKSTAT, PI3K and Ras-GTPase (195). B ecause SjS and SjS-like syndromes are characterized by B cell hyperproliferation, it is important to note that IL-7 also induces the expression of anti-apoptotic molecules such as Bcl-2 (196) thereby enhancing survival of lymphocytes. It is not surprising, then, that Il7 -gene knock-out mice, or normal mice in which the IL-7/IL-7R system is blocked, exhibit reductions in the numbers of both precursor and peripheral T and CD19+ B cells (197 -199), while overexpression of IL-7 in transgenic mice results in an increase in the numbers of preand pro-B cells in blood and seconda ry lymphoid tissues as well as B220+ B cells in the bone marrow. Interestingly, Il7 transgenic animals develop lymphoproliferative disorders (200). These observations are highly consiste nt with our earlier data showing that NOD mice exhibit an up-regulation of IL-7 expres sion and an abnormally high number of IL7R cells (unpublished data).

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124 The second set of genes of interest involves Car1, Car2 and Car3 (located at 10.5, 10.5 and 11.7 cM, respectively) encoding thr ee isoforms of carbonic anhydrase. Carbonic anhydrases are expressed by a variety of tissues and carry ou t a diverse array of functions. Car2 is of particular interest because of its role in the reversible conversion of CO2 to HCO3 -. Bicarbonate ions facilitate fluid secr etions of the salivary glands and, at the same time, serve as a primary mucosal defense by neutralizing the acidic condition in the esophagus and stomach. Antibody to Car2 has been implicated in a number of autoimmune diseases such as autoimmune chronic pancreatitis (201), T1D (202), systemic sclerosis (203), autoimmune cholang itis (204), primary bilia ry cirrhosis and SjS (32). The presence of anticarbonic anhydrase autoantibodie s in SjS patients focuses attention on an important issue. Under normal conditions, acetylcholine binds to the M3R which activates the PIP2 signaling pathway resulting in the release of intracellular Ca++ that, in turn, facilit ates the opening of Cl-/HCO3 channels. The outflow of both HCO3 and Na+ by simple diffusion changes the ioni c gradient across the cytoplasmic membrane, resulting in the release of water either by simple diffusi on or active transport through aquaporin (AQP)-5 molecules (205). Thus, this inter-relati onship between Car2, acetylcholine, M3Rs and anti-carbonic anhydras e autoantibodies requires further analyses to identify what role it might play in th e pathogenesis of SjS and SjS-like disease. The third set of genes of interest present in the Aec1R1 locus is comprised of several loci associated with retroviruses. Retroviruses have been advanced as being involved in SjS, especially leading to onset of MALT-ly mphomas (206), and represent alternatives to Epstein Bar virus (EBV) a nd cytomegalovirus (CMV). Virus-related genes include ecotropic viral in tegration site-1 (Evi1) at position 14.4 cM, Friend MuLV

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125 integration site-3 (Fim3) at position 14.4 cM, modified polytropic leukemia virus-20 (Mpmv20) at position 13.8 cM, mammary tumo r virus locus-56 (Mtv56) located at 7.5 cM and MMTV LTR integration site-4 (Pad4) found at 10.0 cM. Several of these virusassociated sites, especially Evi1, possess th e potential to induce cellular transformation resulting in leukemia (207). Evi1 serves as an integration site fo r ecotropic retrovirus that can lead to murine myeloid leukem ia by up-regulating cell proliferation and expression of anti-apoptotic molecules (208). Interestingly, Evi1 is also an oncogenic transcription factor that regulates and tran sforms hematopoietic stem cells by targeting the GATA-2 promoter (209). The possible importance of viral activity in SjS-like disease is supported by our recent microarr ay data showing an up-regulation of TLRs associated with (intracellular) viral infections rather than bacterial infections. In summary, the newly developed C57BL/6.NODAec1R1Aec2 mouse model, despite narrowing down the genetic region on ch romosome 3 that regulates the onset of the autoimmune reaction to less than 20 cM, continues to mimic the SjS-like disease of the NOD mouse model. The one exception is the apparent lack of detectable disease in the lacrimal gland of female C57BL/6.NODAec1R1Aec2 mice, suggesting either an overall weaker disease or, more likely, the pres ence of an important gene (or set of genes) located within the 19.8 – 48.5 cM segment of chromosome 3 under gender control. The possibility that a gene or set of genes re gulated differentially by sex hormones might lie within the deleted segment of Aec1 is intriguing in light of the strong focus on gender differences associated with SjS (12). While th is observation is inte resting, the critical issue is the fact that we will be able to compare male and female C57BL/6.NODAec1R1Aec2 and parental C57BL/6.NODAec1Aec2 mice to identify differentially

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126 regulated genes that may provide clues to su ch differences. Whether we are able to further subdivide the 19.2 cM Aec1R1 locus will be a matter of chance given its close location to the centromere, but we an ticipate being able to subdivide the Aec2 region of chromosome 1, thereby moving even closer to identifying specific candidate genes involved in the onset and development of SjS-like disease in this model.

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127 Figure 5-1.Generation of the C57BL/6.NODAec1R1Aec2 mouse line. Parental C57BL/6. NODAec1Aec2 mice were bred with C57BL/6J mice. All F1 offspring were genotyped for the deri vation of microsatellite markers on chromosome 3. An offspring with a cross-over at position 19.2-19.8 cM was selected and bred to produce an inbr ed line with a homozygous recombinant chromosome 3.

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128 Figure 5-2.Detection of proteo lytic activity agains t PSP in the salivary gland tissues of C57BL/6.NODAec1R1Aec2 mice. Saliva was collected and assayed for protease activity on an oligopeptide c ontaining the NLNL enzymatic site of PSP. Using HPLC, the whole synthe tic oligopeptide eludes at 13.9 min (A) while the two degradation products of the oligopeptide appear at 9.2 and 12.8 minutes. Saliva from 5 wk old animals (B) is not capable of enzymatically digesting the oligopeptide, while sa liva from 16 (C) and 22 (D) wk old animals showed partial or complete digestion of the oligopeptide. All individual figures are representative.

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129 Figure 5-3.Histological charac terization of sialadenitis and dacryoadenitis of male C57BL/6.NODAec1R1Aec2 mice. Submandibular and lacrimal glands were freshly explanted from male mice euthan ized at 8 (A & D), 10 (B & E) and 19 (C & F) wk of age. The glands were fixed in 10% formalin, embedded in paraffin, sectioned and stained with H/E dye. Infiltrates were present in the submandibular glands as early as 10 wk of age and in the lacrimal glands by 19 wk of age. All individual fi gures are representative. .

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130 Figure 5-4.Histological characte rization of sialadenitis a nd dacryoadenitis of female C57BL/6.NODAec1R1Aec2 mice. Submandibular and lacrimal glands were freshly explanted from female mice euth anized at 8 (A & D), 10 (B & E) and 19 (C & F) wk of age. The glands were prepared similarly as those from the male mice and stained with H/E dye. Infiltrates were present in the submandibular glands as early as 10 wk of age, but no infiltrates were observed in the lacrimal glands.

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131 Figure 5-5.Detection of ANAs in sera of C57BL/6.NODAec1R1Aec2 mice. Serum samples obtained from groups of mice at various ages were pooled, diluted 1:40 and incubated with HEp-2 fixed s ubstrate slides for 30 min at room temperature in a humidified chamber. The substrate slides were then incubated with FITC-conjugated goat anti-mouse IgG diluted 1:50 for 30 min at room temperature and viewed by fluorescence microscopy at 400X magnification.

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132 Figure 5-6.Temporal loss of s ecretory function in C57BL/6.NODAec1R1Aec2 mice. A. Groups of male and female mice were in jected with isopreterenol/pilocarpine, first at 5 wk of age, then at 19-22 wk of age, to stimulate saliva secretion. Saliva was collected from each mouse for 10 min starting 1 minute after injection of the secretagogue. The vol ume of each sample was measured. B. Male and female mice were injected with pilocarpine at 5 wk of age, then at 19-22 wk of age, to stimulate tear excr etion. Tears were collected from each mouse using Zone-Quick*, Phenol Red Th read placed at the intercanthus of the eye for 20 sec. The length of the wet string was measured and the volume converted using the scale provided by the manufacturer. Each measurement was standardized against the weight of the animal. Statistical analysis was performed by Student-Newman-Keuls test.

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133 Figure 5-7.Detection of anti-muscarinic acet ylcholine type-3 receptor antibodies in the sera of C57BL/6.NOD-Aec1R1Aec2 mice. Sera, prepared from groups of 5 wk and 19 wk old male mice, or 5 wk and 22 wk old female mice, were incubated 2 hrs at 4°C with mouse-M3R transfected Flp-In CHO cells. Cells were washed with FACS buffer and incubated for 30 min at 4oC with either FITC-conjugated goat anti-mouse IgM, anti mouse IgG1, anti-mouse IgG2b, anti-mouse IgG2c or anti-mouse IgG3 an tibodies. After a final wash with FACS buffer, the cells were resuspended in FACS buffer and analyzed using a FACScan cytometer. Fluorescence pr ofiles of anti-M3R staining (filled histograms) are plotted against the secondary antibody onl y profiles (solid line).

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134 Figure 5-8.Time-dependent loss of amylase activity in the saliva of C57BL/6.NODAec1R1Aec2 mice. Saliva samples were colle cted from individual female and male mice at the indicated ages, diluted 250-fold in H2O and assayed for amylase activity by incubating each samp le with ethylidenepNP-G7 at 37oC. The absorbances for each reaction were measured at 1 and 2 min of incubation and the activity calculated according to the manufacturer’s formula. Salivary protein concentrations (mg/ml) of the pooled samples were 113.5 ± 19 (5-10 wks), 163.1 ± 23 (16-17 wks) and 162.2 ± 30 (19-22 wks). Enzymatic activities (U/mg), then, were 5.4 (5-10 wks), 5.7 (16-17 wks) and 3.7 (19-22 wks).

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135Table 5-1.Caspase-3 activity in the saliv ary and lacrimal glands of C57BL6.NOD. Aec1R1Aec2 mice. Time-dependent increases in caspase-3 activity in the salivary and lacrimal glands of C57BL/6.NODAec1R1Aec2 mice ________________________________________________________________________________________________________ Age Sex Number of Submandibular Gland Lacrimal Gland Animals _________________________________________________________________________________ Expt 1 Expt 2 Expt 1 Expt 2 ________________________________________________________________________________________________________ 4 wksa F 2 1380 ± 260 (1.0) b 773 ± 69 (1.0) 198 ± 25 (1.0) 241 ± 1 (1.0) 14 wks F 4 2936 ± 323 (2.1) 1999 ± 208 (2.6) 350 ± 15 (1.8) 520 ± 86 (2.2) 17 wks F 4 2492 ± 123 (1.8) 1896 ± 299 (2.5) 549 ± 90 (2.8) 546 ± 175 (2.3) ________________________________________________________________________________________________________ a Values obtained at 4 wks were used as ba seline controls, permitting dete rmination of the fold change in caspase-3/CPP32 activit y within experimental samples at 14 and 17 wks b Values in parentheses are the fold-increase ove r the baseline values determined at 4 wks

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136 CHAPTER 6 OVERALL CONCLUSION Autoimmune disease occurs when a specifi c, adaptive immune response is mounted against self antigens, a consequence of breakdown in sel f-tolerance. Sjögren’s syndrome (SjS) is a chronic human autoimmune diseas e, characterized by the loss of exocrine secretory function resulting from an immunological attack, pr imarily against the acinar tissue of the salivary and lacrim al glands. This attack lead s to the loss of secretion of saliva, termed xerostomia (dry mouth) and/or tears, termed xerophthalm ia (dry eyes). In addition, dryness of mucosal surfaces suppor ted by exocrine glands, such as skin, gastrointestinal tract, lungs, and vagina, has also been observed in patients with SjS. Involvement of the heart, blood vessels, musc les, kidneys, pancreas, liver, and brain is also common in these patients. The diseas e can be deadly in instances where patients develop low grade B cell lymphoma result ing from lymphocyte infiltration in the exocrine glands. According to the European criteria, the fre quency of primary SjS is estimated to be 0.6%-2.1%. In the United States alone, there are approximately 4 million people diagnosed with chronic SjS. There are tem porary reliefs to the symptom of dryness in SjS. Unfortunately, the cure for this dis ease still remains beyond our reach. Due to the wide spread biological dete rioration this disease has on the human body, patients with SjS live a very diminishing quality of life, physically and emotionally. Their compromised way of life also creates a tr emendous pressure on so cial, economical, and familial well-beings.

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137 Focus of the Dissertation The Role of Complement in the Pa thogenesis of SjS-like Autoimmune Exocrinopathy Administration of NOD.B10H2b mice at 10 weeks of age with (CVF), known to deplete C3 from circulation, shown to reduce the severity of lymphocyte infiltration in the salivary glands, decrease in the producti on of autoantibodies, and restore normal salivary gland function. This reduction in clinical disease severity correlated with significant reductions in the number of CD19+ B cells and co-expressions of CD19/CD21. To precisely expand our unders tanding on the role of C3 in SjS-like autoimmune disease, C3 gene knockout mouse de signated as C57BL/6.NODAec1.Aec2.C3-/was created. In-depth examination of C57BL/6.NODAec1.Aec2.C3-/mouse revealed that the clinical manifestat ions of SjS-like disease were diminished. These included the absence of lymphocytic foci within the exocrine glands, lack of ANAs production, and elimination of IgG1-M3R au toantibody with decreasing level of other IgG isotypic M3R autoantibodies. Most importantly, C57BL/6.NODAec1.Aec2.C3-/mice maintained normal salivary secretion. In addition, significant alteration of splenic B cell subpopulations was identified. In the three male models, C57BL/6, C57BL/6.NODAec1Aec2 , and C57BL/6.NODAec1Aec2.C3-/-, of the CD19+ splenic cells examined, the percent of MZ B cells is similar among the th ree models while sple nic CD19+ FO B cells of the C57BL/6.NODAec1Aec2.C3-/mouse showed a significant drop of close to 9% compared to C57BL/6 and C57BL/6.NODAec1Aec2 mice. However, in the three female models, the number of splenic CD19+ MZ and FO B cells app eared to be similar between the C57BL/6 and C57BL/6.NODAec1Aec2.C3-/mice while there is an increase of 8% in the percent of MZ B cells and decrease of 9% in the percent of FO B cells found in

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138 C57BL/6.NODAec1Aec2 mice compare to C57BL/6 and C57BL/6.NODAec1Aec2.C3-/animals. The Role of STAT6 of IL4 Signaling Pathway in the Pathogenesis of SjS-like Autoimmune Exocrinopathy Numerous studies have demonstrated th e importance of certa in cytokines during the autoimmune process. In SLE for example, IFNbehaves like Th1 cytokine that induces the production of IgG2a and IgG3 antibodie s. It is uncertain if the establishment of SLE is mediated by pathogenic ity of the antibodies or the function of the cytokines. In addition, isotypic antibodies against self antigen are highly correlated with the progression and severity of S LE diagnoses. However, in SjS, the role of isotypic autoantibody and its correlation to the progression of the dise ase still remain elusive. There are limited reports suggesting a w eak association between the isotypic autoantibodies and some clin ical aspects of SjS. As discussed extensively in Chapter 1, th ere are three specifi c working hypotheses trying to explain the mechanics of how an tibody reacting against M3R could result in secretory dysfunction. As brie fly review, the first hypothesis involves in the masking of the receptor by antibodies preventing the bind ing of acetylcholine resulted in the downregulation of the cascade. Secondly, antibody-M3R complex could initiate internalization of the complex resulting in de gradation of antibody and receptor. Lastly, chronic binding of antibodies to M3R could re sult in receptor dese nsitization, therefore shutting down the whole cascade of downstream events responsible for fluid secretion. Therefore, the secretion of fluid-rich saliva is mainly regulated by M3R. Any perturbation to this system such as bindi ng of autoantibodies coul d result in glandular dysfunction.

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139 To further examine the role of isotypic switching to IgG1/IgE antibody against M3R, we have generated the NOD.B10H2b.CSTAT6-/mouse. In addition to the inability to produce IgG1 and IgE antibody in serum, NOD.B10H2b.CSTAT6-/mouse exhibited a number of char acteristics observed in dis ease-prone NOD/Lt and NOD.B10H2b mice. These included the presence of leukoc ytic infiltration of the submandibular and lacrimal glands, the production of anti-nuc lear antibodies and autoantibodies reactive with tissues of the salivary gl ands, and the activation of a PSPspecific serine protease. Most importantly, NOD.B10H2b.C. STAT6-/mouse does not -exhibit th e temporal loss of stimulated salivary flow rates and failed to produce an IgG1-M3R specific autoantibody. Genetics of SjS-like Au toimmune Exocrinopathy Genetics linkage to the development of human SjS has always remained controversial. Reports asso ciating disease susceptibility encoded by genes of the major MHC such as HLA-DR and etiology of SjS pr ovide to be highly inconsistent when examined in larger human population. As of now, it is still challenging to determine genetic factors linking human SjS. However, in the mouse model of SjS, genetic manipulations of a variety of mice can either result in the appearance of some phenotypic characteristics of human SjS, or delay / prev ent development and onset of pre-clinical and clinical disease. Conveniently, the mouse system provides a bett er tool to identify genetic regions of the mouse genome that pr edisposes mice to develop SjS-like disease hoping to interpolate these genetic interval s to human population. NOD mouse has been well established as a model for type I diabetes , and as we have characterized it as an appropriate model for SjS. Therefore, ther e must be a genetic f actor presence in the NOD that can recapitulate SjS. We have identified the Idd3 and Idd5 genetic regions

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140 derived from NOD mice that once transfe rred into the SjS nonsusceptible C57BL/6 mouse can recapitulate the entire SjS-like disease phenotypes . The two identified genetic regions, Idd3 and Idd5, provide a great opportunity to explore the candidate genes which lie with in these two regions, thereby possibly contribute to development of SjS. The Idd3 locus span of about 48.5 cM on chromosome 3 of C57BL/6.NODAec1Aec2 mice, which has been known to regulate the immunological aspects of SjS, includes th e initiation of leukocy tic infiltration and autoimmune attack on the exocrine glands. The newly generated mouse designated as C57BL/6.NODAec1R1Aec2 , in which its idd 3 locus has been reduced to a centromeric segment of about 19.2 cM proves to be a very useful mouse model to study genetic of SjS-like disease. Profiling of C57BL/6.NODAec1R1Aec2 mouse showed that both male and female C57BL/6.NODAec1R1Aec2 mice exhibited the pathophysiological abnormalities, including up-regulated caspase-3 activity which is an indicative of cell apoptosis and aberrant expression of PSP. Both male and female NODAec1R1Aec2 mice exhibited the clinical SjS-like diseas e of the salivary gla nd, while male, but not female, C57BL/6.NODAec1R1Aec2 mice manifested the clinical dacryoadenitis. The dichotomy in dacryoadenitis and xer ophthalmia that occurs between male and female is quite intriguing. Once again it reinforces the conno tation regarding the association between gender difference and the initiation of the autoim mune process. It would be very interesting to narrow the region that still exist and the region that has been lost, and see how each contributes to autoimm unity and how it contributes to the gender difference.

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141 The Clinical Implications in Translational Research The aspect that eliminating C3 by tem poral depletion or genetic knockout in animal predisposed to SjS-like disease could ameliorate immunological and clinical manifestation of SjS is amazingly astonishing. As presented in Chapter 2 and 3, C3 plays a very critical role in the autoimmune pr ocess of SjS in animal models. In human population, the role of C3 in SjS remain s quite controversial and debatable. Unfortunately, the debate has gone too long, and the controversy has never been resolved. Due to the lack of research devoted to elucid ating the role of C3 in the pathogenesis of SjS, translational research will be a big chal lenge. There are some reports suggesting that low level of C3 found in SjS patients often asso ciated with an increase risk of developing B cells lymphoma. In addition, hypo-complem entaemia appeared to correlate with unfavorable long term outcome for patients. These preliminary data might argue against the therapy of treating patients by depleting C3 level. The clinical prognosis of the subjected patients mentioned previously c ould be clarified succinctly. All human subjects of the study were at the most advanced stage of the disease. All the C3 and its cleavage molecules have been optimally utilized for the hyperproliferation and hyperactivation of B lymphocytes, especially if that particular B cells population is in the process of becoming transformed B cells. The activity of B lymphocytes at this stage of the disease could explain the hypo-complement aemia phenotype found in some patients. Therefore, patients with a low level of C3 w ould not be clinically benefited if depletion C3 therapy is used. The biggest hurdle that researchers and clinicians will have to face is early detection of the disease. The solution must include universally accepted diagnostic criteria and awareness among the patients to seek professional he lp early. If the

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142 progression of SjS can be de lineated and detected at va rious stages, the temporal depletion of C3 could be very beneficial.

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146 32. Kino-Ohsaki, J., Nishimori, I., Morita , M., Okazaki, K., Yamamoto, Y., Onishi, S., and Hollingsworth, M.A. 1996. Serum antibodies to carbonic anhydrase I and II in patients with idiopathic chroni c pancreatitis and Sjogren's syndrome. Gastroenterology 110:1579-1586. 33. Muller, S., Briand, J.P., Barakat, S., Lagueux, J., Poirier, G.G., De Murcia, G., and Isenberg, D.A. 1994. Autoantibodies reacting with pol y(ADP-ribose) and with a zinc-finger functional domain of poly(ADP-ribose) polymerase involved in the recognition of damaged DNA. Clin. Immunol. Immunopathol. 73:187-196. 34. Tan, E.M. 1989. Antinuclear antibodies: diagnostic markers for autoimmune diseases and probes for cell biology. Adv. Immunol. 44:93-151. 35. Wu, A.J., Chen, Z.J., Tsokos, M., O'Connell, B.C., Ambudkar, I.S., and Baum, B.J. 1996. Interferon-gamma induced cell death in a cultur ed human salivary gland cell line. J. Cell Physiol. 167:297-304. 36. Cavill, D., Waterman, S.A., and Gordon, T.P. 2004. Antibodies raised against the second extracellular loop of the human muscarinic M3 receptor mimic functional autoantibodies in Sjogren's syndrome. Scand. J. Immunol. 59:261-266. 37. Cha, S., Singson, E., Cornelius, J., Yagna, J.P., Knot, H.J., and Peck, A.B. 2006. Muscarinic acetylcholine type-3 receptor desensitization due to chronic exposure to Sjogren's syndrome-associated autoantibodies. J. Rheumatol. 33:296-306. 38. Li, J., Ha, Y.M., Ku, N.Y., Choi, S.Y., L ee, S.J., Oh, S.B., Kim, J.S., Lee, J.H., Lee, E.B., Song, Y.W., et al. 2004. Inhib itory effects of autoantibodies on the muscarinic receptors in Sjogren's syndrome. Lab. Invest. 84:1430-1438. 39. Waterman, S.A., Gordon, T.P., and Risc hmueller, M. 2000. Inhibitory effects of muscarinic receptor autoantibodies on parasympathetic neurotransmission in Sjogren's syndrome. Arthritis Rheum. 43:1647-1654. 40. Dawson, L.J., Allison, H.E., Stanbury, J., Fitzgerald, D., and Smith, P.M. 2004. Putative anti-muscarinic antibodies cannot be detected in patients with primary Sjogren's syndrome using conven tional immunological approaches. Rheumatology (Oxford) 43:1488-1495. 41. Dawson, L.J., Field, E.A., Harmer, A.R., and Smith, P.M. 2001. Acetylcholineevoked calcium mobilization and ion channel activation in human labial gland acinar cells from patients with primary Sjogren's syndrome. Clin. Exp. Immunol. 124:480-485. 42. Smith, A.J., Jackson, M.W., Wang, F., Cavill, D., Rischmueller, M., and Gordon, T.P. 2005. Neutralization of muscarinic receptor autoantibodies by intravenous immunoglobulin in Sjogren syndrome. Hum. Immunol. 66:411-416.

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162 BIOGRAPHICAL SKETCH Cuong Quoc Nguyen was born in Phuoc Long City, Vietnam, in August of 1976. In the summer of 1991, his family was transferred to a refugee camp in Bataam of the Philippines. After eight months of stayi ng in the camp, they immigrated to Crete, Nebraska, where he graduated from Crete Jr. Sr. High School in May 1995. Dr. Nguyen went on to receive his Bachelor of Science degree major in biochemistry and biology from University of Nebraska-Lincoln in th e spring of 1999. After two years working at the University of Nebraska Medical Center on the immortalization of pancreatic islet cells using telomerase, he at tended graduate school at th e University of Florida in August, 2001. Dr. Nguyen took a year off from graduate school to take care of his newborn daughter. In the spring of 2003, He re turned to the University of Florida to complete his Doctor of Philosophy in immunology under the guidance of Dr. Ammon Peck.