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

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Permanent Link: http://ufdc.ufl.edu/UFE0015481/00001

Material Information

Title: Role of Complement, B Cells, and Genetics in Development of Sjogren's-Like Autoimmune Exocrinopathy
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0015481:00001

Permanent Link: http://ufdc.ufl.edu/UFE0015481/00001

Material Information

Title: Role of Complement, B Cells, and Genetics in Development of Sjogren's-Like Autoimmune Exocrinopathy
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0015481:00001


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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.











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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.


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' 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