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Adoptive Transfer Studies to Establish a Model of Phase II Exocrine Gland Dysfunction in the NOD Model of Sjogren's Syndrome

University of Florida Institutional Repository

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ADOPTIVE TRANSFER STUDIES TO ESTABLISH A MODEL OF PHASE II EXOCRINE GLAND DYSFUNCTION IN THE NOD MODEL OF SJGREN'S SYNDROME By VINETTE B. BROWN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2004

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Copyright 2004 by Vinette B. Brown

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This document is dedicated to the graduate students of the University of Florida.

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ACKNOWLEDGMENTS Working on this project has been a wonderful experience. As a master's student I had the opportunity to learn from very knowledgeable professors and researchers. Many people assisted me in the completion of my degree, for which I am very thankful. First and foremost, I would like to thank Dr. Ammon B. Peck who has provided a great deal of support and advice. I am so grateful to have worked under someone who is extremely knowledgeable but is also willing to listen to new ideas. I would like to thank Dr. Michael Humphreys-Beher for allowing me to enter his lab and giving me my very first project. I am very greatful for the help I received from my committee members, Dr. Sally Litherland and Dr. David Ostrov, who opened their labs and offices to me. I would like to thank my entire lab who has provided an immense amount of support. I would like to thank Janet Cornelius and Dr. Seunghee Cha who aided me with their years of experience and lots of their time. I also thank Lori Boggs, Daniel Saban, Eric Sing-Son and Smruti Killedar who have both provided lots of technical assistance and advice. I am very grateful for all of the support I received outside of the Peck lab. I thank Dr. Clare-Salzler's laboratory, Dr. Petersen's laboratory, Dr. Burne's laboratory, Dr. Oh's laboratory, and Dr. Khan's laboratory for all of the assistance. I would also like to thank my friends who provided much needed moral support and a great deal of advice, specifically, Grace Kim and Dr. Kenyon Meadows. iv

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TABLE OF CONTENTS page LIST OF TABLES............................................................................................................vii LIST OF FIGURES.........................................................................................................viii ABSTRACT.......................................................................................................................ix CHAPTER 1 INTRODUCTION........................................................................................................1 Background of Sjgrens Syndrome.............................................................................1 Animal Models.............................................................................................................5 Adoptive Transfer Studies..........................................................................................13 2 MATERIALS AND METHODS...............................................................................16 Animals.......................................................................................................................16 Adoptive Transfer.......................................................................................................16 Saliva Collection.........................................................................................................17 Protein Concentration.................................................................................................18 Amylase Activity Analysis.........................................................................................18 Apoptosis Detection....................................................................................................20 Apoptosis Detection via Flow Cytometry...........................................................20 Akt Expression via Polymerase Chain Reaction.................................................22 Agarose Gel Electrophoresis......................................................................................23 Caspase-3 Activity Detection..............................................................................24 In Situ Apoptosis Detection Kit..........................................................................24 Detection of Infiltration..............................................................................................25 3 RESULTS...................................................................................................................27 Adoptive Transfer.......................................................................................................27 Saliva Collection.........................................................................................................28 Amylase Activity Analysis.........................................................................................29 Amylase Activity Detected in Saliva..................................................................29 Detection of Salivary Gland Amylase Activity...................................................29 Protein Concentration..........................................................................................30 Apoptosis Detection....................................................................................................32 v

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Apoptosis Detection via Flow Cytometry...........................................................32 Caspase-3 Activity Detection..............................................................................34 Akt Expression via Polymerase Chain Reaction.................................................34 In Situ Apoptosis Detection Kit..........................................................................36 Detection of Infiltration..............................................................................................36 4 DISCUSSION.............................................................................................................40 LIST OF REFERENCES...................................................................................................49 BIOGRAPHICAL SKETCH.............................................................................................52 vi

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LIST OF TABLES Table page 3-1 Adoptive transfer, combinations of donor splenocytes transferred to the scid........28 3-2 The saliva from each adoptively transferred scid was collected and quantified......31 3-3 The submandibular glands for the adoptively transferred scid mice and the controls were collected and homogenized................................................................32 3-4 Apoptosis detection using Apo-Direct kit (BD Pharmingen)..................................33 3-5 Akt expression ratios................................................................................................35 3-6 Focus score derived from H+E stained slides of smg glands...................................39 vii

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LIST OF FIGURES Figure page 1-1 Apoptosis pathway. Notice that AKT is an inhibitor early in the pathway and Caspase-3 one of the last activated caspases (7)........................................................9 3-2 Immunohistochemical staining of submandibular glands for lymphocyte-infiltrations...............................................................................................................37 3-3 Hematoxylin/eosin-stained tissue sections of submandibular glands......................38 viii

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science ADOPTIVE TRANSFER STUDIES TO ESTABLISH A MODEL OF PHASE II EXOCRINE GLAND DYSFUNCTION IN THE NOD MODEL OF SJGREN'S SYNDROME By Vinette B. Brown August 2004 Chair: Ammon B. Peck Major Department: Medical Sciences Sjgrens Syndrome (SjS) is an autoimmune exocrinopathy that results in the development of xerostomia (dry mouth) and keratoconjunctivitis (dry eyes). There are two distinct phases of SjS. Phase I which is lymphocyte independent and phase II which is lymphocyte-dependent. The non-obese diabetic (NOD/LtJ) mouse displays symptoms associated with both phases of the disease and, therefore, is a useful model for studying SjS. The insertion of the scid mutation into the NOD/LtJ background has led to the development of the NOD.CB17-PrkdcScid/J (scid). The scid mouse develops phase I of SjS but as a result of the absent lymphocytes, development of phase II does not occur. The lack of mature lymphocyte development in the scid allows it to be used as a recipient in adoptive transfer studies to determine the combination of lymphocytes required to induce phase II of SjS. ix

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The work described in this thesis involves the transfer of lymphocyte combinations derived from specific strains, all of which have the same NOD/LtJ genetic background, into the scid mouse. Adoptively transferred scid mice were analyzed for the progression of disease. Observation of the salivary flow, saliva proteins, presence of apoptosis and infiltration provided information on progression of disease. Results indicate the adoptive transfer of functional lymphocytes can lead to development of phase II. However, the time required for disease progression may vary depending on the origin of the lymphocytes. The development of this adoptive transfer model has provided parameters for future experiments which may clarify the immunopathology important in progression of phase II and onset of SjS-like disease. x

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CHAPTER I INTRODUCTION Many illnesses decrease the quality of life, often making normal daily tasks extremely difficult. Sjgrens syndrome (SjS) is one such disease, which often causes the patient great discomfort. Patients afflicted with SjS have difficulties eating and speaking, due to an inability to secrete saliva. These patients also have dry eyes; therefore, they have a constant feeling of irritation in the eyes. The etiology of SjS is not known and a cure is not available. The current treatment for SjS is salivary and tear stimulants (1). These stimulants are sometimes effective, providing a limited amount of relief, but they can also have undesirable side effects. By understanding the underlying causes and mechanism of SjS, a more complete treatment can be devised. Background of Sjgrens Syndrome In 1932, Henrik Sjgren reported the triad of keratoconjunctivitis sicca (dry eyes), xerostomia (dry mouth), and rheumatoid arthritis (2). This triad of symptoms became known as Sjgrens Syndrome, a chronic autoimmune disorder in which the secretory functions of various exocrine glands are disrupted. The major areas affected are the lacrimal and salivary glands, as well as the skin, upper respiratory, gastrointestinal tract and the genitals. The resulting dryness of mucosal surfaces results in various complications such as gastrointestinal discomfort, difficulty speaking, fatigue and musculoskeletal complaints (3). The disease is not usually fatal, but can be quite distressful for those affected. Furthermore, due to the immunological basis of the disease, patients with SjS are at an increased risk of developing a non-Hodgkins B-cell 1

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2 lymphoma. At this time, the exact etiology is not known, so patients remain uncured. Today, patients are usually treated with secretagogues, which induce lacrimal and salivary secretion, along with other glandular secretion. While secretagogues are readily available, a patient may not receive this treatment due to the difficulties diagnosing SjS. As with many systemic diseases, SjS appears with a wide range of clinical manifestations. These variations, may lead clinicians to misdiagnose symptoms. There are numerous reports describing lung, renal and central nervous system (CNS) involvement (4). Over the years, a number of disease classifications (San Francisco, San Diego, California, Japanese, European original and European revised), each with their own methods and standards for diagnosing Sjgrens syndrome, have been proposed. Among the different classification systems, xerostomia (dry mouth) is usually diagnosed by a lip biopsy. The demonstration of focal lymphocytic infiltrates, on a minor salivary gland (SG) biopsy, has remained the gold standard for the oral component of SjS (2). A salivary gland is considered infiltrated when there are clusters of fifty or more lymphocytes, known as foci, present. For most classification systems the presence of two foci per 4 mm 2 is required to identify the gland as being infiltrated. A less invasive measure of xerostomia is the use of scintigraphy and sialography. Scintigraphy is a technique where an appropriate, short-lived gamma-emitting radioisotope is introduced. Through radiographic imagery the uptake, concentration and excretion of the radioisotope by the major salivary gland (submandibular) is measured. This can be a very sensitive test for glandular function. Sialography is a similar method in which the release of saliva by the salivary glands is evaluated via nuclear imaging

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3 Keratoconjunctivitis (dry eyes) is typically diagnosed by tear volume measured by what has become known as the Schirmer test. This test measures tear stability by the non-invasive break up time and usage of a rose bengal dye to stain the ocular surface, which results in identification of epithelial cell destruction (5). Serologic tests for the detection of autoantibodies, specifically SS-A (anti-Ro), SS-B (anti-La), and anti-nRNP, are also used for correct diagnosis of SjS. In eighty-six Sjgrens syndrome patient sera, more than 96% had SS-A, and 87% had SS-B, compared to 95% of patients with anti-nRNP (6). Serology not only facilitates diagnosis, but also can be useful in predicting the subsequent outcome and complications in patients with primary Sjgrens syndrome (2). The presence of anti-SS-A antibodies may identify patients with systemic disease (7), and in anti-SS-A/ anti-SS-B positive patients, the relative risk of developing non-Hodgkin lymphoma has been reported as high as 49.7%, within 10 years of diagnosis (4). There are two types of Sjgrens syndrome. Individuals that exhibit the classic symptoms (dry eyes and dry mouth), in the absence of another autoimmune disease, are diagnosed as having primary SjS. Secondary SjS includes the presence of the aforementioned symptoms, in addition to another autoimmune rheumatic disease, such as rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE). Due to the variability in classification systems, the exact percentage of patients with SjS is not known, but it is estimated to have a prevalence not exceeding 0.6% of the general population (2). As with most connective tissue autoimmune diseases, there is a sexual dimorphism present in Sjgrens syndrome. There is a 9:1 ratio of women to men in SjS, with most women presenting symptoms of the disease between the ages of 40 and 60 years of age.

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4 A prominent feature of Sjgrens syndrome is the genetic predisposition (2). It is not uncommon for two or more cases of SjS to occur within a family. Polymorphisms in the major histocompatibility complex (MHC) genes are the best documented genetic risk factors for the development of autoimmune diseases overall (3). In Sjgrens syndrome, the most relevant MHC complex genes are the class II genes, specifically the HLA-DR and DQ alleles (8). However, variations in SjS associated haplotypes amongst different ethnicities, makes it difficult to establish which of the genes confers risk. The severity of the disease also seems to be dependent on the combination of the risk-associated alleles. Sjgrens syndrome patients with DQ1/DQ2 alleles have a much more severe autoimmune disease then patients with any other allelic combination at HLA-DQ (6). Even with specific gene combinations, environmental factors may play a role in the disease onset. Among the possible etiologic factors, viral infections are the most often proposed as a possible trigger of autoimmune disease. Potential viral triggers include Epstein-Barr virus and Hepatitis C. Furthermore, a possible relationship between Sjgrens Syndrome and Helicobacter pylori infection has been suspected (2). With these concurrent infections, the risk of mucosa-associated lymphoid tissue lymphoma increases in patients with SjS (2). Dentists and the ophthalmologists encounter patients afflicted by SjS most often, although a few dermatologists are faced with treating patients presenting cutaneous lesions, in addition to the classic symptoms. A dentist may encounter a SjS patient who complains of dryness in the oral cavity, difficulty swallowing, and a burning sensation in the throat. A patient in the ophthalmologists office would complain of dryness in the eye, which could lead to infections, keratitis, and sometimes, melting of the cornea or

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5 ocular perforations (5). The cutaneous lesions in patients with SjS are described by dermatologists as palpable and nonpalpable eruptions, pruritic or non-pruritic, as well as symmetric or nonsymmetric in distribution (2). The dermatologist may not recognize these lesions as being associated with SjS and thus, misdiagnose it as Wegeners granuloma. There is still much to be learned about this disease, and once the information is attained it will aid in recognition and treatment of SjS. Animal Models The current classification systems provide standards to aid clinicians in diagnosing SjS. However, clinicians are inhibited in their ability to make a diagnosis because the exact onset of the disease is difficult to determine. The onset is usually around forty years of age; however, there are cases of juvenile SjS. Research of SjS within the human population is impeded by the ethical and legal limitations of collecting patients salivary glands. To study the pathology and etiology of SjS, a variety of mouse models have been developed, such as the New Zealand Black (NZB) and the MRL/n substrains. Today, the mouse used most often is the non-obese diabetic (NOD/LtJ) mouse. The NOD/LtJ (NOD) is characterized has having insulitis, a leukocytic infiltrate of the pancreatic islets. NOD mice have marked decreases in insulin production by the age of 12 weeks. The NOD/LtJ is considered diabetic when there is moderate glycosuria, and the non-fasting plasma glucose is higher than 250 mg/dl (9). The NOD mouse developed early out of breeding experiments with the Cataract Shionogi (CTS) strain (9). The CTS mice were selected for a high fasting glucose level. From those selected mice a few spontaneously developed overt insulin-dependent diabetes mellitus with insulitis (IDDM), which is now the NOD/LtJ used today. In the NOD mouse, leukocytic infiltrates and antibodies destroy the pancreatic islets, with exocrine glands infiltrated as well. Of particular interest to SjS

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6 researchers is the observation of lymphocytic foci present in the major salivary gland (submandibular) (10), tear-producing gland (lacrimal) and thymus (11). NOD mice have destruction of the submandibular gland, a marked decreased in salivary flow, and changes in the salivary protein composition. Sera from NOD mice is positive for antinuclear antibodies (SS-A and SS-B), which are detectable by nuclear staining. These symptoms are similar to those seen in humans with secondary Sjgrens Syndrome, thus making the NOD a good mouse model for SjS research. The pathogenesis of the SjS-like disease seen in the NOD mouse can be divided into two phases. Phase I is a lymphocyte-independent phase where there are abnormalities intrinsic to the submandibular and lacrimal glands of the NOD. Phase II is marked by the autoimmune response via lymphocytic infiltrates in the submandibular (sialoadenitis) and lacrimal (dacryoadenitis) glands, followed by a decrease in tear and saliva production. The lymphocytic infiltrates appear as periductal foci within the glandular architecture of the salivary and lacrimal glands (12). The cause of the autoimmune reaction may reside in the target organ of the autoimmune response, in the immune system, or in both (13). It is theorized that the loss of secretory function is due to lymphocyte-directed destruction. Increased numbers of apoptotic epithelial cells have been detected in the minor salivary glands of patients with SjS (13). The phase II occurs in the NOD mouse around 10 weeks of age. Current evidence suggests the existence of genetically programmed abnormalities in the exocrine glands of the NOD mouse that may contribute to initiation of the autoimmune reaction (11). Antigen presenting cells, such as dendritic cells, are important mediators of lymphocyte activation, and may respond to organ abnormalities. There is evidence of accumulation of

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7 dendritic cells in the submandibular gland (smg) before the development of lymphocytic infiltrates (13). When these lymphocytic infiltrates form in phase II, dendritic cells interact closely with T-cells, possibly serving to locally activate autoreactive T-cells (11). These organized lymphocytic infiltrates seen in the NOD are also described in the biopsies of SjS patient (14). As stated before, phase II of SjS is lymphocyte-dependent in humans and the NOD. Research indicates that there is an inappropriate response of dendritic cells to abnormalities of the smg and thus an activation of nave autoreactive T-cells. These autoreactive T-cells are then capable of stimulating an inflammatory response and autoantibody production. In addition, the dendritic cells of the NOD were found to have a decreased ability to stimulate T suppressor cells (11). T suppressor cells are important regulators of the immune system and have been shown to posses the capacity to alter the stimulation of autoreactive T-cells (15). Infiltrates of exocrine tissues primarily consist of CD4 + T-cells with a minority of CD8 + T-cell and B-cell populations (12). Studies indicate that activated T-cells, predominantly CD4 + T-cells, are necessary for the initiation of autoimmune disease, as indicated by T-cell transfer studies (12). The infiltrates also exhibit aberrant production of the pro-inflammatory cytokines IL-, IL-2, IL-6, IL-7 IL-10, IL-12, IFN-, and TNF-. Cytokines are important mediators in the immune response, and their altered production can lead to an inappropriate stimulation of lymphocytes. In addition to the changes in proteins seen in phase I of SjS, there are increases in apoptosis of the submandibular gland acinii (13). Apoptosis, otherwise known as programmed cell death, is an important inhibitory mechanism of cell growth, aiding in

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8 organogenesis, maintaining tissue morphology, and deletion of autoreactive lymphocytes (16). As seen in Fig. 1-1, apoptosis is an complex pathway with many stimulators, such as Caspase-3, and inhibitors, such as AKT. Apoptosis is highly regulated due its destructive potential. Evidence shows that apoptotic cells can induce inflammation and/or result in the formation of cryptic epitopes (13). Previous studies show increase levels of apoptosis in the scid mouse and the NOD, compared to the BalB/c and young NOD (17). The increased levels of apoptosis in the scid mouse, indicates that apoptosis may be one of the regulatory abnormalities of the submandibular gland present in phase I (18). The increased cysteine proteases in the scid mouse implies that apoptosis is lymphocyte-independent in phase I. However, cysteine proteases have been shown to increase in activity as the NOD aged from 8 weeks to 20 weeks, with the scid mouse showing the highest level of activity (17). The humoral response plays an important role in phase II of SjS. In addition to the SLE antibodies (SS-A and SS-B), anti-M 3 muscarinic acetylcholine receptor autoantibodies have been identified in humans (19) and the NOD mouse (17). SS-A and SS-B are antibodies targeted to antigens that are usually confined to the nucleus (20); however, due to certain events in phase I of SjS these antigens may be presented to the immune system. Muscarinic acetylcholine receptors are important in the secretory function of mammalian exocrine glands. The muscarinic-cholinergic receptor is stimulated by neurotransmitters, resulting in a signal transduction that allows fluid secretion from the salivary gland acinar cells (17). The interaction between an anti-M 3 antibody and the M 3 receptor may result in down-regulation of receptor density, as well as postreceptor second messenger pathway signaling events necessary for proper

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9 activation of fluid movement through epithelial cells (20). In studies where the anti-M 3 antibody and other autoantibodies were introduced into mice lacking the adaptive immune response (scid mutation), the anti-M 3 antibody was the only antibody capable of inhibiting secretory function (20). The development of autoantibodies could be a primary immunological response or a secondary effect. In the case of SjS, it is most plausible that the presence of autoantibodies is a secondary effect of the immune system activation (17). Interestingly, many of the autoantibodies are targeted to cell surface proteins important in the secretory response (20), possibly explains why other areas (skin, lungs, GI tract and vaginal tissues), besides the smg and lacrimal glands, are affected in SjS. Figure 1-1. Apoptosis pathway. Notice that AKT is an inhibitor early in the pathway and Caspase-3 one of the last activated caspases (16).

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10 The NOD mouse has allowed researchers insight into the phase I of SjS, plus the subsequent development of clinical manifestations. However, a multitude of factors important in the initiation and progression of the disease are not known. Through the development of congenic NOD strains, specific aspects of SjS can be studied. The NOD.CB17-PrkdcScid/J (scid) mouse is a congenic mouse that was developed by backcrossing the immunodeficiency locus (scid) onto the NOD genetic background. The scid locus confers a functional loss of the B and T lymphocytes, otherwise the NOD-scid mouse retains the genetic profile of the NOD/LtJ. The NOD-scid mouse clarifies the significance of the genetic background in the initiation of SjS, as well as the role of lymphocytes in the clinical manifestations. In addition, the scid mouse is capable of accepting allogenic and xenogenic grafts, making it a good mouse model for adoptive cell transfer studies. There is evidence of intrinsic factors present in the salivary glands of the NOD mouse that are capable of triggering an autoimmune response (14). Analysis of the NOD-scid mouse can clarify the role genetics has in the onset and progression of the disease. During phase I, the NOD-scid mouse shows a morphological change in the salivary glands, involving a loss of acinar cells within the submandibular glands. The loss of acinar cells increases with age and may be due to either hyperproliferation of the submandibular ductal cells, or apoptotic events (21). However, there are dramatic increases in cysteine protease activity (enzymes important in programmed cell death) in the submandibular glands of older NOD mice and NOD-scid mice (12). While the scid mouse depicts the same glandular changes as the NOD mouse, the salivary flow rate does not decrease in the scid. It is possible; therefore, that there is compensation by the minor

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11 salivary glands (parotid, sublingual, and those in the oral mucosa), resulting in a stable saliva production (21). Nevertheless, data strongly suggest that lymphocytes are necessary for disease progression and the loss of secretory function. Although salivary flow remains stable, there are changes in its composition. The composition of salivary proteins, as shown by epidermal growth factor (EGF) and amylase concentrations in NOD-scid saliva, show significant changes with age (21). There is also a novel expression of PRP and an internally cleaved PSP isoform (27 kDa), prominent in 15-week-old NOD-scid mouse saliva, but not detected in normal BALB/c mice or younger NOD-scid mice (21). These protein changes are similar to those observed in the NOD/LtJ. Interestingly, the time at which this new isoform of PSP appears in the saliva and submandibular glands of NOD-scid mice coincides with the appearance of lymphocytic infiltrates in the salivary glands of NOD mice (21). It is possible that the new isoform of PSP, intrinsic to the NOD mouse and its progenitors, may present as a foreign antigen to the NOD immune system, thus initiating the autoimmune response. In the NOD mouse, there is stimulation of autoreactive T-cells and autoantibodies. The stimulation of both branches of adaptive immunity may result in the progression of the disease to phase II of SjS. The NOD-scid aids in clarifying the roles of genetics and lymphocytes in the initiation and progression of SjS. It is been made clear, by observing the scid mouse, that lymphocytes are important in the loss of saliva secretion. However, the mechanism by which lymphocytes affect the salivary gland function is unclear. There are a few factors of the immune system that may separately, or collectively, inhibit saliva secretion. There is evidence of apoptotic activity in the salivary glands in the NOD mouse (13). The NOD

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12 shows the presence of antibodies specific to exocrine gland receptors, which are capable of reducing salivary flow (20). As stated previously, there are a variety of aberrantly expressed cytokines. The possible pathological effects of these immune responses, provide a range of areas to be researched. In order to study the different immunological aspects, we examined congenic, NOD-derived strains. Two interesting strains used in this study are the NOD.Igh6 tmlCgn commonly known as NOD.Ig null and for convenience in this thesis, Ig null and NOD.129P2(B6)-Il4 tmtCgn /DvsJ, commonly know as NOD.IL-4 -/and for convenience in this thesis, IL-4 -/. The NOD.Ig null mouse is a congenic partner strain of the NOD mouse that is deficient in B-cells. This mouse was developed by disrupting one of the membrane exons of the gene encoding the -chain constant region by gene targeting in mouse embryonic stem cells (13). NOD.Ig null mice exhibit glandular abnormalities of the NOD. Aberrant PSP production is present, but exocrine gland dysfunction (xerostomia, keratoconjunctivitis) does not occur (21). These data imply that B-cells have a significant role in the appearance of xerostomia and keratoconjunctivitis sicca. The B-cell may be an important antigen presenting cell, capable of stimulating autoreactive T-cells. The B-cell also produces autoantibodies present in the NOD mouse and patients with SjS. Transfer of serum from human patients into the Ig null mice results in a decrease in salivary flow, thus providing evidence that the B-cell is important as an antibody producer, rather than as an antigen presenter (22). This also implies a role for autoantibodies as the effector mechanism for secretory inhibition. The NOD mouse has a cytokine profile that is specific to the appearance of infiltrates in the salivary glands. The cytokines present consist of interferon (IFN)-,

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13 tumor necrosis factor (TNF)-, IL-1, IL-2, IL-6, IL-12 and IL-18, but usually lacking detection of IL-4 (23). Due to its absence, IL-4 has been disregarded as important in progression of the disease. However, the increasing evidence of autoantibodies as an effector mechanism of secretory loss supports a possible role for IL-4 in development of phase II of SjS. The IL-4 cytokine regulates the B-cell maturation and the switch from the IgM to IgG 1 antibody. NOD.IL 4 -/mice exhibit aberrant glandular formations, the novel PSP isoforms, and glandular infiltrations. However, the IL-4 -/mouse does not exhibit the loss of salivary flow. The B-cells, unable to class switch, can not produce autoantibodies specific to exocrine gland receptor, such as the M 3 R. This findings support the theory that the humoral response is the mechanism by which exocrine gland secretion is disrupted. Adoptive Transfer Studies The use of various gene knockout NOD strains allow different aspects of the disease to be investigated. The NOD-scid clarifies the roles of genetics and the immune response. In addition, the roles of B-cells and the IL-4 cytokine are elucidated by observing the NOD.Ig null and NOD.IL-4 -/. All of these strains have the glandular abnormalities and protein production characteristic of phase I of SjS, but development of xerostomia or keratoconjunctivitis does not occur. These observations, as well as serologic analysis of the NOD mouse and patients with SjS, suggest that the humoral response is a pathologic mediator of SjS. By utilizing the adoptive transfer model, the introduction of a combination of lymphocytes into the NOD-scid mouse, we hope to identify immune cells important in the role of autoantibody production in the immunopathology of SjS. The adoptive transfer model, using the NOD-scid mouse, was originally developed to study the etiology of diabetes in the NOD/LtJ. Islet-infiltrating lymphocytes, as well as

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14 splenic lymphocytes from NOD mice, can initiate diabetes when transferred into NOD-scid mice (24). In those studies, lymphocytes were collected from pre-diabetic and diabetic mice. The pre-diabetic and diabetic lymphocytes were transferred separately, and also as a heterogeneous pool, into NOD-scid recipients. The incidence of diabetes in the recipient NOD-scid was approximately 70%, similar to the NOD/LtJ control, when splenic lymphocytes from diabetic NOD mice were transferred into NOD neonate recipients (pre-diabetic and pre-SjS) (18). At 12 weeks of age, 50% of the recipient NOD mice showed submandibulary gland infiltrations (18). Lymphocytes from the NOD/LtJ also initiate sialoadenitis in the scid mouse, but these findings do not identify the role of the humoral response in the cessation of salivary secretion. The congenic NOD.IL-4 -/mouse does not develop SjS. T-cells from the IL-4 -/mouse are not capable of stimulating isotype switching in B-cells and consequently the IgG 1 anti-M 3 R antibody is absent from its sera. In a preliminary study, NOD.IL-4 -/mice were intraperitoneally injected with splenic T lymphocytes from NOD.Ig null or NOD.IL 4 -/mice (23). This resulted in a decrease of salivary flow in 66% of the Ig null T cell recipients, in contrast to the stable salivary flow of the IL 4 -/T cell recipient (23). The inherent inability of IL 4 -/mice to develop SjS implies that the T-cells main role in disease progression is as an activator of isotype-switching in B-cells. This theory is further supported by the progression of xerostomia elicited by adoptively transferred splenic T-cells. As shown in a previously mentioned study, NOD.Ig null T-cells are capable of stimulating autoantibody production. However, the Ig null mouse does not develop xerostomia. The absence of xerostomia was reversed by transfer of human serum IgG, from Sjgrens Syndrome patients, into the Ig null mouse (22). Therefore, a logical

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15 conclusion from these data, is that the humoral response appears to be a necessary and sufficient mediator of xerostomia. In order to clarify the causal effect of B-cells and T-cells in Sjgrens Syndrome, the research of this thesis seeks to fulfill the following specific aim: 1. Adoptively transfer different combinations of splenic lymphocytes from the NOD/LtJ mouse and its congenic strains, NOD.Ig null and NOD.IL 4 -/into the NOD-scid mouse in order to establish the adoptive transfer model for phase II of SjS. To verify the establishment of SjS in these adoptive transfer mice, I have examined: The salivary flow and enzyme activity within the saliva of the treated scid mice. The submandibular gland of the treated scid mice to detect the presence of apoptotic events, using flow cytometry, cysteine protease activity, in situ staining, and protein expression via polymerase chain reaction.

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CHAPTER 2 MATERIALS AND METHODS Animals Animals used in this study were NOD-scid (experimental recipient), NOD/LtJ (experimental donor), NOD.IL 4 -/(experimental donor), NOD.Ig null (experimental donor), and NOD-scid (negative control), and NOD/LtJ (positive control). Female mice were used in all experiments. The donor mice were 16 weeks of age, while the recipient and control mice were 12 weeks old at the time of adoptive transfer. Each treated set includes 4 recipient mice (NOD-scid), 2 positive control mice (NOD/LtJ), and 2 negative control mice (NOD-scid). Most of the NOD-scid mice were purchased from Jackson Laboratory (Bar Harbor, Maine). The other NOD-scid mice and the NOD/LtJ, NOD.IL 4 -/, NOD.Ig null were purchased from the University of Florida, Department of Pathology Mouse Colony (Gainesville, FL) and were housed in the Department of Pathology Mouse Colony under specific, pathogen free conditions. Adoptive Transfer The donor mice were euthanized by cervical dislocation and their spleens extracted. The spleens were then pressed through a wire mesh in order to separate the splenic lymphocytes. The lymphocytes were collected and the erythrocytes lysed with 0.84% NaCl. The cells were then washed and incubated for 30 min with the appropriate antibody. Antibodies used were anti CD19 FITC, anti CD3 APC and anti CD25 PE. All antibodies were from BD Pharmingen. After incubation the cells were washed and diluted to a concentration of 8 x 10 6 cells/ml in 2% FBS in PBS. The lymphocytes were 16

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17 then sorted by Douglas Smith at the University of Florida, Flow Cytometry Core Laboratory. CD19 + B-cells and CD3 + CD25 T-cells were collected. The fractionated splenic lymphocytes were then washed and placed in 100l 1X Phosphate Buffered Saline. The fractionated splenic lymphocytes were intravenously injected into the recipient scid mice in a 1 to 1 ratio (B-cells to T-cells). Each recipient mouse received approximately 3 x 10 6 cells. The experimental and control mice were divided into two sets of 8 mice (4 experimental scid mice, 2 positive control NOD mice, and 2 negative control scid mice). One set was observed for 4 weeks, post-transfer (housed until 16 weeks of age), before being euthanized. The other set was observed for 8 weeks, post-transfer (housed until 20 weeks of age) and then euthanized. The submandibular glands, spleens and pancreas of the euthanized mice were collected. The submandibular glands were sectioned into 3 fractions. One section was placed in 2 ml microcentrifuge tubes on ice, for flow cytometry analysis. The next section was placed in cassettes, stored in 4% Formalin overnight, and then placed in 70% EtOH to be formed into paraffin-embedded blocks. The last section was placed in 2 ml microcentrifuge tubes and frozen with dry ice. The frozen tubes were then stored in at -80C for future analysis. Saliva Collection Saliva was collected from the experimental (NOD-scid), positive control (NOD/LtJ), and negative control (NOD.IL 4 -/). The salivary glands were stimulated with secretagogues to induce salivation. The secretagogue solution is composed of Isoproterenol (1mg/ml) and Pilocarpine (2mg/ml) (Sigma) in 1X Phosphate Buffered Saline. Each mouse received 100 l of the secretagogue via an intraperitoneally injection. The saliva was stimulated for 1 minute and then collected by pipetting for 10 minutes. The collected saliva was placed in 1.5 ml microcentrifuge tubes. The volume of saliva

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18 was quantified using pipettors and recorded. The saliva was then stored in a -80C freezer for future protein analysis. Protein Concentration The protein concentration of the collected saliva and submandibular gland lysate was determined, using the Bradford assay. 1 mg of Bovine Serum Albumin (Sigma) was diluted in 1 ml deionized H2O. The Bradford protein assay dye was then diluted 1:5 in deionizd H 2 O. The standards and samples were made and later read in in 2 ml cuvettes. The 1 mg/ml BSA was diluted with H 2 O, to a total volume of 25 l, to make the following standards: 0 mg/ml, 5 mg/ml, 10 mg/ml, 15 mg/ml, and 20 mg/ml. For each sample, 5l of sample and 20 l of H2O was added to each cuvette. 1 ml of the diluted Bradford dye was added to each cuvette, and mixed with a pipette. The standards and samples were allowed to incubate for 5 minutes at room temperature. The protein concentration was detected using the protein concentration program available in the spectrophotometer (Bradford). The standards were used to make a standard curve and then the protein concentrations of each sample extrapolated from the standard curve. Amylase Activity Analysis Detection of amylase present in collected saliva and submandibular gland lysate was accomplished by using the Infinity Amylase Reagent (Thermotrace). Amylase activity is detected via utilization of Ethylidene-pNP-G7 (EPS) as the substrate. The cleaved EPS reacts with -glucosidase, resulting in release of a chromophore. The color change is then detected at 405nm in a spectrophotometer (BioRad). Amylase activity is defined as the rate of formation of EPS fragments per liter of sample (U/L). The saliva samples were diluted 1:200 in deionized H 2 0. The submandibular gland lysate were analyzed undiluted However a few gland lysate samples had an amylase activity that

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19 was unmeasurable when not diluted. These samples were diluted 1:100 in H 2 O. 1 ml of Infinity Amylase reagent was added to a cuvette and incubated in a waterbath set at 37C for 1 min. 25 l of diluted sample was then added to the heated reagent and incubated for 1 min. After the 1 min incubation was completed, the timer for the next incubation minute is started. The reagent/sample was mixed with a pipette and read in the spectrophotometer to obtain the initial reading. Afterwards the reagent/sample was placed in the waterbath until the incubation time is complete. The timer was started for the final incubation time of 1 min, immediately after the second incubation time was completed. The reagent/sample was read in the spectrophotometer to obtain the second reading. The reagent/sample was placed in the waterbath. At the end of the final incubation time, the reagent/sample was read. The spectrophotometer was blanked with H 2 O in the Infinity Reagent. The positive and negative controls were obtained from the provider of the amylase reagent (Thermotrace) and treated the same as the samples. From this assay, three O.D. readings are obtained. The change in amylase activity is obtained by subtracting the initial O.D. reading from the final O.D. reading and dividing by the amount of incubation time between the initial and the final reading (2 min). The obtained value is then multiplied by the conversion factor (5140).The conversion factor is calculated based on manufacturer instructions. The amylase activity of each sample (U/L) is then divided by the known protein concentration (g/L), and then multiplied by 1 x 10 4 to standardize. The resulting amylase activity is measured as the rate of formation of EPS per gram of protein (U/gram of protein). The assay was performed three times in order to obtain statistical analysis.

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20 Apoptosis Detection Apoptosis Detection via Flow Cytometry The day the collected submandibular glands are retrieved from the experimental and control mice, the glands are digested. The submandibular glands are minced and placed in a vial with 5 ml of Collagenase Solution I (2 mg/ml of collagenase IV (Sigma) in 1X Hanks Balanced Salt Solution (HBSS) and 200 l of Dnase). The vials were then placed in a shaking waterbath, set at 37C, for 15 minutes. The partially-digested glands were passed through a series of syringes with needles increasing in gauge, from18 to 23. The digested glands were then placed in a vial with Collagenase Solution II (1 mg/ml of collagenase in 1X HBSS with 200 l of Dnase). The glands were incubated in a shaking waterbath for 5 min. The digested gland mixture was passed through a series of syringes again. At this point the glands should be fully digested. The submandibular cells were placed in 50 ml conical tubes containing 2% FBS in 1X HBSS and placed on ice. The submandibular cells were collected after centrifugation and counted. The submandibular cells were diluted with 1X PBS to a concentration of 1X 10 6 cells/ml. In sterile 12 ml plastic tubes, 5 mls of 55% Percol was added and 2 mls of the diluted, digested glands were carefully layered on top with siliconized pipettes. The tubes were centrifuged for 30 min at a speed of 2,000 rpms. After the gradient separation the acinar cells and infiltrates were collected with siliconized pipettes and placed in 12 ml plastic tubes. The cells were washed and counted. The acinar and infiltrates were then placed in a sterile culture plate with a solution containing 2% FBS in RPMI (Sigma) and 1X antibiotics. The cells were cultured overnight to allow the cells to recuperate. The following day, the acinar and infiltrate cells were fixed in 1 % paraformaldehyde in 1X PBS for 15 min on ice. The cells were washed and placed in 5 ml flow cytometry tubes with 70% ETOH. The tubes

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21 were placed in a -20C freezer to be stored for 24 hrs. The fixed cells were washed in preparation of staining by reagents provided in the APO-Direct kit (BD Pharmingen). This assay utilizes a Tunel labeling system, where terminal deoxynucleotidyl transferase enzyme (TdT) enables the template independent addition of FITC labeled deoxyuridine triphosphate nucleotides (FITC-dUTP) to dna strand breaks present in apoptotic cells. Propidium Iodide (PI)/RNase solution was used to stain total DNA. Apoptotic cells were provided by the kit as a positive control, as well as non-apoptotic cells as a negative control. After staining, following the procedure outlined by manufacturer specifications, the cells were incubated in Propidium Iodide and analyzed by flow cytometry. Manufacturer suggestions were used as a guide to determine appropriate parameters for apoptosis detection. Cells were identified as apoptotic if they expressed FITC-dUTP. The paramaters for positive apoptosis was based on the level of positivity determined by the positive and negative control. A window was selected that included positive control cells that express dUTP, but excluded negative control cells. In order to analyze and standardized the results, the following formula was used: (%pos. cells sample x % pos. cells in the neg. control)/% pos. cells in pos.control. Using the aforementioned formula, substituting the %pos. cell sample x with the %pos.cells positive control (provided in the kit), will standardize the positive control as 100% positive. The same formula is applied to the negative control (provided in the kit) so that the level of positivity is 0%. The equation is applied to each sample and the positivity determined. For comparison, the relative positivity in each treated set is assigned a representative value on a scale from 0 to 10 (0 is equal to 0% positive and 10 is equal to100% positive).

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22 Akt Expression via Polymerase Chain Reaction The stored experimental and control submandibular glands were removed from the freezer and 25 mg of tissue removed. The sectioned piece of submandibular gland was then treated according to manufacturer specifications to extract total RNA (Qiagen). The extracted total RNA was then quantified in the spectrophotometer. A quartz cuvette was used and the spectrophotometer was blanked with deionized water. The total RNA was diluted 1: 50 in deionized water and read in the spectrophotometer. Only total RNA with at least a concentration of 800 g/ml were used to produce cdna. To a .02 ml nuclease-free centrifuge tube the reagents were added in the following order; 1 l of pd(T) 12-18 1 l of 10 mM dNTP, 1 g of total RNA, and enough water so that the total volume in the tubes equal 10 l. After mixing, the tubes were heated in a waterbath for 5 min at 65C and quick chilled on ice. The tubes were then centrifuged and the reagents added in the following order; 4 l of 5X First-Strand buffer, 2 l 0.1M DTT, 1 l RNasin, and 2 l acetylated BSA. After gentle mixing, the tubes were incubated in a waterbath, and set at 42 C for 2 minutes. To each tube 1l of SuperScript II was added and mixed gently. The tubes were incubated at 42 C for 50 minutes and then 70 C for 15 minutes in the thermocycler. The cdna concentration was quantified by the spectrophotometer. A quartz cuvette was used and the spectrophotometer blanked with deionized water. The cdna was diluted 1 : 50 in deionized water and read in the spectrophotometer. A master mix was made by adding the following reagents to a 2 ml nuclease-free centrifuge tube; 5.1 l 10X Buffer, 1.1 l 10 mM dNTPs, and 1.6 l of MgCl 2 (formula is for one pcr reaction). 7.5l of the master mix was added to a .02ml nuclease-free centrifuge tube. To each .02 ml pcr tube the following reagents were added; 1.5 l of the forward primer, 1,5 l of the reverse primer, 2 g of cdna, and enough water so that the volume of the

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23 microcentrifuge tube equals 49.75 l. The primer sets used were 18S (housekeeping gene) and AKT. Both primer sets were individually developed by Invitrogen. The tubes were mixed, centrifuged and incubated in a thermocycler, following the standard program for polymerase chain reaction. The annealing temperature was set at 57.2C. After the dentaure step, but before the 1 st annealing temperature is reached, .25 l of Taq DNA Polymerase (Invitrogen) is added to each tube. The pcr reaction is then continued in the thermocycler. Agarose Gel Electrophoresis A 2% TBE, agarose gel was made and 5 l of ethidium bromide added to the gel while it was still warm. After the gel set, the previously made pcr products were defrosted. 12 l of 18s amplicons and 3 l of 6X Blue/Orange Dye (Promega) were added per well. In the lane above, 24 l of akt amplicons and 4 l of 6X Blue/Orange Dye (Promega) were loaded into each well. 1 l of the PGEM DNA marker (Promega), 4 l of dye and 23 l of 1X TBE was loaded into a well. The TBE gel was then run at 70 volts for 1.5 hr. Afterwards, the gel was visualized with a gel document system, and quantitative analysis done using the AlphaEase FC program (Alpha Innotech). The level of akt expression per sample is based on the samples corresponding level of 18s expression. 18s is ubiquitous in all cells and should; therefore, all samples should have the same amount of expression. However slight variations in the level of expression occurs. Within each lane of a gel are many samples. To standardized the values for 18s expression, a ratio is formed by dividing highest 18s value within that lane by the18s value for each sample within the same lane. The inverse of the calculated 18s ratio (highest 18s/18s of sample x) is then multiplied by the corresponding akt expression

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24 value (inverse of 18s ratio of sample x, multiplied by akt of sample x). From these calculations a relative, qualitative expression value is obtained. Caspase-3 Activity Detection Stored submandibular glands were defrosted on ice and homogenized with 600 l of Tris-HCL, ph 8.0 in 5 ml plastic tubes. The gland lysate was transferred to 2.0 ml microcentrifuge tubes. The gland lysate was then assayed following manufacturer specifications (Calbiochem). The activity assay was read in a microplate reader for 20 minutes and the values calculated in Excel. The data points were plotted on a graph, activity (x axis) vs. time (y axis), and a trendline generated. The slope of the trendline was then multiplied by the conversion factor (conversion factor tabulated as suggested by kit). In Situ Apoptosis Detection Kit. The previously paraffinized submandibular glands were sectioned and placed on slides by Histology Technical Services. The slides were then deparaffinized by incubation in a series of solutions in the following order: two incubations in Xylene for 5 min, two incubations in 100% EtOH for 2 min and two incubations in 95% EtOH for 2 min, two incubations in 70% EtOH for 2 min, two incubations in H 2 O for 2 min. The slides were then stained, following manufacturer specifications (Trevigen), in a humidity chamber. After the slides were dehydrated, the tissues were covered with a non-aqueous mount and a coverslip added. The slides were allowed to dry overnight and then viewed under brightfield microscopy.

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25 Detection of Infiltration Immunohistochemistry The paraffinized submandibular slides were obtained as stated previously. They were deparaffinized (as stated previously) and placed in a heated coplin jar, containing 1X antigen unmasking solution (Trevigen). The coplin jar was then incubated in a waterbath for 30 min at 95C. The slides were then washed with H 2 O and placed in a humidity chamber. The slides were equilibrated with the buffer, 1X TBST (Dako) for 5 min. 200 l of diluted (1:67) goat serum (Sigma) was pipetted onto each slide and incubated for 20 min at room temperature. The goat serum was carefully wiped off the slide, so that the tissue remained undisturbed. The slides were then incubated for 1 hr with 200 l diluted (1:25) B2-20 antibody (BD Pharmingen), at room temperature. The slides were then washed with 1X TBST for 5 min and incubated with 200 l of diluted (1:200) anti-rat antibody (Sigma), for 30 min. Before the diluted anti-rat antibody is added to the slides approximately 5 l of mouse serum is added to the 2 antibody solution. The slides are washed with 1X TBST and treated with an ABC alkaline phosphates kit from Vector Labs. The slides were treated with reagents as specified by the manufacturer (Vector Labs). The substrate used in the alkaline phosphates system was Vector Red (Vector Labs). The substrate was mixed following manufacturer specifications and incubated on the slides for 20 min. The slides were then washed in deionized H 2 0 for 1 min and then washed with 1X TBST for 5 min. The slides were incubated with 200 l of diluted (1:67) rabbit serum (Sigma) for 20 min. The slides were then incubated with 200 l, diluted (1:125) CD3 antibody (Santa Cruz), overnight at 4 C. The slides were then washed with 1X TBST for 5 min, and incubated with 200 l of diluted (1:200) -goat antibody (Sigma) for 30 min. The slides were then treated with the

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26 reagents provided in the ABC immunoperoxidase system (Vector Labs). The slides were treated as specified by the manufacturer. DAB (Vector Labs) was used as the substrate, and was mixed as specified by the manufacturer. The slides were incubated with the DAB for 5 min. The slides are then washed in H 2 O for 1 min. The slides are immersed in a working solution of the Light Green Counterstain (Sigma) for 1 min. The slides are then immersed twice in H 2 0 for 1 min. The slides are then dehydrated by being immersed in a series of solutions for 2 min; 70% EtOH, 95% EtOH, 2 times in 100% EtOH and followed by immersion in Xylene for 5 min, 2 times. Afterwards the slides are coverslipped and visualized by brightfield microscopy. The presence of lymphocytes is determined as cells that positively stained with Vector Red (T-cells) and DAB (B-cells)

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CHAPTER 3 RESULTS Adoptive Transfer Purified splenic lymphocytes from certain donor mice were labeled with the appropriate fluorescent conjugated antibody (except for the unfractionated donor NOD/LtJ splenocytes) and sorted by flow cytometry. The collected splenocytes were selected based on the level of binding to the conjugated antibody. The splenocytes were collected in two separated fractions. Selected B-cells depicted a high level of CD19 + FITC expression. Selected T-cells depicted a high level of CD3 + APC while showing no CD25 + PE expression. CD25 + T-cells have shown to have a protective quality in adoptive transferred of diabetes studies (25). To circumvent inhibition of disease transfer, the CD25 + phenotype was set as a negative selection parameter. The purity of the collected splenocytes was analyzed by flow cytometry. Each fraction had a purity of approximately 93%. The collected splenocytes were then washed and intravenously injected into the recipient NOD-scid. Each scid received an average of 3 x 10 6 B and/or T-cells. The donor splenocytes were selected based on their expected contribution to the development of SjS. The T-cells and B-cells from the NOD/LtJ were selected because they should transfer the disease to the scid. The T-cells of the Ig null was selected due to its shown ability to stimulate an autoreactive B-cell, thus transferring the disease. The B-cells from the NOD.IL 4 -/should interact with a normal T-cell, resulting in transfer of the disease. Those scid mice that received T-cells or B-cells, but not both, are expected to exhibit no disease transfer. 27

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28 Table 3-1. Adoptive transfer, combinations of donor splenocytes transferred to the scid. Recipient Donor T-cells Donor B-cells Pos. Control Neg. Control NOD-scid Total spleen Total spleen NOD/LtJ NOD-scid NOD-scid Total spleen Total spleen NOD/LtJ NOD-scid NOD-scid NOD/LtJ NOD/LtJ NOD/LtJ NOD-scid NOD-scid NOD/LtJ NOD/LtJ NOD/LtJ NOD-scid NOD-scid NOD/LtJ NOD.IL4 -/NOD/LtJ NOD-scid NOD-scid NOD/LtJ NOD.IL4 -/NOD/LtJ NOD-scid NOD-scid NOD.IL4 -/NOD/LtJ NOD-scid NOD-scid NOD.IL4 -/NOD/LtJ NOD-scid NOD-scid NOD.Ig null NOD.IL4 -/NOD/LtJ NOD-scid NOD-scid NOD.Ig null NOD.IL4 -/NOD/LtJ NOD-scid NOD-scid NOD.Ig null NOD/LtJ NOD-scid NOD-scid NOD.Ig null NOD/LtJ NOD-scid NOD-scid NOD.Ig null NOD/LtJ NOD/LtJ NOD-scid NOD-scid NOD.Ig null NOD/LtJ NOD/LtJ NOD-scid Saliva Collection A significant manifestation of SjS in the NOD/LtJ mouse is the presence of xerostomia. Xerostomia in the recipient scid was detected by quantifying the volume of saliva produced in 10 minutes. Salivation was stimulated by secretagogues and the saliva collected from 4 treated scid mice, 2 positive control NOD mice and 2 negative control scids. The collected saliva from the 4 treated mice, per group, was pooled. The collected saliva from the pos. control was pooled, and the neg. control saliva was pooled as well. Saliva was collected once a week for 7 wks, but many mice were euthanized early. Diabetes, which is present in the NOD, was transferred along with the SjS. The mice that became diabetic were treated with insulin, but many expired early. To circumvent the loss of all experimental mice; many diabetic scids were euthanized early. The collected saliva from 4 week post transfer was compared to the volume of saliva from the last week of collection. As seen in Table 3-2, the scid (Unfractionated NOD) did not show a change in salivary flow. The scid (B and T NOD) expressed a decrease of 46%, which is comparable to previous data on the NOD. The

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29 scid (B IL4 -/and T NOD) showed a 68% decrease in flow. The scid (B IL4 -/) showed a small decrease of 13%. The scid (T Ig null and B IL4 -/) and scid (T Ig null ) showed a increase of about 35%. The scid (T Ig null and B NOD) had a small increase of 9%. The scid mice that were recipients of either T or B cells, but not both, had a stable flow, similar to the NOD-scid. Surprisingly, none of the NOD mice depicted xerostomia. The NOD, in previous studies, has proven to be a good positive control for SjS, so this was certainly an unexpected occurrence. Amylase Activity Analysis Amylase Activity Detected in Saliva. An important aspect of SjS is the aberrant production of salivary components such as amylase. The collected saliva was analyzed to determine the amylase activity of the saliva. As shown in Table 3-2, the amylase activity of mice 4 weeks post-transfer (16 week old mice) was compared to the activity of mice 8 weeks post-transfer (20 week old mice). The calculated readings showed that the scid (Unfractionated NOD) had an increase of 62%, the scid (T and B NOD) amylase activity decreased by 8% and the scid (T NOD and B IL4 -/) had an increase of 46%. The scid (B IL4 -/) had a decrease in activity of 57%. The scid recipients of T-cells from the Ig null all showed a decrease in amylase activity that ranged from 5% in the scid(T Ig null and B IL4 -/) to 33% in the scid(T Ig null and B NOD). The NOD control shows a 21% decrease and a 43% decrease in the scid. Detection of Salivary Gland Amylase Activity An important aspect of the initiator phase of SjS is the change in composition of the submandibular glands. In order to determine if there are changes, the amount of amylase present was analyzed. The submandibular glands were homogenized and the gland lysate

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30 incubated with Infinity Amylase Reagent (Thermotrace) for the appropriate time. The activity was detected in spectrophotometer. As seen in Table 3-3, the scid (Unfractionated NOD) and scid (T NOD and B IL4 -/) exhibited an increase in activity or 300% and 970%, respectively. The scid (T and B NOD) had a decrease of 80%. The scid (B IL4 -/) showed an increase of 500%. The scid (T Ig null and B IL4 -/) and the scid (T Ig null and B NOD) had a decrease in amylase activity of 90% and 34%, respectively. The scid (T Ig null ) showed a less than 6% increase. The NOD and scid showed a decrease in amylase activity of 33% and 25%. Protein Concentration A significant aspect of SjS is the aberrant protein production, specifically the production of two novel forms of parotid secretory protein (PSP). The deviant PSP production allows the protein concentration to remain stable, even as other major proteins decrease. Using the Bradford protein assay the protein concentration of saliva and submandibular gland lysate was determined. The protein concentration of the saliva did not show a change in concentration in any of the adoptively transferred scid mice or the controls. The adoptively transferred scid mice and the controls was had an average protein concentration of 3.6 mg/ml, 4 weeks post-transfer. The average protein concentration at 8 weeks post-transfer was 3.8 mg/ml. The submandibular gland lysate depicted a decrease in protein concentration for the scid mice that received a combination of T and B-cells. This decrease ranged from 78% in the scid (T NOD and B IL4 -/) to 22% in the scid (T Ig null and B Il4 -/). The scid (T Ig null ) depicted a decrease of 55%. The scid (B IL4 -/), scid (Unfractionated NOD) and the controls did not show a change in protein concentration.

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31 Table 3-2. The saliva from each adoptively transferred scid was collected and quantified. The saliva was then assayed to determine the amount of amylase present and the concentration of proteins. Age of treated mouse Total salivary volume (l/10 min) Amylase activity (U/gram of protein) Total protein (mg/ml) 4 wks NOD (n = 10) 185 5881 671 d 3.72 .77 >8 wks NOD (n = 7) 236 4643 541 3.48 .3 4 wks scid (n = 12) 180 7082 547 3.01 .8 >8 wks scid (n = 12) 216 4051 879 3.21 .1 4 wks scid (Unfractionated NOD) (n = 4) 168 2786 173 3.92 .02 >8 wks scid (Unfractionated NOD) (n = 4) 170 4509 410 4.85 .03 4 wks scid (T and B NOD) (n = 4) 168 4881 446 3.72 .02 >8 wks scid (T and B NOD) (n = 2) 90 a e 4486 107 4.60 .02 4 wks scid (T NOD and B IL-4 -/) (n = 4) 125 6055 106 3.63 .003 >8 wks scid (T NOD and B IL-4 -/) (n = 2) 40 b 8846 288 e ** 2.55 .02 4 wks scid (B IL-4 -/) (n = 4) 200 4282 126 5.02 .04 >8 wks scid (B IL-4 -/) (n = 4) 175 1824 450 5.25 .03 4 wks scid (TIg null and B IL-4 -/) (n = 4) 63 e 7202 443 e 2.93 .01 >8 wks scid (TIg null and B IL-4 -/) (n = 2) 100 c 6788 136 3.42 .01 4 wks scid (TIg null ) (n = 4) 113 4154 667 3.51 .03 >8 wks scid (TIg null ) (n = 4) 150 3160 519 3.06 .02 4 wks scid (TIg null and B NOD) (n = 4) 110 7091 442 3.02 .02 >8 wks scid (TIg null and B NOD) (n = 3) 120 4735 397 3.44 .01 a Due to the transfer of diabetes, this set euthanized at week 5. b Due to the transfer of diabetes, this set euthanized at week 6. c Due to the transfer of diabetes, this set euthanized at week 5. d Values are given as the mean standard error. e Statistical comparison of adopotively transferred NOD-scid mouse groups to the age-matched NOD-scid parental control by the one way ANOVA test: (*P < 0.05, **P < .001).

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32 Table 3-3. The submandibular glands for the adoptively transferred scid mice and the controls were collected and homogenized. The level of caspase activity was determined using the Caspase-3 activity kit. The gland lysate was assayed to determine the amylase present and protein concentration. Age of treated mouse Total protein (mg/ml) Amylase activity (U/gram of protein) Caspase 3 Activity (pmol/min/gram of protein) 4 wks NOD (n = 10) 3.4 .08 101.2 94 39.8 10 a >8 wks NOD (n = 7) 4.3 .41 67.9 .4 31 5.8 4 wks scid (n = 12) 3.4 .02 159.4 30.8 28.4 3.8 >8 wks scid (n = 12) 4.4 .02 119.1 10.2 30.1 6.7 4 wks scid (Unfractionated NOD) (n = 4) 4.0 .04 194.7 94 59.6 3.3 b ** >8 wks scid (Unfractionated NOD) (n = 4) 4.2 .01 863 433 b ** 26.5 0.3 4 wks scid (T and B NOD) (n = 4) 5.2 .02 b 415 53 29.1 .6 >8 wks scid (T and B NOD) (n = 2) 1.6 .02 b ** 77 4 40.5 0.1 4 wks scid (T NOD and B IL-4 -/) (n = 4) 5.4 .01 b 34 13 34.6 .2 >8 wks scid (T NOD and B IL-4 -/) (n = 2) 1.2 .01 b ** 365 84 265.8 23.8 b ** 4 wks scid (B IL-4 -/) (n = 4) 2.9 .03 137 29 38.6 .2 >8 wks scid (B IL-4 -/) (n = 4) 4.6 .02 955 257 44.1 .9 4 wks scid (TIg null and B IL-4 -/) (n = 4) 4.3 .01 b ** 409 148 36.5 1.3 >8 wks scid (TIg null and B IL-4 -/) (n = 2) 1.6 .01 43 14 46.8 4.6 4 wks scid (TIg null ) (n = 4) 3.4 .02 161 30 28.6 1.1 >8 wks scid (TIg null ) (n = 4) 1.5 .01 b ** 170 114 62.8 11.4 b ** 4 wks scid (TIg null and B NOD) (n = 4) 5.1 .02 b 862 153 b 31.0 4.7 >8 wks scid (TIg null and B NOD) (n = 3) 4.0 .01 573 129 b 48.3 .8 a Values are given as the mean standard error. b Statistical comparison of adopotively transferred NOD-scid mouse groups to the age-matched NOD-scid parental control by the one way ANOVA test: (*P < 0.05, **P < .001). Apoptosis Detection Apoptosis Detection via Flow Cytometry The presence of apoptosis is a significant aspect of xerostomia and is a noted event in disease progression. Apoptosis in the submandibulary acinii cells was detected following the protocol provided in the Apo-Direct kit (BD Pharmingen). Apoptotic and non-apoptotic cells were provided as a positive and negative control. The level of positivity for apoptosis was based on the controls. As seen in Table 3-4, apoptosis was not detected in the scid (B IL4 -/) at 4 weeks and 8 weeks post-transfer. Apoptosis was also detected in the scid (T Ig null ) at 8 weeks. The NOD mice exhibited a low level of apoptosis at 4 weeks post-transfer with no apoptosis present at 8 weeks. There was no

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33 apoptotic events detected in the scid negative control or the experimental scid mice at 4 weeks or 8 weeks. Table 3-4. Apoptosis detection using Apo-Direct kit (BD Pharmingen). The positivity of each sample is based on the positive and negative controls provided in the kit. The samples were standardized by the following formula: (% Pos of sample X % Pos. of Neg. control)/ (% Pos. of Pos. Control). Numerical values are assigned so that 0/10 is equal to 0% positive for apoptosis and 10/10 is equal to 100% positive. Recipient NOD-scid (age) Donor cells Apoptosis Activity (AU) 4 weeks 1/10 N OD/LtJ Positive Control 8 weeks 0/10 4 weeks 0/10 N OD-scid Negative Control 8 weeks 0/10 4 weeks Unfractionated NOD 0/10 N OD-scid 8 weeks Unfractionated NOD 0/10 4 weeks TNOD and BIl4 -/0/10 N OD-scid 8 weeks TNOD and BIl4 -/0/10 4 weeks T and B NOD 0/10 N OD-scid 8 weeks T and B NOD 0/10 4 weeks B IL4 -/7/10 N OD-scid 8 weeks B IL4 -/10/10 4 weeks T Ig null 0/10 N OD-scid 8 weeks T Ig null 10/10 4 weeks TIg null and B Il4 -/0/10 N OD-scid 8 weeks TIg null and BIl4 -/0/10 4 weeks T Ig null and B NOD 0/10 N OD-scid 8 weeks T Ig null and B NOD 0/10

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34 Caspase-3 Activity Detection Apoptosis, otherwise known as programmed cell death, is an important inhibitory mechanism of cell growth. Apoptosis aids in organogenesis, maintenance of tissue morphology, and deletion of autoreactive lymphocytes. As seen in Fig. 3-1, there are a series of events that occur in the apoptotic pathway. One of the last events is the activation of caspases. Caspase-3 is a member of the interleukin 1 converting enzyme (ICE) family of cysteine proteases. It is activated by a series of upstream proteases and is one of the last signaling events before apoptosis occurs. The submandibular gland lysate samples were analyzed in an enzymatic assay. For each treated set caspase activity was compared at 4 weeks post-transfer to 8 weeks post-transfer. As seen in Table 3-3, the scid (Unfractionated NOD) depicted a 55% decrease in caspase activity. The scid (T and B NOD) showed an increase in activity of 40%. The scid (T-NOD and B-IL 4 -/) depicted an increase of over 200%. The scid (B-IL 4 -/) had an increase of 14% in caspase activity. The scid (T-Ig null ) and the scid (T-Ig null and B-NOD) both depicted an increase in activity of about 50%. The scid (T-Ig null and B-IL 4 -/) depicted a slight decrease of 18%. The NOD had a decrease in activity of 22% and the scid had a less than 6% increase in caspase activity. Akt Expression via Polymerase Chain Reaction As a critical mechanism of homeostasis maintenance, apoptosis also has a large destructive potential, if not closely controlled. As seen in Fig 3-1, there are many proteins capable of inhibiting apoptosis. Akt, also known as protein kinase B, is the protein of interest for this thesis. Akt inhibits apoptosis by phosphorylating, and thus inactivating procaspases, Bad, and other transcription factors. The level of akt expression is determined by amplifying cdna, from each sample, by polymerase chain reaction. The

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35 polymerase chain reaction products are visualized by gel electrophoresis. The level of expression is tabulated for 18s pcr products (housekeeping gene) and the akt pcr products, using the AlphaEase FC program. The relative expression of akt of each treated sample is then compared to the NOD (pos. control) and the scid (neg. control), to form ratios. The sample/scid ratio is then compared to the sample/NOD ratio to determine the relative increase in expression from 4 weeks to 8 weeks post-transfer. As shown in Table 3-5, the scid (Unfractionated NOD) depicted an increase in akt expression when compared to the NOD and the scid. The scid (T and B NOD), scid (T NOD and B IL4 -/) showed a decrease in akt expression compared to the NOD and the scid. The scid (T Ig null and B IL4 -/) decreased in akt expression compared to the scid, and remained stable compared to the NOD. The scid (B IL4 -/) depicted an increase in akt expression compared to the scid but remained stable compared to the NOD. The scid consistently had an increased level of akt expression from 4 weeks to 8 weeks, when compared to the NOD. Table 3-5. Akt expression ratios. Age of treated mouse Akt expression ratio a (compared to NOD) Akt expression ratio(compared to scid) Akt expression ratio (scid compared to NOD) 4 wks scid (Unfractionated NOD) (n = 4) 1X >1X b 2X >8 wks scid (Unfractionated NOD) (n = 4) 2X >1X 2X 4 wks scid (T and B NOD) (n = 4) 1X 1X 1X >8 wks scid (T and B NOD) (n = 2) >1X >1X b 3X 4 wks scid (T NOD and B IL4 -/) (n = 4) 15X 2X 4X >8 wks scid (T NOD and B IL4 -/) (n = 2) 4X >1X b 5X 4 wks scid (B IL4 -/) (n = 4) 2X 1X 3X >8 wks scid (B IL4 -/) (n = 4) 2X 2X 1X 4 wks scid (TIg null and B IL4 -/) (n = 4) 2X 2X 2X >8 wks scid (TIg null and B IL4 -/) (n = 2) 2X >1X b 4X 4 wks scid (TIg null ) (n = 4) 1X 2X >1X >8 wks scid (TIg null ) (n = 4) NA NA NA 4 wks scid (TIg null and B NOD) (n = 4) NA NA NA >8 wks scid (TIg null and B NOD) (n = 3) 1X 2X 1X a The fold increase ratio based on the akt expression detected by gel electrophoresis. b The amount of akt expressed is 2x or more than the scid control when compared to the adoptively-transferred scid.

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36 In Situ Apoptosis Detection Kit. The presence of apoptotic cells was detected by TUNEL staining using fixed section of smg. As seen in Fig. 3-3, apoptosis has detected in untreated scid mice at 4 wks pos-transfer and 8 wks post-transfer. Apoptotic cells were also detected in the scid (T and B NOD) mouse, scid (T Ig null and B IL-4 -/) mouse and the scid (T Ig null and B NOD) mouse. The average number of apoptotic cells per five fields was determined. As seen in Table 3-6, the adoptively transferred scid mice showed a higher number of apoptotic cells compared to the scid parental controls. Detection of Infiltration Immunohistochemistry Many classification systems use the presence of infiltration in the salivary glands as a definitive marker of SjS. Lymphocytic infiltrates are seen in patients with SjS and the NOD/LtJ. After euthanization the submandibular glands of the adoptively-transferred and control mice were removed. A fraction of those submandibular glands were fixed and set in paraffin blocks. Afterwards, sections of paraffinized tissue were placed onto glass slides. The tissue was then treated with immunohistochemistry reagents to visualize infiltrates present in the submandibular gland. These slides were stained with Vector Red and DAB to visualize T and B-cells, respectively. A stained T-cell appears red and the B-cell appears as brown. A light green counterstain was used as well. As seen in Figs. 3-3 A and 3-3 A, there is no infiltrates present in the NOD-scid. Figs. 3-3 B and 3-3 B show infiltration of NOD/LtJ submandibular glands. There is a predominance of B-cells in the population. Figs. 3-3 C and 3-3 C show infiltration is present in the scid (T Ig null + B IL4 -/), with T-cells being the predominate cell.

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37 Sections of paraffinized tissue were also made into hematoxylin and eosin stained slide. The slides were visualized by brightfield microscopy in order to detect the presence of infiltrates. Infiltrated lymphocytes were counted to determine the focus score. The NOD at both 4 weeks and 8 weeks post-transfer had a focus score of >2 (Table 3-6). The scid positive control did not show any infiltration at week 4 or week 8. Figure 3-2: Immunohistochemical staining of submandibular glands for lymphocyte-infiltrations. Staining was performed on paraffin-embedded sections of submandibular glands. B-cells stained with DAB (brown), T-cells stained with VectorRed (red) and counterstained with light green. A) 20 wk old NOD-scid smg gland; focus score = 0. B) 20 wk old NOD/LtJ smg gland; focus score = 1. C) 16 wk old (4 wks post-transfer) NOD-scid (T Ig null + B IL4 -/); focus score = 1. Magnification = 10x.

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38 Figure 3-3. Hematoxylin/eosin-stained tissue sections of submandibular glands. Staining was performed on paraffin-embedded sections of submandibular glands. A) 20 wk old NOD-scid smg gland; focus score = 0. B) 20 wk old NOD/LtJ smg gland; focus score = 1. C) 16 wk old (4 wks post-transfer) NOD-scid (T Ig null + B IL4 -/); focus score = 1. Magnification = 10x. The scid (T and B NOD) had a focus score of >2 at 4 weeks with no infiltration seen by week 8.The scid (T Ig null and B IL4 -/) at 4 weeks post transfer had a focus score of 1 (moderate infiltration), but by 8 weeks no infiltration was detected. The scid (T Ig null and B NOD) showed no infiltration at 4 weeks with a focus score of >2 at 8 weeks. The other adoptively transferred mouse groups did not show infiltration at 4 weeks or 8 weeks post-transfer.

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39 Table 3-6. Focus score derived from H+E stained slides of smg glands. A focus score of 1 is equivalent to 50 lymphocytes in a view. 50 lymphocytes in a view is given a score of 1; 100 lymphocytes is given a score of 2; and over 100 lymphocytes is given a score of >2. Recipient NOD-scid (age) Donor cells Focus score 4 weeks >2 N OD/LtJ Positive Control 8 weeks >2 4 weeks 0 N OD-scid Negative Control 8 weeks 0 4 weeks Unfractionated NOD 0 N OD-scid 8 weeks Unfractionated NOD 0 4 weeks T and B NOD >2 N OD-scid 8 weeks T and B NOD 0 4 weeks TNOD and BIl4 -/0 N OD-scid 8 weeks TNOD and BIl4 -/0 4 weeks B IL4 -/0 N OD-scid 8 weeks B IL4 -/0 4 weeks TIg null and B Il4 -/1 N OD-scid 8 weeks TIg null and BIl4 -/0 4 weeks T Ig null 0 N OD-scid 8 weeks T Ig null 0 4 weeks T Ig null and B NOD 0 N OD-scid 8 weeks T Ig null and B NOD >2

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CHAPTER 4 DISCUSSION Studies using the NOD-scid mouse have led to advancement in the research of SjS. Observations on the scid mouse have provided clarification of the glandular and protein changes that occur in the salivary gland in phase I of SjS disease. Although the scid mouse has these glandular changes, its lack of progression to phase II identifies the critical role lymphocytes have in development of phase II of the disease. Providing the scid mouse with appropriate lymphocytes was hypothesized to result in progression of phase II. The results of this thesis provide details on the ability to adoptively transfer various combinations of lymphocytes from the NOD/LtJ, NOD.IL-4 -/, and NOD.Ig null into the NOD-scid mouse, thereby, transferring phase II of SjS to this non-autoimmune host. The NOD mouse is considered a good mouse model for SjS research and is the positive control used in the research of this thesis. It is expected that lymphocytes from the NOD, placed in the scid mouse, should result in SjS development. The data show a 46% decrease in the salivary flow, of adoptively transferred scid mice from week 4 post-transfer to week 8, receiving T and B-cells from the NOD. However, there is no change in the salivary flow of scid recipients of unfractionated NOD splenocytes. The stable salivary flow observed in the scid (Unfractionated NOD) mouse implies that there is a population of cells in the splenic lymphocytes capable of inhibiting disease transfer. The removal of this population allows for SjS progression, as seen in the 40

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41 scid (T and B NOD) mouse. Presumably, the T-cells from the NOD activates antibody production by the B NOD, thus resulting in the observed salivary loss. The T NOD also show an ability to transfer disease when combined with B-cells from the NOD.IL 4 -/. The NOD.IL-4 -/mouse does not develop phase II disease, even though disease precursors are present. The absence of IL 4 cytokine, which is necessary for class switching, prevents disease progression. However, T-cells from NOD mice provide the IL-4 cytokine, thus allowing antibody production and the resulting decrease in salivation. The scid (B IL-4 -/) mouse had little change in saliva volume, possibly due to the absence of an appropriate T-cell. B cells from the NOD mouse, as seen in the scid (T and B NOD) mouse, have an ability to transfer disease when activated by an appropriate T-cell. The NOD.Ig null which lacks B-cells, has functional T-cells that produce IL-4 and are capable of activating B-cells. The recipients of T-cells from the Ig null mouse and B-cells from the NOD mouse did not show a decrease in salivary flow as might be predicted, yet transfer of phase II is possible as shown by the scid (T and B NOD) mouse and the scid (T NOD and B IL-4 -/) mouse. Thus, transferred disease may be delayed in certain combinations, such as the scid (T Ig null and B NOD) mouse. In the NOD environment, antigen presentation by B-cells to T-cells, or other APC, may occur before the splenocytes are fractionated. Under such conditions, T-cells are already producing the cytokines required for class switching before transfer into the scid mouse. This would allow for rapid transfer of disease. However the Ig null without B-cells capable of presenting antigens, may have nave T-cells. Transfer of these nave T-cells may then require a longer incubation for development of phase II. Thus, it is possible that scid recipients of T Ig null and

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42 B IL-4 -/did not show a decrease in salivary flow at 8 weeks post-transfer due to the nave nature T-cells from the Ig null mouse. In a previous study, T-cells from the Ig null were transferred into the IL-4 -/mouse, resulting in development of phase II (23). However, the researchers of this study allotted more incubation time for progression of phase II (12 weeks compared to the 8 weeks allotted in this thesis). It is also possible that there is a loss in salivary flow in the scid (T Ig null and B NOD) mouse and the scid (T Ig null and B IL-4 -/), but this loss is belied by the method of collection. Pooling of saliva during collection and then quantifying may mask any loss of salivary flow that is occurring in some of the treated mice. In the scid (T and B NOD) mouse and the scid (T NOD and B IL-4 -/) mouse the loss of salivary flow for the 4 scid mice was not simultaneous. A variation in timing of the appearance of phase II was also observed in the T Ig null transfer to NOD.IL-4 -/study. The scid recipients of T-cells from the Ig null mouse had normal salivary flow, indicating no disease progression. The NOD has been shown previously to be a good positive control, typically having a 45 75% loss in salivary flow by 20 weeks of age (17,22,25). However, a loss in saliva volume was not detected in these experiments. Nevertheless, the parental NOD-scid mouse had stable salivary flow. Diabetes, a disease present in the NOD, appeared in all of the scid mice that received B and T-cells from the NOD, but not unfractionated splenocytes. It is apparent that diabetes was co-transferred with phase II. Due to the presence of diabetes, 0.1 cc of insulin was administered to each mouse, once a day. Despite treatment, a few of the experimental and positive controls expired early. In order

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43 to prevent the loss of all of the treated mice, some were euthanized prior to 8 weeks post transfer. This early euthanization could have affected the results. Concomitant with the loss of saliva secretion, there are significant changes in the composition and activity of salivary proteins. Probably indicating multiple mechanisms of action. The decline in amylase may be a result of glandular changes that results from defects in glandular homeostasis (21). Amylase, an enzyme that hydrolyzes starch, is one of the glandular proteins that have an age-related decline in NOD and scid mice, and therefore, a marker of phase I disease. The NOD and scid controls, as well as the adoptively transferred scid mice, all had a decline in salivary amylase activity with the exception of the scid (Unfractionated NOD) mouse and scid (T NOD and B IL-4 -/) mouse, which had increases of 46%. The decline in amylase activity indicates that there are glandular changes occurring, to which the adoptively transferred lymphocytes may respond. Although the scid (T NOD and B IL-4 -/) mouse has an increase in amylase activity, the decrease in salivary flow suggests transfer of disease. It is not clear why there is a discrepancy in this particular adoptive transfer combination. Observation of the results shows variability in the amylase activity of the submandibular glands amongst the treated scid mice. The NOD positive control and scid negative control both showed a decline in amylase activity in the submandibular gland lysate. The scid (T and B NOD) mouse, scid (T Ig null and B IL-4 -/) mouse and scid (T Ig null and B NOD) mouse showed variable levels of declining amylase activity in the submandibular gland. However, the scid (Unfractionated NOD) mouse, scid (T NOD and B IL-4 -/) mouse, scid (B IL-4 -/) mouse and scid (T Ig null ) mouse showed an increase in amylase activity. It is not certain what causes such variability in detection of amylase activity in

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44 the submandibular gland, when this same method is useful for detection of salivary amylase activity. There seemed to be significantly less amylase in the submandibular gland lysate compared to the saliva. Many samples were analyzed undiluted (a few were diluted 1:100). From a technical point of view, though, it is possible that this assay was not sensitive enough to detect amylase activity in the smg lysate. Phase I of SjS is marked by aberrant production of salivary proteins. There is production of two neoteric forms of PSP, which enables the protein concentration to remain constant, despite declines in other major proteins (21). Results show that there is no change in salivary protein concentration for the adoptively transferred mice or the controls. However, there were decreases in submandibular gland lysate protein concentration from scid mice that received a combination of T and B-cells, irrespective of the origin of the lymphocytes. These decreases in protein concentration from 4 weeks to 8 weeks post-transfer may be indicative of degradation of the submandibular gland by apoptosis. The scid (T Ig null ) mouse also exhibited a decrease in protein concentration. Apoptosis was measured to determine the effect the transferred lymphocytes may have on inducing cell death in the submandibular gland. Apoptosis occurs in the NOD and the scid during phase I of SjS. This apoptosis is lymphocyte-independent and is most likely a result of the aberrant glandular homeostasis (27,28,29). As SjS progresses to the effector stage, the NOD mouse exhibits increased levels of apoptosis in the submandibular gland, which has been shown to be lymphocyte-dependent (13). The results of the caspase-3 activity analysis revealed an increase of activity in all of the scid recipients receiving combinations of T and B-cells, except for the

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45 scid (Unfractionated NOD) mouse. The increase in Caspase-3 activity in scid recipients of T and B-cell transfer, indicate that the transferred lymphocytes have an effect on apoptosis. The scid (T Ig null ) mouse had a large increase (120%) in caspase activity. This may indicate that there is T-cell dependent role in apoptosis via cytokine production or cytotoxic action of CD8 + T-cells. Caspase-3 activation is one of the last required events before apoptosis is completed, thus the observed increase in activity. However, AKT, as an inhibitor of apoptosis, would exhibit a decrease of expression. The results indicate a decrease in AKT expression in the scid (T and B NOD) mouse and scid (T NOD and B IL-4 -/) mouse compared to the level of AKT expression in the NOD. These groups also had a decrease of AKT expression when compared to the scid negative control. These findings suggest that AKT production has been decreased and may be allowing activation of apoptosis in the submandibular gland. This correlates with the increased level of caspase-3 activity seen in these two groups. Interestingly, the scid (Unfractionated NOD) mouse depicted an increase of AKT expression when compared to the NOD and scid, as well as a decrease in capase-3 activity. This is possibly due to what is a still an unidentified, inhibitory factor in the unfractionated NOD splenocyte population. The scid (B IL-4 -/) mouse did not show a decrease in AKT expression when compared to the NOD mouse and had a slight increase in expression, when compared to the scid mouse. The increase of AKT expression in the scid (B IL-4 -/) mouse and concurrent decrease of caspase-3 activity, would indicate a cessation in apoptosis activity. The scid (T Ig null and B IL-4 -/) mouse did not show a decrease in AKT expression compared to the NOD, but had a decrease in AKT expression compared to the scid

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46 mouse. This group also showed an increased level of caspase-3 activity, which is indicative of apoptosis. The scid positive control had an average 2-fold increase of AKT expression compared to the NOD mouse. The presence of elevated capase-3 activity and decreased AKT expression suggests the presence of apoptotic cells. Visualization of apoptotic cells by flow cytometry was attempted. However, apoptosis was only detected in the scid (T Ig null ) mouse and scid (B IL-4 -/) mouse. It is possible that apoptotic cells were lost in the preparation of the acinar cells and unable to be detected by flow cytometry. The high level of apoptosis detected in the scid (T Ig null ) mouse by flow cytometry may be the result of cytokine production. It is not clear why a high level of apoptosis was detectable in the scid (B IL-4 -/) mouse. The results of the TUNEL stain show apoptotic cells in the scid mouse, parental control and the adoptively treated scid mice. Previous studies show that apoptotic cells are present in phase II, thereby indicating the development of phase II in the adoptively transferred scid mice. Previous findings show that the number of apoptotic cells increases as the scid mouse ages (17,19). However the NOD-scid parental control had a decrease in the number of apoptotic cells present. A definitive aspect of SjS is the appearance of infiltrates in the submandibular gland. The results from the stained histology slides show the presence of infiltrating lymphocytes in the scid (T and B NOD) mouse, scid (T Ig null and B IL-4 -/) mouse, and the scid (T Ig null and B IL-4 -/) mouse. This localization of lymphocytes in the submandibular gland is a precursor to the loss of salivary flow, thus indicating a transfer of SjS-like disease. Interestingly, the scid (T and B NOD) mouse depict a high level of infiltration (focus score >2) at 4 weeks, and also exhibited a decrease in salivation.

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47 However, the scid (T Ig null and B NOD) mouse did not have infiltration until 8 weeks post-transfer, and did not lose salivary flow within that time. This supports the theory that adoptive transfer of T-cells from the Ig null mouse into the scid recipient may require a longer incubation for SjS to develop. A similar situation is observed in the scid (T Ig null and B IL-4 -/) mouse, which had a lesser amount of infiltration then the scid (T and B NOD) mouse and did not have a decrease in salivation at 8 weeks post-transfer. Infiltration in the scid (T NOD and B IL-4 -/) mouse was not observed at 4 or 8 weeks post-transfer; however, there was a loss of salivary flow. This raises the possibility of a great effect by cytokine or other soluble factors. In conclusion, transfer of a clinical Sjgrens Syndrome-like disease into the scid mouse by adoptive transfer is possible under certain conditions. The results of this thesis show that a transfer of T-cells from the NOD with functional B-cells will allow transfer of phase II to the scid recipient. Seemingly, the transfer of entire NOD splenocyte population may allow for a delay in SjS or even disease prevention. The results of the different analysis show that the scid (Unfractionated NOD) mouse consistently had results that were contradictory to the results observed in the scid (T and B NOD) mouse. This provides more evidence of a possible inhibitory factor present in the unfractionated NOD splenocytes. There is a population of T-cells that can inhibit development of diabetes suggesting an important role of regulatory cells within the NOD mouse. This population may be present in the unfractionated population, and capable of preventing disease progression. Although the loss of salivary flow was not detected in adoptive transfer of T-cells from the Ig null mouse together with functional B-cells, adoptive transfer of phase II may be possible. It is possible that T-cells from the Ig null

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48 may not be presented with the necessary antigens until interacting with a functional B-cell. In this situation, the development of disease would require a longer period of time. It is also probable that pooling of saliva during collection may mask individual changes in flow rates and protein content, especially since the loss of salivation may occur at different times in individual mice. Future directions of this research would be to use the NOD.B10.H-2 b mouse as one of the donor mice. The NOD.B10.H-2 b develops SjS, but does not develop diabetes as per the NOD (30), thus preventing diabetes complications. Utilization of the NOD.B10.H-2 b should allow for longer observation of the treated scid mice. In addition to the analysis performed in this study, detection of autoantibodies in the adoptively transferred mouse sera would be beneficial. Completion of future experiments may lead to a clarification of the etiopathology of Sjgrens Syndrome, which may result in better treatment for human patients.

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LIST OF REFERENCES 1. Vivino F. The treatment of Sjgrens syndrome patients with Pilocarpine-tablets. Scand J Rheumatol 2001;115:1-13. 2. Jonsson R, Moen K, Vestrheim D, Szodoray P. Current issues in Sjgrens syndrome. Oral Diseases 2002;8:130-140. 3. Nepom GT. MHC and autoimmune diseases. Immunol Ser. 1993;59:143-64. 4. Davidson BK, Kelly CA, Griffiths ID. Primary Sjogren's syndrome in the North East of England: a long-term follow-up study. Rheumatology (Oxford). 1999 Mar;38(3):245-53. 5. Rolando M. Sjogren's syndrome as seen by an ophthalmologist. Scand J Rheumatol Suppl. 2001;(115):27-31; discussion 31-3. 6. Harley JB, Alexander EL, Bias WB, Fox OF, Provost TT, Reichlin M, Yamagata H, Arnett FC. Anti-Ro (SS-A) and anti-La (SS-B) in patients with Sjogren's syndrome. Arthritis Rheum. 1986 Feb;29(2):196-206. 7. Kelly CA, Foster H, Pal B, Gardiner P, Malcolm AJ, Charles P, Blair GS, Howe J, Dick WC, Griffiths ID. Primary Sjogren's syndrome in north east England--a longitudinal study. Br J Rheumatol. 1991 Dec;30(6):437-42. 8. Reveille JD. The molecular genetics of systemic lupus erythematosus and Sjogren's syndrome. Curr Opin Rheumatol. 1992 Oct;4(5):644-56. 9. The Jackson Laboratory. http://jaxmice.jax.org/jaxmice-cgi/jaxmicedb.cgi?objtype=pricedetail&stock=001976 (Accessed 24 March 2003). 10. Humphreys-Beher MG, Brinkley L, Purushotham KR, Wang PL, Nakagawa Y, Dusek D, Kerr M, Chegini N, Chan EK. Characterization of antinuclear autoantibodies present in the serum from nonobese diabetic (NOD) mice. Clin Immunol Immunopathol. 1993 Sep;68(3):350-6. 11. van Blokland SC, Versnel MA.Pathogenesis of Sjogren's syndrome: characteristics of different mouse models for autoimmune exocrinopathy. Clin Immunol. 2002 May;103(2):111-24. 49

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50 12. Humphreys-Beher MG, Yamachika S, Yamamoto H, Maeda N, Nakagawa Y, Peck AB, Robinson CP. Salivary gland changes in the NOD mouse model for Sjogren's syndrome: is there a non-immune genetic trigger? Eur J Morphol. 1998 Aug;36 Suppl:247-51. 13. van Blokland SC, van Helden-Meeuwsen CG, Wierenga-Wolf AF, Drexhage HA, Hooijkaas H, van de Merwe JP, Versnel MA. Two different types of sialoadenitis in the NODand MRL/lpr mouse models for Sjogren's syndrome: a differential role for dendritic cells in the initiation of sialoadenitis? Lab Invest. 2000 Apr;80(4):575-85. 14. Stott DI, Hiepe F, Hummel M, Steinhauser G, Berek C. Antigen-driven clonal proliferation of B-cells within the target tissue of an autoimmune disease. The salivary glands of patients with Sjogren's syndrome. J Clin Invest. 1998 Sep 1;102(5):938-46. 15. Crispin JC, Vargas MI, Alcocer-Varela J. Immunoregulatory T-cells in autoimmunity. Autoimmun Rev. 2004 Feb;3(2):45-51. 16. Rudin CM and Thompson CB. Apoptosis and disease; Regulation and clinical revelance of programmed cell death. Annu Rev Med. 1997;48:267-81. 17. Kong L, Robinson CP, Peck AB, Vela-Roch N, Sakata KM, Dang H, Talal N, Humphreys-Beher MG. Inappropriate apoptosis of salivary and lacrimal gland epithelium of immunodeficient NOD-scid mice. Clin Exp Rheumatol. 1998 Nov-Dec;16(6):675-81. 18. Goillot E, Mutin M, Touraine JL. Sialadenitis in nonobese diabetic mice: transfer into syngeneic healthy neonates by splenic T lymphocytes. Clin Immunol Immunopathol. 1991 Jun;59(3):462-73. 19. Bacman S, Sterin-Borda L, Camusso JJ, Arana R, Hubscher O, Borda E. Circulating antibodies against rat parotid gland M3 muscarinic receptors in primary Sjogren's syndrome. Clin Exp Immunol. 1996 Jun;104(3):454-9. 20. Nguyen KH, Brayer J, Cha S, Diggs S, Yasunari U, Hilal G, Peck AB, Humphreys-Beher MG. Evidence for antimuscarinic acetylcholine receptor antibody-mediated secretory dysfunction in nod mice. Arthritis Rheum. 2000 Oct;43(10):2297-306. 21. Robinson CP, Yamamoto H, Peck AB, Humphreys-Beher MG. Genetically programmed development of salivary gland abnormalities in the NOD (nonobese diabetic)-scid mouse in the absence of detectable lymphocytic infiltration: a potential trigger for sialoadenitis of NOD mice. Clin Immunol Immunopathol. 1996 Apr;79(1):50-9.

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51 22. Robinson CP, Brayer J, Yamachika S, Esch TR, Peck AB, Stewart CA, Peen E, Jonsson R, Humphreys-Beher MG. Transfer of human serum IgG to nonobese diabetic Igmu null mice reveals a role for autoantibodies in the loss of secretory function of exocrine tissues in Sjogren's syndrome. Proc Natl Acad Sci U S A. 1998 Jun 23;95(13):7538-43. 23. Brayer JB, Cha S, Nagashima H, Yasunari U, Lindberg A, Diggs S, Martinez J, Goa J, Humphreys-Beher MG, Peck AB. IL-4-dependent effector phase in autoimmune exocrinopathy as defined by the NOD.IL-4-gene knockout mouse model of Sjogren's syndrome. Scand J Immunol. 2001 Jul-Aug;54(1-2):133-40. 24. Rohane PW, Shimada A, Kim DT, Edwards CT, Charlton B, Shultz LD, Fathman CG. Islet-infiltrating lymphocytes from prediabetic NOD mice rapidly transfer diabetes to NOD-scid/scid mice. Diabetes. 1995 May;44(5):550-4. 25. Szanya V, Ermann J, Taylor C, Holness C, Fathman CG. The subpopulation of CD4+CD25+ splenocytes that delays adoptive transfer of diabetes expresses L-selectin and high levels of CCR7. J Immunol. 2002 Sep 1;169(5):2461-5. 26. Serreze DV, Chapman HD, Varnum DS, Hanson MS, Reifsnyder PC, Richard SD, Fleming SA, Leiter EH, Shultz LD. B lymphocytes are essential for the initiation of T cell-mediated autoimmune diabetes: analysis of a new "speed congenic" stock of NOD.Ig mu null mice. J Exp Med. 1996 Nov 1;184(5):2049-53. 27. van Blokland SC, Van Helden-Meeuwsen CG, Wierenga-Wolf AF, Tielemans D, Drexhage HA, Van De Merwe JP, Homo-Delarche F, Versnel MA. Apoptosis and apoptosis-related molecules in the submandibular gland of the nonobese diabetic mouse model for Sjogren's syndrome: limited role for apoptosis in the development of sialoadenitis. Lab Invest. 2003 Jan;83(1):3-11. 28. Humphreys-Beher MG, Peck AB, Dang H, Talal N. The role of apoptosis in the initiation of the autoimmune response in Sjgrens Syndrome. Clin Exp Immunol. 1999 Jan; 116: 383-387. 29. Yamamoto H, Sims NE, Macauley SP, Nguyen KH, Nakagawa Y, Humphreys-Beher MG. Alterations in the secretory response of non-obese diabetic (NOD) mice to muscarinic receptor stimulation. Clin Immunol Immunopathol. 1996 Mar;78(3):245-55. 30. Cha S, van Blockland SC, Versnel MA, Homo-Delarche F, Nagashima H, Brayer J, Peck AB, Humphreys-Beher MG. Abnormal organogenesis in salivary gland development may initiate adult onset of autoimmune exocrinopathy. Exp Clin Immunogenet. 2001;18(3):143-60.

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BIOGRAPHICAL SKETCH Vinette B. Brown was born in Brooklyn, New York, in 1979. She comes from a very large extended Jamaican family. When she was 10 years old, she and her parents moved to Hollywood, Florida, where she graduated from Hollywood Hills High School in 1997. Afterwards, she received a Bachelor of Science in zoology, with a minor in business from the University of Florida in 2001. She went on to receive a Master of Science in molecular genetics and microbiology in August 2004 from the University of Florida. Vinette will work towards earning a Doctor of Dental Science degree at the School of Dental and Oral Surgery at Columbia University. 52


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Permanent Link: http://ufdc.ufl.edu/UFE0007027/00001

Material Information

Title: Adoptive Transfer Studies to Establish a Model of Phase II Exocrine Gland Dysfunction in the NOD Model of Sjogren's Syndrome
Physical Description: Mixed Material
Language: English
Creator: Brown, Vinette B. 1979- ( Dissertant )
Peck, Ammon B. ( Thesis advisor )
Litherland, Sally ( Reviewer )
Humphreys-Beher, Michael ( Reviewer )
Ostrov, David ( Reviewer )
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2004
Copyright Date: 2004

Subjects

Subjects / Keywords: Department of Medical Sciences thesis, M.S
Mice, SCID   ( msh )
Models, Animal   ( msh )
Research   ( msh )
Dissertations, Academic -- UF -- College of Medicine -- Department of Medical Sciences
Sjogren's Syndrome -- Genetics   ( msh )
Sjogren's Syndrome -- Immunology   ( msh )
Sjogren's Syndrome -- Physiopathology   ( msh )

Notes

Abstract: Sjo umlautgren?s Syndrome (SjS) is an autoimmune exocrinopathy that results in the development of xerostomia (dry mouth) and keratoconjunctivitis (dry eyes). There are two distinct phases of SjS. Phase I which is lymphocyte independent and phase II which is lymphocyte-dependent. The non-obese diabetic (NOD/LtJ) mouse displays symptoms associated with both phases of the disease and, therefore, is a useful model for studying SjS. The insertion of the scid mutation into the NOD/LtJ background has led to the development of the NOD.CB17-PrkdcScid/J (scid). The scid mouse develops phase I of SjS but as a result of the absent lymphocytes, development of phase II does not occur. The lack of mature lymphocyte development in the scid allows it to be used as a recipient in adoptive transfer studies to determine the combination of lymphocytes required to induce phase II of SjS. The work described in this thesis involves the transfer of lymphocyte combinations derived from specific strains, all of which have the same NOD/LtJ genetic background, into the scid mouse. Adoptively transferred scid mice were analyzed for the progression of disease. Observation of the salivary flow, saliva proteins, presence of apoptosis and infiltration provided information on progression of disease. Results indicate the adoptive transfer of functional lymphocytes can lead to development of phase II. However, the time required for disease progression may vary depending on the origin of the lymphocytes. The development of this adoptive transfer model has provided parameters for future experiments which may clarify the immunopathology important in progression of phase II and onset of SjS-like disease.
Subject: adoptive, model, mouse, sjogren, transfer
General Note: Title from title page of source document.
General Note: Document formatted into pages; contains 62 pages.
General Note: Includes vita.
Thesis: Thesis (M.S.)--University of Florida, 2004.
Bibliography: Includes bibliographical references.
General Note: Text (Electronic thesis) in PDF format.

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
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Material Information

Title: Adoptive Transfer Studies to Establish a Model of Phase II Exocrine Gland Dysfunction in the NOD Model of Sjogren's Syndrome
Physical Description: Mixed Material
Language: English
Creator: Brown, Vinette B. 1979- ( Dissertant )
Peck, Ammon B. ( Thesis advisor )
Litherland, Sally ( Reviewer )
Humphreys-Beher, Michael ( Reviewer )
Ostrov, David ( Reviewer )
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2004
Copyright Date: 2004

Subjects

Subjects / Keywords: Department of Medical Sciences thesis, M.S
Mice, SCID   ( msh )
Models, Animal   ( msh )
Research   ( msh )
Dissertations, Academic -- UF -- College of Medicine -- Department of Medical Sciences
Sjogren's Syndrome -- Genetics   ( msh )
Sjogren's Syndrome -- Immunology   ( msh )
Sjogren's Syndrome -- Physiopathology   ( msh )

Notes

Abstract: Sjo umlautgren?s Syndrome (SjS) is an autoimmune exocrinopathy that results in the development of xerostomia (dry mouth) and keratoconjunctivitis (dry eyes). There are two distinct phases of SjS. Phase I which is lymphocyte independent and phase II which is lymphocyte-dependent. The non-obese diabetic (NOD/LtJ) mouse displays symptoms associated with both phases of the disease and, therefore, is a useful model for studying SjS. The insertion of the scid mutation into the NOD/LtJ background has led to the development of the NOD.CB17-PrkdcScid/J (scid). The scid mouse develops phase I of SjS but as a result of the absent lymphocytes, development of phase II does not occur. The lack of mature lymphocyte development in the scid allows it to be used as a recipient in adoptive transfer studies to determine the combination of lymphocytes required to induce phase II of SjS. The work described in this thesis involves the transfer of lymphocyte combinations derived from specific strains, all of which have the same NOD/LtJ genetic background, into the scid mouse. Adoptively transferred scid mice were analyzed for the progression of disease. Observation of the salivary flow, saliva proteins, presence of apoptosis and infiltration provided information on progression of disease. Results indicate the adoptive transfer of functional lymphocytes can lead to development of phase II. However, the time required for disease progression may vary depending on the origin of the lymphocytes. The development of this adoptive transfer model has provided parameters for future experiments which may clarify the immunopathology important in progression of phase II and onset of SjS-like disease.
Subject: adoptive, model, mouse, sjogren, transfer
General Note: Title from title page of source document.
General Note: Document formatted into pages; contains 62 pages.
General Note: Includes vita.
Thesis: Thesis (M.S.)--University of Florida, 2004.
Bibliography: Includes bibliographical references.
General Note: Text (Electronic thesis) in PDF format.

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: UFE0007027:00001


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ADOPTIVE TRANSFER STUDIES TO ESTABLISH A
MODEL OF PHASE II EXOCRINE GLAND DYSFUNCTION IN THE NOD MODEL
OF SJOGREN'S SYNDROME
















By

VINETTE B. BROWN


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2004


































Copyright 2004

by

Vinette B. Brown


































This document is dedicated to the graduate students of the University of Florida.















ACKNOWLEDGMENTS

Working on this project has been a wonderful experience. As a master's student I

had the opportunity to learn from very knowledgeable professors and researchers. Many

people assisted me in the completion of my degree, for which I am very thankful.

First and foremost, I would like to thank Dr. Ammon B. Peck who has provided a

great deal of support and advice. I am so grateful to have worked under someone who is

extremely knowledgeable but is also willing to listen to new ideas. I would like to thank

Dr. Michael Humphreys-Beher for allowing me to enter his lab and giving me my very

first project. I am very greatful for the help I received from my committee members, Dr.

Sally Litherland and Dr. David Ostrov, who opened their labs and offices to me.

I would like to thank my entire lab who has provided an immense amount of

support. I would like to thank Janet Cornelius and Dr. Seunghee Cha who aided me with

their years of experience and lots of their time. I also thank Lori Boggs, Daniel Saban,

Eric Sing-Son and Smruti Killedar who have both provided lots of technical assistance

and advice.

I am very grateful for all of the support I received outside of the Peck lab. I thank

Dr. Clare-Salzler's laboratory, Dr. Petersen's laboratory, Dr. Burne's laboratory, Dr. Oh's

laboratory, and Dr. Khan's laboratory for all of the assistance.

I would also like to thank my friends who provided much needed moral support

and a great deal of advice, specifically, Grace Kim and Dr. Kenyon Meadows.
















TABLE OF CONTENTS

page

L IST O F T A B L E S ...... .. .. ....... ... ......... ... .................................................... .. vii

LIST OF FIGURES ............. ............. ........ ....... .......................... viii

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

CHAPTER

1 IN TR OD U CTION ............................................... .. ......................... ..

B background of Sj gren's Syndrom e..................................... ......................... ...........
A nim al M models ................................................ .......................... 5
A adoptive Transfer Studies .......................................................... ............... 13

2 M ATERIALS AND M ETHOD S ........................................ ......................... 16

A n im a ls ........................................................................ 1 6
A adoptive Transfer .................. ................................... .. .............. ... 16
S aliv a C collection ................................................. ................ 17
Protein Concentration ............................................... .. ..... ................. 18
A m ylase A activity A analysis ................................................. ............. ............... 18
A p opto sis D election ................ .. .. ...... ................ .......................... .. ................... 2 0
Apoptosis Detection via Flow Cytom etry ................................ ..................... 20
Akt Expression via Polymerase Chain Reaction..............................................22
A garose G el E lectrophoresis ........................................................... .....................23
Caspase-3 A activity D election .................................... ............................. ....... 24
In Situ Apoptosis Detection Kit. .............................................. ............... 24
D election of Infiltration ................................................................... .....................2 5

3 R E S U L T S .......................................................................... 2 7

A adoptive Transfer .................. ...................................... .......... .. ..27
S aliv a C collection ................................................. ................ 2 8
A m ylase A activity A analysis .................................................. ............ ............... 29
Amylase Activity Detected in Saliva. ...................................... ............... 29
Detection of Salivary Gland Amylase Activity .............................................29
Protein Concentration ........ ............................ ........ ...... .. ................30
A poptosis D election ......... .............................................. .............. .. .... ...... 32


v









Apoptosis Detection via Flow Cytom etry ................................ ..................... 32
Caspase-3 A activity D election ......... ................. ........................ ............... 34
Akt Expression via Polymerase Chain Reaction.............................................34
In Situ Apoptosis Detection Kit. .............................................. ............... 36
D election of Infi ltration ...................... .. .. ......... .. ......................... ...................... 36

4 D ISC U S SIO N ...................... .. .. ......... .. .. ......... ...................................40

LIST OF REFEREN CES ................................................................... ............... 49

B IO G R A PH IC A L SK E TCH ..................................................................... ..................52
















LIST OF TABLES

Table pge

3-1 Adoptive transfer, combinations of donor splenocytes transferred to the scid. .......28

3-2 The saliva from each adoptively transferred scid was collected and quantified...... 31

3-3 The submandibular glands for the adoptively transferred scid mice and the
controls were collected and homogenized....................... ..................... 32

3-4 Apoptosis detection using Apo-Direct kit (BD Pharmingen) ................................33

3-5 A kt ex pression ratio s ...................................................................... .............. 3 5

3-6 Focus score derived from H+E stained slides of smg glands..............................39
















LIST OF FIGURES


Figure pge

1-1 Apoptosis pathway. Notice that AKT is an inhibitor early in the pathway and
Caspase-3 one of the last activated caspases (7). ...................................................9

3-2 Immunohistochemical staining of submandibular glands for lymphocyte-
in fi ltratio n s ...............................................................................................................3 7

3-3 Hematoxylin/eosin-stained tissue sections of submandibular glands ....................38















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

ADOPTIVE TRANSFER STUDIES TO ESTABLISH A
MODEL OF PHASE II EXOCRINE GLAND DYSFUNCTION IN THE NOD MODEL
OF SJOGREN'S SYNDROME

By

Vinette B. Brown

August 2004

Chair: Ammon B. Peck
Major Department: Medical Sciences

Sjogren's Syndrome (Sj S) is an autoimmune exocrinopathy that results in the

development of xerostomia (dry mouth) and keratoconjunctivitis (dry eyes). There are

two distinct phases of Sj S. Phase I which is lymphocyte independent and phase II which

is lymphocyte-dependent. The non-obese diabetic (NOD/LtJ) mouse displays symptoms

associated with both phases of the disease and, therefore, is a useful model for studying

Sj S. The insertion of the scid mutation into the NOD/LtJ background has led to the

development of the NOD.CB 17-PrkdcScid/J (scid). The scid mouse develops phase I of

Sj S but as a result of the absent lymphocytes, development of phase II does not occur.

The lack of mature lymphocyte development in the scid allows it to be used as a recipient

in adoptive transfer studies to determine the combination of lymphocytes required to

induce phase II of Sj S.









The work described in this thesis involves the transfer of lymphocyte combinations

derived from specific strains, all of which have the same NOD/LtJ genetic background,

into the scid mouse. Adoptively transferred scid mice were analyzed for the progression

of disease. Observation of the salivary flow, saliva proteins, presence of apoptosis and

infiltration provided information on progression of disease.

Results indicate the adoptive transfer of functional lymphocytes can lead to

development of phase II. However, the time required for disease progression may vary

depending on the origin of the lymphocytes. The development of this adoptive transfer

model has provided parameters for future experiments which may clarify the

immunopathology important in progression of phase II and onset of Sj S-like disease.














CHAPTER I
INTRODUCTION

Many illnesses decrease the quality of life, often making normal daily tasks

extremely difficult. Sj gren's syndrome (Sj S) is one such disease, which often causes the

patient great discomfort. Patients afflicted with SjS have difficulties eating and speaking,

due to an inability to secrete saliva. These patients also have dry eyes; therefore, they

have a constant feeling of irritation in the eyes. The etiology of Sj S is not known and a

cure is not available. The current treatment for Sj S is salivary and tear stimulants (1).

These stimulants are sometimes effective, providing a limited amount of relief, but they

can also have undesirable side effects. By understanding the underlying causes and

mechanism of Sj S, a more complete treatment can be devised.

Background of Sj6gren's Syndrome

In 1932, Henrik Sjogren reported the triad of keratoconjunctivitis sicca (dry eyes),

xerostomia (dry mouth), and rheumatoid arthritis (2). This triad of symptoms became

known as Sjogren's Syndrome, a chronic autoimmune disorder in which the secretary

functions of various exocrine glands are disrupted. The major areas affected are the

lacrimal and salivary glands, as well as the skin, upper respiratory, gastrointestinal tract

and the genitals. The resulting dryness of mucosal surfaces results in various

complications such as gastrointestinal discomfort, difficulty speaking, fatigue and

musculoskeletal complaints (3). The disease is not usually fatal, but can be quite

distressful for those affected. Furthermore, due to the immunological basis of the disease,

patients with Sj S are at an increased risk of developing a non-Hodgkin's B-cell









lymphoma. At this time, the exact etiology is not known, so patients remain uncured.

Today, patients are usually treated with secretagogues, which induce lacrimal and

salivary secretion, along with other glandular secretion.

While secretagogues are readily available, a patient may not receive this treatment

due to the difficulties diagnosing Sj S. As with many systemic diseases, Sj S appears with

a wide range of clinical manifestations. These variations, may lead clinicians to

misdiagnose symptoms. There are numerous reports describing lung, renal and central

nervous system (CNS) involvement (4). Over the years, a number of disease

classifications (San Francisco, San Diego, California, Japanese, European original and

European revised), each with their own methods and standards for diagnosing Sjogren's

syndrome, have been proposed. Among the different classification systems, xerostomia

(dry mouth) is usually diagnosed by a lip biopsy. The demonstration of focal lymphocytic

infiltrates, on a minor salivary gland (SG) biopsy, has remained the gold standard for the

oral component of Sj S (2). A salivary gland is considered infiltrated when there are

clusters of fifty or more lymphocytes, known as foci, present. For most classification

systems the presence of two foci per 4 mm2 is required to identify the gland as being

infiltrated. A less invasive measure of xerostomia is the use of scintigraphy and

sialography. Scintigraphy is a technique where an appropriate, short-lived gamma-

emitting radioisotope is introduced. Through radiographic imagery the uptake,

concentration and excretion of the radioisotope by the major salivary gland

submandibularr) is measured. This can be a very sensitive test for glandular function.

Sialography is a similar method in which the release of saliva by the salivary glands is

evaluated via nuclear imaging.









Keratoconjunctivitis (dry eyes) is typically diagnosed by tear volume measured by

what has become known as the Schirmer test. This test measures tear stability by the non-

invasive break up time and usage of a rose bengal dye to stain the ocular surface, which

results in identification of epithelial cell destruction (5). Serologic tests for the detection

of autoantibodies, specifically SS-A (anti-Ro), SS-B (anti-La), and anti-nRNP, are also

used for correct diagnosis of SjS. In eighty-six Sjogren's syndrome patient sera, more

than 96% had SS-A, and 87% had SS-B, compared to 95% of patients with anti-nRNP

(6). Serology not only facilitates diagnosis, but also can be useful in predicting the

subsequent outcome and complications in patients with primary Sjogren's syndrome (2).

The presence of anti-SS-A antibodies may identify patients with systemic disease (7), and

in anti-SS-A/ anti-SS-B positive patients, the relative risk of developing non-Hodgkin

lymphoma has been reported as high as 49.7%, within 10 years of diagnosis (4).

There are two types of Sjogren's syndrome. Individuals that exhibit the classic

symptoms (dry eyes and dry mouth), in the absence of another autoimmune disease, are

diagnosed as having primary Sj S. Secondary Sj S includes the presence of the

aforementioned symptoms, in addition to another autoimmune rheumatic disease, such as

rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE). Due to the variability

in classification systems, the exact percentage of patients with Sj S is not known, but it is

estimated to have a prevalence not exceeding 0.6% of the general population (2). As with

most connective tissue autoimmune diseases, there is a sexual dimorphism present in

Sjogren's syndrome. There is a 9:1 ratio of women to men in SjS, with most women

presenting symptoms of the disease between the ages of 40 and 60 years of age.









A prominent feature of Sjogren's syndrome is the genetic predisposition (2). It is

not uncommon for two or more cases of Sj S to occur within a family. Polymorphisms in

the major histocompatibility complex (MHC) genes are the best documented genetic risk

factors for the development of autoimmune diseases overall (3). In Sjogren's syndrome,

the most relevant MHC complex genes are the class II genes, specifically the HLA-DR

and DQ alleles (8). However, variations in SjS associated haplotypes amongst different

ethnicities, makes it difficult to establish which of the genes confers risk. The severity of

the disease also seems to be dependent on the combination of the risk-associated alleles.

Sjogren's syndrome patients with DQ1/DQ2 alleles have a much more severe

autoimmune disease then patients with any other allelic combination at HLA-DQ (6).

Even with specific gene combinations, environmental factors may play a role in the

disease onset. Among the possible etiologic factors, viral infections are the most often

proposed as a possible trigger of autoimmune disease. Potential viral triggers include

Epstein-Barr virus and Hepatitis C. Furthermore, a possible relationship between

Sjogren's Syndrome and Helicobacter pylori infection has been suspected (2). With these

concurrent infections, the risk of mucosa-associated lymphoid tissue lymphoma increases

in patients with Sj S (2).

Dentists and the ophthalmologists encounter patients afflicted by Sj S most often,

although a few dermatologists are faced with treating patients presenting cutaneous

lesions, in addition to the classic symptoms. A dentist may encounter a Sj S patient who

complains of dryness in the oral cavity, difficulty swallowing, and a burning sensation in

the throat. A patient in the ophthalmologist's office would complain of dryness in the

eye, which could lead to infections, keratitis, and sometimes, melting of the cornea or









ocular perforations (5). The cutaneous lesions in patients with Sj S are described by

dermatologists as palpable and nonpalpable eruptions, pruritic or non-pruritic, as well as

symmetric or nonsymmetric in distribution (2). The dermatologist may not recognize

these lesions as being associated with SjS and thus, misdiagnose it as Wegener's

granuloma. There is still much to be learned about this disease, and once the information

is attained it will aid in recognition and treatment of SjS.

Animal Models

The current classification systems provide standards to aid clinicians in diagnosing

SjS. However, clinicians are inhibited in their ability to make a diagnosis because the

exact onset of the disease is difficult to determine. The onset is usually around forty years

of age; however, there are cases of juvenile Sj S. Research of Sj S within the human

population is impeded by the ethical and legal limitations of collecting patients' salivary

glands. To study the pathology and etiology of Sj S, a variety of mouse models have been

developed, such as the New Zealand Black (NZB) and the MRL/n substrains. Today, the

mouse used most often is the non-obese diabetic (NOD/LtJ) mouse. The NOD/LtJ (NOD)

is characterized has having insulitis, a leukocytic infiltrate of the pancreatic islets. NOD

mice have marked decreases in insulin production by the age of 12 weeks. The NOD/LtJ

is considered diabetic when there is moderate glycosuria, and the non-fasting plasma

glucose is higher than 250 mg/dl (9). The NOD mouse developed early out of breeding

experiments with the Cataract Shionogi (CTS) strain (9). The CTS mice were selected

for a high fasting glucose level. From those selected mice a few spontaneously developed

overt insulin-dependent diabetes mellitus with insulitis (IDDM), which is now the

NOD/LtJ used today. In the NOD mouse, leukocytic infiltrates and antibodies destroy the

pancreatic islets, with exocrine glands infiltrated as well. Of particular interest to Sj S









researchers is the observation of lymphocytic foci present in the major salivary gland

submandibularr) (10), tear-producing gland (lacrimal) and thymus (11). NOD mice have

destruction of the submandibular gland, a marked decreased in salivary flow, and changes

in the salivary protein composition. Sera from NOD mice is positive for antinuclear

antibodies (SS-A and SS-B), which are detectable by nuclear staining. These symptoms

are similar to those seen in humans with secondary Sjogren's Syndrome, thus making the

NOD a good mouse model for Sj S research.

The pathogenesis of the Sj S-like disease seen in the NOD mouse can be divided

into two phases. Phase I is a lymphocyte-independent phase where there are

abnormalities intrinsic to the submandibular and lacrimal glands of the NOD. Phase II is

marked by the autoimmune response via lymphocytic infiltrates in the submandibular

(sialoadenitis) and lacrimal (dacryoadenitis) glands, followed by a decrease in tear and

saliva production. The lymphocytic infiltrates appear as periductal foci within the

glandular architecture of the salivary and lacrimal glands (12). The cause of the

autoimmune reaction may reside in the target organ of the autoimmune response, in the

immune system, or in both (13). It is theorized that the loss of secretary function is due to

lymphocyte-directed destruction. Increased numbers of apoptotic epithelial cells have

been detected in the minor salivary glands of patients with Sj S (13).

The phase II occurs in the NOD mouse around 10 weeks of age. Current evidence

suggests the existence of genetically programmed abnormalities in the exocrine glands of

the NOD mouse that may contribute to initiation of the autoimmune reaction (11).

Antigen presenting cells, such as dendritic cells, are important mediators of lymphocyte

activation, and may respond to organ abnormalities. There is evidence of accumulation of









dendritic cells in the submandibular gland (smg) before the development of lymphocytic

infiltrates (13). When these lymphocytic infiltrates form in phase II, dendritic cells

interact closely with T-cells, possibly serving to locally activate autoreactive T-cells (11).

These organized lymphocytic infiltrates seen in the NOD are also described in the

biopsies of Sj S patient (14).

As stated before, phase II of Sj S is lymphocyte-dependent in humans and the NOD.

Research indicates that there is an inappropriate response of dendritic cells to

abnormalities of the smg and thus an activation of naive autoreactive T-cells. These

autoreactive T-cells are then capable of stimulating an inflammatory response and

autoantibody production. In addition, the dendritic cells of the NOD were found to have a

decreased ability to stimulate T suppressor cells (11). T suppressor cells are important

regulators of the immune system and have been shown to posses the capacity to alter the

stimulation of autoreactive T-cells (15). Infiltrates of exocrine tissues primarily consist of

CD4+ T-cells with a minority of CD8 T-cell and B-cell populations (12). Studies

indicate that activated T-cells, predominantly CD4+ T-cells, are necessary for the

initiation of autoimmune disease, as indicated by T-cell transfer studies (12). The

infiltrates also exhibit aberrant production of the pro-inflammatory cytokines IL-P, IL-2,

IL-6, IL-7 IL-10, IL-12, IFN-y, and TNF-a. Cytokines are important mediators in the

immune response, and their altered production can lead to an inappropriate stimulation of

lymphocytes.

In addition to the changes in proteins seen in phase I of Sj S, there are increases in

apoptosis of the submandibular gland acinii (13). Apoptosis, otherwise known as

programmed cell death, is an important inhibitory mechanism of cell growth, aiding in









organogenesis, maintaining tissue morphology, and deletion of autoreactive lymphocytes

(16). As seen in Fig. 1-1, apoptosis is an complex pathway with many stimulators, such

as Caspase-3, and inhibitors, such as AKT. Apoptosis is highly regulated due its

destructive potential. Evidence shows that apoptotic cells can induce inflammation and/or

result in the formation of cryptic epitopes (13). Previous studies show increase levels of

apoptosis in the scid mouse and the NOD, compared to the BalB/c and young NOD (17).

The increased levels of apoptosis in the scid mouse, indicates that apoptosis may be one

of the regulatory abnormalities of the submandibular gland present in phase I (18). The

increased cysteine proteases in the scid mouse implies that apoptosis is lymphocyte-

independent in phase I. However, cysteine proteases have been shown to increase in

activity as the NOD aged from 8 weeks to 20 weeks, with the scid mouse showing the

highest level of activity (17).

The humoral response plays an important role in phase II of Sj S. In addition to the

SLE antibodies (SS-A and SS-B), anti-M3 muscarinic acetylcholine receptor

autoantibodies have been identified in humans (19) and the NOD mouse (17). SS-A and

SS-B are antibodies targeted to antigens that are usually confined to the nucleus (20);

however, due to certain events in phase I of Sj S these antigens may be presented to the

immune system. Muscarinic acetylcholine receptors are important in the secretary

function of mammalian exocrine glands. The muscarinic-cholinergic receptor is

stimulated by neurotransmitters, resulting in a signal transduction that allows fluid

secretion from the salivary gland acinar cells (17). The interaction between an anti-M3

antibody and the M3 receptor may result in down-regulation of receptor density, as well

as postreceptor second messenger pathway signaling events necessary for proper








activation of fluid movement through epithelial cells (20). In studies where the anti-M3

antibody and other autoantibodies were introduced into mice lacking the adaptive

immune response (scid mutation), the anti-M3 antibody was the only antibody capable of

inhibiting secretary function (20). The development of autoantibodies could be a primary

immunological response or a secondary effect. In the case of Sj S, it is most plausible that

the presence of autoantibodies is a secondary effect of the immune system activation

(17). Interestingly, many of the autoantibodies are targeted to cell surface proteins

important in the secretary response (20), possibly explains why other areas (skin, lungs,

GI tract and vaginal tissues), besides the smg and lacrimal glands, are affected in Sj S.






S Survival ,
PP2C AM \

FLIP PP2A






C. .spi- '
Bax





Apoptosis g






Figure 1-1. Apoptosis pathway. Notice that AKT is an inhibitor early in the pathway and
Caspase-3 one of the last activated caspases (16).









The NOD mouse has allowed researchers insight into the phase I of Sj S, plus the

subsequent development of clinical manifestations. However, a multitude of factors

important in the initiation and progression of the disease are not known. Through the

development of congenic NOD strains, specific aspects of Sj S can be studied. The

NOD.CB 17-PrkdcScid/J (scid) mouse is a congenic mouse that was developed by

backcrossing the immunodeficiency locus (scid) onto the NOD genetic background. The

scid locus confers a functional loss of the B and T lymphocytes, otherwise the NOD-scid

mouse retains the genetic profile of the NOD/LtJ. The NOD-scid mouse clarifies the

significance of the genetic background in the initiation of Sj S, as well as the role of

lymphocytes in the clinical manifestations. In addition, the scid mouse is capable of

accepting allogenic and xenogenic grafts, making it a good mouse model for adoptive cell

transfer studies.

There is evidence of intrinsic factors present in the salivary glands of the NOD

mouse that are capable of triggering an autoimmune response (14). Analysis of the NOD-

scid mouse can clarify the role genetics has in the onset and progression of the disease.

During phase I, the NOD-scid mouse shows a morphological change in the salivary

glands, involving a loss of acinar cells within the submandibular glands. The loss of

acinar cells increases with age and may be due to either hyperproliferation of the

submandibular ductal cells, or apoptotic events (21). However, there are dramatic

increases in cysteine protease activity (enzymes important in programmed cell death) in

the submandibular glands of older NOD mice and NOD-scid mice (12). While the scid

mouse depicts the same glandular changes as the NOD mouse, the salivary flow rate does

not decrease in the scid. It is possible; therefore, that there is compensation by the minor









salivary glands parotidd, sublingual, and those in the oral mucosa), resulting in a stable

saliva production (21). Nevertheless, data strongly suggest that lymphocytes are

necessary for disease progression and the loss of secretary function. Although salivary

flow remains stable, there are changes in its composition. The composition of salivary

proteins, as shown by epidermal growth factor (EGF) and amylase concentrations in

NOD-scid saliva, show significant changes with age (21). There is also a novel

expression of PRP and an internally cleaved PSP isoform (27 kDa), prominent in 15-

week-old NOD-scid mouse saliva, but not detected in normal BALB/c mice or younger

NOD-scid mice (21). These protein changes are similar to those observed in the

NOD/LtJ. Interestingly, the time at which this new isoform of PSP appears in the saliva

and submandibular glands of NOD-scid mice coincides with the appearance of

lymphocytic infiltrates in the salivary glands of NOD mice (21). It is possible that the

new isoform of PSP, intrinsic to the NOD mouse and its progenitors, may present as a

"foreign antigen" to the NOD immune system, thus initiating the autoimmune response.

In the NOD mouse, there is stimulation of autoreactive T-cells and autoantibodies. The

stimulation of both branches of adaptive immunity may result in the progression of the

disease to phase II of Sj S.

The NOD-scid aids in clarifying the roles of genetics and lymphocytes in the

initiation and progression of Sj S. It is been made clear, by observing the scid mouse, that

lymphocytes are important in the loss of saliva secretion. However, the mechanism by

which lymphocytes affect the salivary gland function is unclear. There are a few factors

of the immune system that may separately, or collectively, inhibit saliva secretion. There

is evidence of apoptotic activity in the salivary glands in the NOD mouse (13). The NOD









shows the presence of antibodies specific to exocrine gland receptors, which are capable

of reducing salivary flow (20). As stated previously, there are a variety of aberrantly

expressed cytokines. The possible pathological effects of these immune responses,

provide a range of areas to be researched. In order to study the different immunological

aspects, we examined congenic, NOD-derived strains. Two interesting strains used in this

study are the NOD.Igh6tm Cg, commonly known as NOD.Ignt"l and for convenience in

this thesis, Ig~null, and NOD. 129P2(B6)-I/4 tmtCgnDvsJ, commonly know as NOD.IL-4--

and for convenience in this thesis, IL-4-.

The NOD.Igng"l mouse is a congenic partner strain of the NOD mouse that is

deficient in B-cells. This mouse was developed by disrupting one of the membrane exons

of the gene encoding the p-chain constant region by gene targeting in mouse embryonic

stem cells (13). NOD.Ignuit1 mice exhibit glandular abnormalities of the NOD. Aberrant

PSP production is present, but exocrine gland dysfunction (xerostomia,

keratoconjunctivitis) does not occur (21). These data imply that B-cells have a significant

role in the appearance of xerostomia and keratoconjunctivitis sicca. The B-cell may be an

important antigen presenting cell, capable of stimulating autoreactive T-cells. The B-cell

also produces autoantibodies present in the NOD mouse and patients with Sj S. Transfer

of serum from human patients into the Iggnul mice results in a decrease in salivary flow,

thus providing evidence that the B-cell is important as an antibody producer, rather than

as an antigen presenter (22). This also implies a role for autoantibodies as the effector

mechanism for secretary inhibition.

The NOD mouse has a cytokine profile that is specific to the appearance of

infiltrates in the salivary glands. The cytokines present consist of interferon (IFN)-y,









tumor necrosis factor (TNF)-P, IL-10, IL-2, IL-6, IL-12 and IL-18, but usually lacking

detection of IL-4 (23). Due to its absence, IL-4 has been disregarded as important in

progression of the disease. However, the increasing evidence of autoantibodies as an

effector mechanism of secretary loss supports a possible role for IL-4 in development of

phase II of Sj S. The IL-4 cytokine regulates the B-cell maturation and the switch from the

IgM to IgGi antibody. NOD.IL 4-/' mice exhibit aberrant glandular formations, the novel

PSP isoforms, and glandular infiltrations. However, the IL-4-- mouse does not exhibit the

loss of salivary flow. The B-cells, unable to class switch, can not produce autoantibodies

specific to exocrine gland receptor, such as the M3R. This findings support the theory that

the humoral response is the mechanism by which exocrine gland secretion is disrupted.

Adoptive Transfer Studies

The use of various gene knockout NOD strains allow different aspects of the

disease to be investigated. The NOD-scid clarifies the roles of genetics and the immune

response. In addition, the roles of B-cells and the IL-4 cytokine are elucidated by

observing the NOD.Ign""l and NOD.IL-4-/-. All of these strains have the glandular

abnormalities and protein production characteristic of phase I of Sj S, but development of

xerostomia or keratoconjunctivitis does not occur. These observations, as well as

serologic analysis of the NOD mouse and patients with Sj S, suggest that the humoral

response is a pathologic mediator of Sj S. By utilizing the adoptive transfer model, the

introduction of a combination of lymphocytes into the NOD-scid mouse, we hope to

identify immune cells important in the role of autoantibody production in the

immunopathology of Sj S.

The adoptive transfer model, using the NOD-scid mouse, was originally developed

to study the etiology of diabetes in the NOD/LtJ. Islet-infiltrating lymphocytes, as well as









splenic lymphocytes from NOD mice, can initiate diabetes when transferred into NOD-

scid mice (24). In those studies, lymphocytes were collected from pre-diabetic and

diabetic mice. The pre-diabetic and diabetic lymphocytes were transferred separately, and

also as a heterogeneous pool, into NOD-scid recipients. The incidence of diabetes in the

recipient NOD-scid was approximately 70%, similar to the NOD/LtJ control, when

splenic lymphocytes from diabetic NOD mice were transferred into NOD neonate

recipients (pre-diabetic and pre-SjS) (18). At 12 weeks of age, 50% of the recipient NOD

mice showed submandibulary gland infiltrations (18). Lymphocytes from the NOD/LtJ

also initiate sialoadenitis in the scid mouse, but these findings do not identify the role of

the humoral response in the cessation of salivary secretion.

The congenic NOD.IL-4-- mouse does not develop Sj S. T-cells from the IL-4--

mouse are not capable of stimulating isotype switching in B-cells and consequently the

IgGi, anti-M3R antibody is absent from its sera. In a preliminary study, NOD.IL-4/- mice

were intraperitoneally injected with splenic T lymphocytes from NOD.Igtnulor NOD.IL

4-/- mice (23). This resulted in a decrease of salivary flow in 66% of the Ig"lU -ll T cell

recipients, in contrast to the stable salivary flow of the IL 4-" T cell recipient (23). The

inherent inability of IL 4-- mice to develop Sj S implies that the T-cell's main role in

disease progression is as an activator of isotype-switching in B-cells. This theory is

further supported by the progression of xerostomia elicited by adoptively transferred

splenic T-cells. As shown in a previously mentioned study, NOD.Ig"U[11 T-cells are

capable of stimulating autoantibody production. However, the Igt"11 mouse does not

develop xerostomia. The absence of xerostomia was reversed by transfer of human serum

IgG, from Sjogren's Syndrome patients, into the Igat" mouse (22). Therefore, a logical









conclusion from these data, is that the humoral response appears to be a necessary and

sufficient mediator of xerostomia. In order to clarify the causal effect of B-cells and T-

cells in Sjogren's Syndrome, the research of this thesis seeks to fulfill the following

specific aim:

1. Adoptively transfer different combinations of splenic lymphocytes from the

NOD/LtJ mouse and its congenic strains, NOD.Ignal" and NOD.IL 4-/- into the

NOD-scid mouse in order to establish the adoptive transfer model for phase II

of Sj S.

To verify the establishment of Sj S in these adoptive transfer mice, I have

examined:

The salivary flow and enzyme activity within the saliva of the treated

scid mice.

The submandibular gland of the treated scid mice to detect the presence

of apoptotic events, using flow cytometry, cysteine protease activity, in

situ staining, and protein expression via polymerase chain reaction.














CHAPTER 2
MATERIALS AND METHODS

Animals

Animals used in this study were NOD-scid (experimental recipient), NOD/LtJ

(experimental donor), NOD.IL 4-/- (experimental donor), NOD.Ign""l (experimental -

donor), and NOD-scid (negative control), and NOD/LtJ (positive control). Female mice

were used in all experiments. The donor mice were 16 weeks of age, while the recipient

and control mice were 12 weeks old at the time of adoptive transfer. Each treated set

includes 4 recipient mice (NOD-scid), 2 positive control mice (NOD/LtJ), and 2 negative

control mice (NOD-scid). Most of the NOD-scid mice were purchased from Jackson

Laboratory (Bar Harbor, Maine). The other NOD-scid mice and the NOD/LtJ,

NOD.IL 4--, NOD.Ig""ul were purchased from the University of Florida, Department of

Pathology Mouse Colony (Gainesville, FL) and were housed in the Department of

Pathology Mouse Colony under specific, pathogen free conditions.

Adoptive Transfer

The donor mice were euthanized by cervical dislocation and their spleens extracted.

The spleens were then pressed through a wire mesh in order to separate the splenic

lymphocytes. The lymphocytes were collected and the erythrocytes lysed with 0.84%

NaC1. The cells were then washed and incubated for 30 min with the appropriate

antibody. Antibodies used were anti CD19 FITC, anti CD3 APC and anti CD25 PE.

All antibodies were from BD Pharmingen. After incubation the cells were washed and

diluted to a concentration of 8 x 106 cells/ml in 2% FBS in PBS. The lymphocytes were









then sorted by Douglas Smith at the University of Florida, Flow Cytometry Core

Laboratory. CD19 B-cells and CD3+CD25- T-cells were collected. The fractionated

splenic lymphocytes were then washed and placed in 1001p IX Phosphate Buffered

Saline. The fractionated splenic lymphocytes were intravenously injected into the

recipient scid mice in a 1 to 1 ratio (B-cells to T-cells). Each recipient mouse received

approximately 3 x 106 cells. The experimental and control mice were divided into two

sets of 8 mice (4 experimental scid mice, 2 positive control NOD mice, and 2 negative

control scid mice). One set was observed for 4 weeks, post-transfer (housed until 16

weeks of age), before being euthanized. The other set was observed for 8 weeks, post-

transfer (housed until 20 weeks of age) and then euthanized. The submandibular glands,

spleens and pancreas of the euthanized mice were collected. The submandibular glands

were sectioned into 3 fractions. One section was placed in 2 ml microcentrifuge tubes on

ice, for flow cytometry analysis. The next section was placed in cassettes, stored in 4%

Formalin overnight, and then placed in 70% EtOH to be formed into paraffin-embedded

blocks. The last section was placed in 2 ml microcentrifuge tubes and frozen with dry ice.

The frozen tubes were then stored in at -800C for future analysis.

Saliva Collection

Saliva was collected from the experimental (NOD-scid), positive control

(NOD/LtJ), and negative control (NOD.IL 4-/-). The salivary glands were stimulated with

secretagogues to induce salivation. The secretagogue solution is composed of

Isoproterenol (Img/ml) and Pilocarpine (2mg/ml) (Sigma) in IX Phosphate Buffered

Saline. Each mouse received 100 .il of the secretagogue via an intraperitoneally injection.

The saliva was stimulated for 1 minute and then collected by pipetting for 10 minutes.

The collected saliva was placed in 1.5 ml microcentrifuge tubes. The volume of saliva









was quantified using pipettors and recorded. The saliva was then stored in a -80C freezer

for future protein analysis.

Protein Concentration

The protein concentration of the collected saliva and submandibular gland lysate

was determined, using the Bradford assay. 1 mg of Bovine Serum Albumin (Sigma) was

diluted in 1 ml deionized H20. The Bradford protein assay dye was then diluted 1:5 in

deionizd H20. The standards and samples were made and later read in in 2 ml cuvettes.

The 1 mg/ml BSA was diluted with H20, to a total volume of 25 il, to make the

following standards: 0 mg/ml, 5 mg/ml, 10 mg/ml, 15 mg/ml, and 20 mg/ml. For each

sample, 5 ll of sample and 20 pl of H20 was added to each cuvette. 1 ml of the diluted

Bradford dye was added to each cuvette, and mixed with a pipette. The standards and

samples were allowed to incubate for 5 minutes at room temperature. The protein

concentration was detected using the protein concentration program available in the

spectrophotometer (Bradford). The standards were used to make a standard curve and

then the protein concentrations of each sample extrapolated from the standard curve.

Amylase Activity Analysis

Detection of amylase present in collected saliva and submandibular gland lysate

was accomplished by using the Infinity Amylase Reagent (Thermotrace). Amylase

activity is detected via utilization of Ethylidene-pNP-G7 (EPS) as the substrate. The

cleaved EPS reacts with a-glucosidase, resulting in release of a chromophore. The color

change is then detected at 405nm in a spectrophotometer (BioRad). Amylase activity is

defined as the rate of formation of EPS fragments per liter of sample (U/L). The saliva

samples were diluted 1:200 in deionized H20. The submandibular gland lysate were

analyzed undiluted However a few gland lysate samples had an amylase activity that









was unmeasurable when not diluted. These samples were diluted 1:100 in H20. 1 ml of

Infinity Amylase reagent was added to a cuvette and incubated in a waterbath set at 37C

for 1 min. 25 il of diluted sample was then added to the heated reagent and incubated for

1 min. After the 1 min incubation was completed, the timer for the next incubation

minute is started. The reagent/sample was mixed with a pipette and read in the

spectrophotometer to obtain the initial reading. Afterwards the reagent/sample was placed

in the waterbath until the incubation time is complete. The timer was started for the final

incubation time of 1 min, immediately after the second incubation time was completed.

The reagent/sample was read in the spectrophotometer to obtain the second reading. The

reagent/sample was placed in the waterbath. At the end of the final incubation time, the

reagent/sample was read. The spectrophotometer was blanked with H20 in the Infinity

Reagent. The positive and negative controls were obtained from the provider of the

amylase reagent (Thermotrace) and treated the same as the samples. From this assay,

three O.D. readings are obtained. The change in amylase activity is obtained by

subtracting the initial O.D. reading from the final O.D. reading and dividing by the

amount of incubation time between the initial and the final reading (2 min). The obtained

value is then multiplied by the conversion factor (5140).The conversion factor is

calculated based on manufacturer instructions. The amylase activity of each sample (U/L)

is then divided by the known protein concentration (pg/L), and then multiplied by 1 x 104

to standardize. The resulting amylase activity is measured as the rate of formation of EPS

per gram of protein (U/gram of protein). The assay was performed three times in order to

obtain statistical analysis.









Apoptosis Detection

Apoptosis Detection via Flow Cytometry

The day the collected submandibular glands are retrieved from the experimental

and control mice, the glands are digested. The submandibular glands are minced and

placed in a vial with 5 ml of Collagenase Solution I (2 mg/ml of collagenase IV (Sigma)

in 1X Hanks Balanced Salt Solution (HBSS) and 200 Cl of Dnase). The vials were then

placed in a shaking waterbath, set at 370C, for 15 minutes. The partially-digested glands

were passed through a series of syringes with needles increasing in gauge, from 18 to 23.

The digested glands were then placed in a vial with Collagenase Solution II (1 mg/ml of

collagenase in IX HBSS with 200 Cl of Dnase). The glands were incubated in a shaking

waterbath for 5 min. The digested gland mixture was passed through a series of syringes

again. At this point the glands should be fully digested. The submandibular cells were

placed in 50 ml conical tubes containing 2% FBS in IX HBSS and placed on ice. The

submandibular cells were collected after centrifugation and counted. The submandibular

cells were diluted with 1X PBS to a concentration of 1X 106 cells/ml. In sterile 12 ml

plastic tubes, 5 mls of 55% Percol was added and 2 mls of the diluted, digested glands

were carefully layered on top with siliconized pipettes. The tubes were centrifuged for 30

min at a speed of 2,000 rpms. After the gradient separation the acinar cells and infiltrates

were collected with siliconized pipettes and placed in 12 ml plastic tubes. The cells were

washed and counted. The acinar and infiltrates were then placed in a sterile culture plate

with a solution containing 2% FBS in RPMI (Sigma) and IX antibiotics. The cells were

cultured overnight to allow the cells to recuperate. The following day, the acinar and

infiltrate cells were fixed in 1 % paraformaldehyde in 1X PBS for 15 min on ice. The

cells were washed and placed in 5 ml flow cytometry tubes with 70% ETOH. The tubes









were placed in a -20C freezer to be stored for 24 hrs. The fixed cells were washed in

preparation of staining by reagents provided in the APO-Direct kit (BD Pharmingen).

This assay utilizes a Tunel labeling system, where terminal deoxynucleotidyl transferase

enzyme (TdT) enables the template independent addition of FITC labeled deoxyuridine

triphosphate nucleotides (FITC-dUTP) to dna strand breaks present in apoptotic cells.

Propidium Iodide (PI)/RNase solution was used to stain total DNA. Apoptotic cells were

provided by the kit as a positive control, as well as non-apoptotic cells as a negative

control. After staining, following the procedure outlined by manufacturer specifications,

the cells were incubated in Propidium Iodide and analyzed by flow cytometry.

Manufacturer suggestions were used as a guide to determine appropriate parameters for

apoptosis detection. Cells were identified as apoptotic if they expressed FITC-dUTP. The

parameters for positive apoptosis was based on the level of positivity determined by the

positive and negative control. A window was selected that included positive control cells

that express dUTP, but excluded negative control cells. In order to analyze and

standardized the results, the following formula was used: (%pos. cells sample x % pos.

cells in the neg. control)/% pos. cells in pos.control. Using the aforementioned formula,

substituting the %pos. cell sample x with the %pos.cells positive control (provided in the

kit), will standardize the positive control as 100% positive. The same formula is applied

to the negative control (provided in the kit) so that the level of positivity is 0%. The

equation is applied to each sample and the positivity determined. For comparison, the

relative positivity in each treated set is assigned a representative value on a scale from 0

to 10 (0 is equal to 0% positive and 10 is equal to100% positive).









Akt Expression via Polymerase Chain Reaction

The stored experimental and control submandibular glands were removed from the

freezer and 25 mg of tissue removed. The sectioned piece of submandibular gland was

then treated according to manufacturer specifications to extract total RNA (Qiagen). The

extracted total RNA was then quantified in the spectrophotometer. A quartz cuvette was

used and the spectrophotometer was blanked with deionized water. The total RNA was

diluted 1: 50 in deionized water and read in the spectrophotometer. Only total RNA with

at least a concentration of 800 .g/ml were used to produce cdna. To a .02 ml nuclease-

free centrifuge tube the reagents were added in the following order; 1 tl of pd(T)12-18, 1

pl of 10 mM dNTP, 1 pg of total RNA, and enough water so that the total volume in the

tubes equal 10 pl. After mixing, the tubes were heated in a waterbath for 5 min at 65C

and quick chilled on ice. The tubes were then centrifuged and the reagents added in the

following order; 4 pl of 5X First-Strand buffer, 2 p0l 0.1M DTT, 1 pl RNasin, and 2 pl

acetylated BSA. After gentle mixing, the tubes were incubated in a waterbath, and set at

42 C for 2 minutes. To each tube 1 p1 of SuperScript II was added and mixed gently. The

tubes were incubated at 420 C for 50 minutes and then 700 C for 15 minutes in the

thermocycler. The cdna concentration was quantified by the spectrophotometer. A quartz

cuvette was used and the spectrophotometer blanked with deionized water. The cdna was

diluted 1 : 50 in deionized water and read in the spectrophotometer. A master mix was

made by adding the following reagents to a 2 ml nuclease-free centrifuge tube; 5.1 pl

10X Buffer, 1.1 pl 10 mM dNTPs, and 1.6 pl of MgC12 (formula is for one per reaction).

7.5l of the master mix was added to a .02ml nuclease-free centrifuge tube. To each .02

ml per tube the following reagents were added; 1.5 pl of the forward primer, 1,5 pl of

the reverse primer, 2 pg of cdna, and enough water so that the volume of the









microcentrifuge tube equals 49.75 [il. The primer sets used were 18S (housekeeping

gene) and AKT. Both primer sets were individually developed by Invitrogen. The tubes

were mixed, centrifuged and incubated in a thermocycler, following the standard program

for polymerase chain reaction. The annealing temperature was set at 57.20C. After the

dentaure step, but before the 1st annealing temperature is reached, .25 tl of Taq DNA

Polymerase (Invitrogen) is added to each tube. The per reaction is then continued in the

thermocycler.

Agarose Gel Electrophoresis

A 2% TBE, agarose gel was made and 5 itl of ethidium bromide added to the gel

while it was still warm. After the gel set, the previously made per products were

defrosted. 12 pl of 18s amplicons and 3 pl of 6X Blue/Orange Dye (Promega) were

added per well. In the lane above, 24 pl of akt amplicons and 4 pl of 6X Blue/Orange

Dye (Promega) were loaded into each well. 1 tl of the PGEM DNA marker (Promega),

4 tl of dye and 23 pl of 1X TBE was loaded into a well. The TBE gel was then run at 70

volts for 1.5 hr. Afterwards, the gel was visualized with a gel document system, and

quantitative analysis done using the AlphaEase FC program (Alpha Innotech). The level

of akt expression per sample is based on the samples corresponding level of 18s

expression. 18s is ubiquitous in all cells and should; therefore, all samples should have

the same amount of expression. However slight variations in the level of expression

occurs. Within each lane of a gel are many samples. To standardized the values for 18s

expression, a ratio is formed by dividing highest 18s value within that lane by the 18s

value for each sample within the same lane. The inverse of the calculated 18s ratio

(highest 18s/18s of sample x) is then multiplied by the corresponding akt expression









value (inverse of 18s ratio of sample x, multiplied by akt of sample x). From these

calculations a relative, qualitative expression value is obtained.

Caspase-3 Activity Detection

Stored submandibular glands were defrosted on ice and homogenized with 600 ptl

of Tris-HCL, ph 8.0 in 5 ml plastic tubes. The gland lysate was transferred to 2.0 ml

microcentrifuge tubes. The gland lysate was then assayed following manufacturer

specifications (Calbiochem). The activity assay was read in a microplate reader for 20

minutes and the values calculated in Excel. The data points were plotted on a graph,

activity (x axis) vs. time (y axis), and a trendline generated. The slope of the trendline

was then multiplied by the conversion factor (conversion factor tabulated as suggested by

kit).

In Situ Apoptosis Detection Kit.

The previously paraffinized submandibular glands were sectioned and placed on

slides by Histology Technical Services. The slides were then deparaffinized by

incubation in a series of solutions in the following order: two incubations in Xylene for 5

min, two incubations in 100% EtOH for 2 min and two incubations in 95% EtOH for 2

min, two incubations in 70% EtOH for 2 min, two incubations in H20 for 2 min. The

slides were then stained, following manufacturer specifications (Trevigen), in a humidity

chamber. After the slides were dehydrated, the tissues were covered with a non-aqueous

mount and a coverslip added. The slides were allowed to dry overnight and then viewed

under brightfield microscopy.









Detection of Infiltration

Immunohistochemistry

The paraffinized submandibular slides were obtained as stated previously. They

were deparaffinized (as stated previously) and placed in a heated coplin jar, containing

IX antigen unmasking solution (Trevigen). The coplin jar was then incubated in a

waterbath for 30 min at 950C. The slides were then washed with H20 and placed in a

humidity chamber. The slides were equilibrated with the buffer, 1X TBST (Dako) for 5

min. 200 pl of diluted (1:67) goat serum (Sigma) was pipetted onto each slide and

incubated for 20 min at room temperature. The goat serum was carefully wiped off the

slide, so that the tissue remained undisturbed. The slides were then incubated for 1 hr

with 200 pl diluted (1:25) B2-20 antibody (BD Pharmingen), at room temperature. The

slides were then washed with IX TBST for 5 min and incubated with 200 pl of diluted

(1:200) anti-rat antibody (Sigma), for 30 min. Before the diluted anti-rat antibody is

added to the slides approximately 5 pl of mouse serum is added to the 2 antibody

solution. The slides are washed with IX TBST and treated with an ABC alkaline

phosphates kit from Vector Labs. The slides were treated with reagents as specified by

the manufacturer (Vector Labs). The substrate used in the alkaline phosphates system

was Vector Red (Vector Labs). The substrate was mixed following manufacturer

specifications and incubated on the slides for 20 min. The slides were then washed in

deionized H20 for 1 min and then washed with 1X TBST for 5 min. The slides were

incubated with 200 pl of diluted (1:67) rabbit serum (Sigma) for 20 min. The slides were

then incubated with 200 1l, diluted (1:125) CD3 antibody (Santa Cruz), overnight at 40

C. The slides were then washed with 1X TBST for 5 min, and incubated with 200 Cl of

diluted (1:200) a-goat antibody (Sigma) for 30 min. The slides were then treated with the









reagents provided in the ABC immunoperoxidase system (Vector Labs). The slides

were treated as specified by the manufacturer. DAB (Vector Labs) was used as the

substrate, and was mixed as specified by the manufacturer. The slides were incubated

with the DAB for 5 min. The slides are then washed in H20 for 1 min. The slides are

immersed in a working solution of the Light Green Counterstain (Sigma) for 1 min. The

slides are then immersed twice in H20 for 1 min. The slides are then dehydrated by being

immersed in a series of solutions for 2 min; 70% EtOH, 95% EtOH, 2 times in 100%

EtOH and followed by immersion in Xylene for 5 min, 2 times. Afterwards the slides are

coverslipped and visualized by brightfield microscopy. The presence of lymphocytes is

determined as cells that positively stained with Vector Red (T-cells) and DAB (B-cells)














CHAPTER 3
RESULTS

Adoptive Transfer

Purified splenic lymphocytes from certain donor mice were labeled with the

appropriate fluorescent conjugated antibody (except for the unfractionated donor

NOD/LtJ splenocytes) and sorted by flow cytometry. The collected splenocytes were

selected based on the level of binding to the conjugated antibody. The splenocytes were

collected in two separated fractions. Selected B-cells depicted a high level of CD19

FITC expression. Selected T-cells depicted a high level of CD3+APC while showing no

CD25+ PE expression. CD25+ T-cells have shown to have a protective quality in adoptive

transferred of diabetes studies (25). To circumvent inhibition of disease transfer, the

CD25+ phenotype was set as a negative selection parameter. The purity of the collected

splenocytes was analyzed by flow cytometry. Each fraction had a purity of approximately

93%. The collected splenocytes were then washed and intravenously injected into the

recipient NOD-scid. Each scid received an average of 3 x 106 B and/or T-cells.

The donor splenocytes were selected based on their expected contribution to the

development of Sj S. The T-cells and B-cells from the NOD/LtJ were selected because

they should transfer the disease to the scid. The T-cells of the Ig n"ul was selected due to

its shown ability to stimulate an autoreactive B-cell, thus transferring the disease. The B-

cells from the NOD.IL 4-- should interact with a normal T-cell, resulting in transfer of the

disease. Those scid mice that received T-cells or B-cells, but not both, are expected to

exhibit no disease transfer.










Table 3-1. Adoptive transfer, combinations of donor splenocytes transferred to the scid.


Recipient Donor T-cells Donor B-cells Pos. Control Neg. Control
NOD-scid Total spleen Total spleen NOD/LtJ NOD-scid
NOD-scid Total spleen Total spleen NOD/LtJ NOD-scid
NOD-scid NOD/LtJ NOD/LtJ NOD/LtJ NOD-scid
NOD-scid NOD/LtJ NOD/LtJ NOD/LtJ NOD-scid
NOD-scid NOD/LtJ NOD.IL4/' NOD/LtJ NOD-scid
NOD-scid NOD/LtJ NOD.IL4/' NOD/LtJ NOD-scid
NOD-scid NOD.IL4' NOD/LtJ NOD-scid
NOD-scid NOD.IL4/' NOD/LtJ NOD-scid
NOD-scid NOD.Igjn1l NOD.IL4' NOD/LtJ NOD-scid
NOD-scid NOD.Igtn l NOD.IL4/' NOD/LtJ NOD-scid
NOD-scid NOD.Igynn1 NOD/LtJ NOD-scid
NOD-scid NOD.Igynn1 NOD/LtJ NOD-scid
NOD-scid NOD.Igjn1l NOD/LtJ NOD/LtJ NOD-scid
NOD-scid NOD.Igtn l NOD/LtJ NOD/LtJ NOD-scid

Saliva Collection

A significant manifestation of Sj S in the NOD/LtJ mouse is the presence of

xerostomia. Xerostomia in the recipient scid was detected by quantifying the volume of

saliva produced in 10 minutes. Salivation was stimulated by secretagogues and the saliva

collected from 4 treated scid mice, 2 positive control NOD mice and 2 negative control

scids. The collected saliva from the 4 treated mice, per group, was pooled. The collected

saliva from the pos. control was pooled, and the neg. control saliva was pooled as well.

Saliva was collected once a week for 7 wks, but many mice were euthanized early.

Diabetes, which is present in the NOD, was transferred along with the Sj S. The mice that

became diabetic were treated with insulin, but many expired early. To circumvent the

loss of all experimental mice; many diabetic scids were euthanized early.

The collected saliva from 4 week post transfer was compared to the volume of

saliva from the last week of collection. As seen in Table 3-2, the scid (Unfractionated

NOD) did not show a change in salivary flow. The scid (B and T NOD) expressed a

decrease of 46%, which is comparable to previous data on the NOD. The









scid (B IL4-- and T NOD) showed a 68% decrease in flow. The scid (B IL4 )

showed a small decrease of 13%. The scid (T Igniull and B IL4 -) and

scid (T Igni"l") showed a increase of about 35%. The scid (T Ign"ull and B NOD)

had a small increase of 9%. The scid mice that were recipients of either T or B cells, but

not both, had a stable flow, similar to the NOD-scid. Surprisingly, none of the NOD mice

depicted xerostomia. The NOD, in previous studies, has proven to be a good positive

control for Sj S, so this was certainly an unexpected occurrence.

Amylase Activity Analysis

Amylase Activity Detected in Saliva.

An important aspect of Sj S is the aberrant production of salivary components such

as amylase. The collected saliva was analyzed to determine the amylase activity of the

saliva. As shown in Table 3-2, the amylase activity of mice 4 weeks post-transfer (16

week old mice) was compared to the activity of mice 8 weeks post-transfer (20 week old

mice). The calculated readings showed that the scid (Unfractionated NOD) had an

increase of 62%, the scid (T and B NOD) amylase activity decreased by 8% and the

scid (T NOD and B IL4- -) had an increase of 46%. The scid (B IL4 -) had a

decrease in activity of 57%. The scid recipients of T-cells from the Igu""ll all showed a

decrease in amylase activity that ranged from 5% in the scid(T Ignf"ll and B IL4-'-) to

33% in the scid(T Ign"ull and B NOD). The NOD control shows a 21% decrease and a

43% decrease in the scid.

Detection of Salivary Gland Amylase Activity

An important aspect of the initiator phase of Sj S is the change in composition of the

submandibular glands. In order to determine if there are changes, the amount of amylase

present was analyzed. The submandibular glands were homogenized and the gland lysate









incubated with Infinity Amylase Reagent (Thermotrace) for the appropriate time. The

activity was detected in spectrophotometer. As seen in Table 3-3, the scid

(Unfractionated NOD) and scid (T NOD and B IL4--) exhibited an increase in activity

or 300% and 970%, respectively. The scid (T and B NOD) had a decrease of 80%. The

scid (B IL4-/-) showed an increase of 500%. The scid (T Ig"ull" and B IL4- -) and the

scid (T Ig"ull and B NOD) had a decrease in amylase activity of 90% and 34%,

respectively. The scid (T IgnL"ll) showed a less than 6% increase. The NOD and scid

showed a decrease in amylase activity of 33% and 25%.

Protein Concentration

A significant aspect of Sj S is the aberrant protein production, specifically the

production of two novel forms of parotid secretary protein (PSP). The deviant PSP

production allows the protein concentration to remain stable, even as other major proteins

decrease. Using the Bradford protein assay the protein concentration of saliva and

submandibular gland lysate was determined. The protein concentration of the saliva did

not show a change in concentration in any of the adoptively transferred scid mice or the

controls. The adoptively transferred scid mice and the controls was had an average

protein concentration of 3.6 mg/ml, 4 weeks post-transfer. The average protein

concentration at 8 weeks post-transfer was 3.8 mg/ml. The submandibular gland lysate

depicted a decrease in protein concentration for the scid mice that received a combination

of T and B-cells. This decrease ranged from 78% in the scid (T NOD and B IL4-/-) to

22% in the scid (T Igni"u and B I14--). The scid (T Ign""ll) depicted a decrease of

55%. The scid (B IL4-/-), scid (Unfractionated NOD) and the controls did not show a

change in protein concentration.










Table 3-2. The saliva from each adoptively transferred scid was collected and quantified.
The saliva was then assayed to determine the amount of amylase present and
the concentration of proteins.



Age of treated mouse Total Amylase activity Total protein
salivary (U/gram of protein) (mg/ml)
volume
(gl/10 min)
4 wks NOD (n = 10) 185 5881 671d 3.72 .77
>8 wks NOD (n = 7) 236 4643 541 3.48 .3
4wks-scid (n=12) 180 7082 547 3.01 .8
>8 wks scid (n= 12) 216 4051 879 3.21 .1
4 wks scid (Unfractionated NOD) (n = 4) 168 2786 + 173 3.92 + .02
>8 wks scid (Unfractionated NOD) (n= 4) 170 4509 + 410 4.85 .03
4 wks scid (T and B NOD) (n = 4) 168 4881 + 446 3.72 .02
>8 wks scid (T and B NOD) (n = 2) 90a e* 4486 + 107 4.60 + .02
4 wks scid (T NOD and B IL-4-) (n = 4) 125 6055 + 106 3.63 + .003
>8 wks scid (T NOD and B IL-4- ) (n = 2) 40b 8846 + 288e** 2.55 + .02
4 wks scid (B IL-4-) (n = 4) 200 4282 + 126 5.02 + .04
>8 wks scid (B IL-4-) (n = 4) 175 1824 + 450 5.25 + .03
4 wks scid(T- Iggnul and B IL-4-) (n= 4) 63e* 7202 + 443e* 2.93 + .01
>8wks scid(T- Iggnu andB IL-4--)(n= 2) 100 6788 + 136 3.42 + .01
4 wks scid (T- Iggn") (n= 4) 113 4154 + 667 3.51+ .03
>8 wks scid (T- Iggnl) (n= 4) 150 3160 + 519 3.06 + .02
4 wks scid (T- Iggnl" and B NOD) (n= 4) 110 7091 + 442 3.02 + .02
>8 wks scid (T- Iggn aand B NOD) (n = 3) 120 4735 + 397 3.44 + .01
a Due to the transfer of diabetes, this set euthanized at week 5.
b Due to the transfer of diabetes, this set euthanized at week 6.
c Due to the transfer of diabetes, this set euthanized at week 5.
d Values are given as the mean standard error.
e Statistical comparison of adopotively transferred NOD-scid mouse groups to the age-matched NOD-scid parental control by the one
way ANOVA test: (*P < 0.05, **P < .001).










Table 3-3. The submandibular glands for the adoptively transferred scid mice and the
controls were collected and homogenized. The level of caspase activity was
determined using the Caspase-3 activity kit. The gland lysate was assayed to
determine the amylase present and protein concentration.


Age of treated mouse


Total protein
(mg/ml)


Amylase activity
(U/gram of protein)


Caspase 3
Activity
(pmol/min/gram
of protein)


4 wks NOD (n = 10) 3.4 .08 101.2 94 39.8 1
>8 wks NOD (n = 7) 4.3 .41 67.9 .4 31 5
4wks-scid (n=12) 3.4 .02 159.4 30.8 28.4 3
>8 wks scid (n = 12) 4.4 .02 119.1 + 10.2 30.1 6
4 wks scid (Unfractionated NOD) (n = 4) 4.0 + .04 194.7 + 94 59.6 3.3
>8 wks scid (Unfractionated NOD) (n = 4) 4.2 .01 863 433b** 26.5 C
4 wks scid (T and B NOD) (n= 4) 5.2 .02 b* 415 53 29.1
>8 wks scid (T and B NOD) (n = 2) 1.6 .02b** 77 4 40.5 (
4 wks scid (T NOD and B IL-4-) (n= 4) 5.4 .Olb* 34 13 34.6
>8 wks scid (T NOD and B IL-4--) (n = 2) 1.2 .Olb** 365 84 265.8 23.:
4 wks scid (B IL-4--) (n = 4) 2.9 + .03 137 + 29 38.6
>8 wks scid (B IL-4--) (n = 4) 4.6 .02 955 257 44.1
4wks scid(T-Ignull andB IL-4--)(n= 4) 4.3 .Olb** 409 148 36.5
>8wks scid(T- Ignu llandB IL-4--)(n = 2) 1.6 .01 43 14 46.8
4 wks scid (T- Ign"11) (n = 4) 3.4 + .02 161 30 28.6
>8 wks scid (T- Iggn") (n = 4) 1.5 + .Olb** 170 114 62.8 11
4wks scid(T-Ignull andB -NOD) (n = 4) 5.1 + .02b* 862 + 153b* 31.0 -
>8wks scid(T- IgInullandB -NOD)(n= 3) 4.0 + .01 573 129b* 48.3 +
aValues are given as the mean standard error.
b Statistical comparison of adopotively transferred NOD-scid mouse groups to the age-matched NOD-scid parental
control by the one way ANOVA test: (*P < 0.05, **P < .001).


0a
.8
.8
>.7
3b**
'.3
.6
).1
.2
8b**
.2
.9
1.3
4.6
1.1
.4b**
t.7
.8


Apoptosis Detection

Apoptosis Detection via Flow Cytometry

The presence of apoptosis is a significant aspect of xerostomia and is a noted event

in disease progression. Apoptosis in the submandibulary acinii cells was detected

following the protocol provided in the Apo-Direct kit (BD Pharmingen). Apoptotic and

non-apoptotic cells were provided as a positive and negative control. The level of

positivity for apoptosis was based on the controls. As seen in Table 3-4, apoptosis was

not detected in the scid (B IL4--) at 4 weeks and 8 weeks post-transfer. Apoptosis was

also detected in the scid (T Ign""ll) at 8 weeks. The NOD mice exhibited a low level of

apoptosis at 4 weeks post-transfer with no apoptosis present at 8 weeks. There was no











apoptotic events detected in the scid negative control or the experimental scid mice at 4


weeks or 8 weeks.


Table 3-4. Apoptosis detection using Apo-Direct kit (BD Pharmingen). The positivity of
each sample is based on the positive and negative controls provided in the kit.
The samples were standardized by the following formula: (% Pos of sample X
% Pos. of Neg. control)/ (% Pos. of Pos. Control). Numerical values are
assigned so that 0/10 is equal to 0% positive for apoptosis and 10/10 is equal
to 100% positive.

Recipient NOD-scid (age) Donor cells Apoptosis Activity (AU)


4 weeks
NOD/LtJ
8 weeks

4 weeks
NOD-scid
8 weeks

4 weeks
NOD-scid
8 weeks

4 weeks
NOD-scid
8 weeks

4 weeks
NOD-scid
8 weeks

4 weeks
NOD-scid
8 weeks

4 weeks
NOD-scid
8 weeks

4 weeks
NOD-scid
8 weeks

4 weeks
NOD-scid
8 weeks


Positive Control



Negative Control


Unfractionated NOD

Unfractionated NOD

T- NOD and B- I14-/-

T- NOD and B- 114-/-

T and B NOD

T and B NOD

B IL4-/

B IL4-/

T Ig[nu""

T Ig "ull

T- Ig[ nul and B I14-

T- Ig[ nul and B- I14/

T Igiunl and B NOD

T Ignunl and B NOD


1/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

7/10

10/10

0/10

10/10

0/10

0/10

0/10

0/10









Caspase-3 Activity Detection

Apoptosis, otherwise known as programmed cell death, is an important inhibitory

mechanism of cell growth. Apoptosis aids in organogenesis, maintenance of tissue

morphology, and deletion of autoreactive lymphocytes. As seen in Fig. 3-1, there are a

series of events that occur in the apoptotic pathway. One of the last events is the

activation of caspases. Caspase-3 is a member of the interleukin 11 converting enzyme

(ICE) family of cysteine proteases. It is activated by a series of upstream proteases and is

one of the last signaling events before apoptosis occurs. The submandibular gland lysate

samples were analyzed in an enzymatic assay. For each treated set caspase activity was

compared at 4 weeks post-transfer to 8 weeks post-transfer. As seen in Table 3-3, the

scid (Unfractionated NOD) depicted a 55% decrease in caspase activity. The

scid (T and B NOD) showed an increase in activity of 40%. The

scid (T-NOD and B-IL 4- -) depicted an increase of over 200%. The scid (B-IL 4/-) had an

increase of 14% in caspase activity. The scid (T-Ignu"ll) and the

scid (T-Ignull" and B-NOD) both depicted an increase in activity of about 50%. The

scid (T-Ignull and B-IL 4- -) depicted a slight decrease of 18%. The NOD had a decrease

in activity of 22% and the scid had a less than 6% increase in caspase activity.

Akt Expression via Polymerase Chain Reaction

As a critical mechanism of homeostasis maintenance, apoptosis also has a large

destructive potential, if not closely controlled. As seen in Fig 3-1, there are many proteins

capable of inhibiting apoptosis. Akt, also known as protein kinase B, is the protein of

interest for this thesis. Akt inhibits apoptosis by phosphorylating, and thus inactivating

procaspases, Bad, and other transcription factors. The level of akt expression is

determined by amplifying cdna, from each sample, by polymerase chain reaction. The










polymerase chain reaction products are visualized by gel electrophoresis. The level of

expression is tabulated for 18s per products (housekeeping gene) and the akt per

products, using the AlphaEase FC program. The relative expression of akt of each treated

sample is then compared to the NOD (pos. control) and the scid (neg. control), to form

ratios. The sample/scid ratio is then compared to the sample/NOD ratio to determine the

relative increase in expression from 4 weeks to 8 weeks post-transfer. As shown in Table

3-5, the scid (Unfractionated NOD) depicted an increase in akt expression when

compared to the NOD and the scid. The scid (T and B NOD),

scid (T NOD and B IL4--) showed a decrease in akt expression compared to the NOD

and the scid. The scid (T Igfnull and B IL4--) decreased in akt expression compared to

the scid, and remained stable compared to the NOD. The scid (B IL4--) depicted an

increase in akt expression compared to the scid but remained stable compared to the

NOD. The scid consistently had an increased level of akt expression from 4 weeks to 8

weeks, when compared to the NOD.

Table 3-5. Akt expression ratios.


Age of treated mouse


4 wks scid (Unfractionated NOD) (n = 4)
>8 wks scid (Unfractionated NOD) (n = 4)
4 wks scid (T and B NOD) (n = 4)
>8 wks scid (T and B NOD) (n = 2)
4 wks scid (T NOD and B IL4-) (n = 4)
>8 wks scid (T NOD and B IL4--) (n = 2)
4 wks scid (B IL4--) (n = 4)
>8 wks scid (B IL4-) (n = 4)
4 wks scid (T- Iggn1l and B IL4-) (n = 4)
>8 wks scid (T- Ignl"" and B IL4-) (n = 2)
4 wks scid (T- Ign"l1) (n = 4)
>8 wks scid (T- Ignul") (n = 4)
4 wks scid (T- Ignull and B NOD) (n = 4)
>8 wks scid (T- Iggn"" and B NOD) (n = 3)


Akt expression
ratio (compared
to NOD)
1X
2X
1X
>lX
>1X
15X
4X
2X
2X
2X
2X
1X
NA
NA
1X


Akt expression
ratio(compared to
scid)
>IX
>1X


2X
>IXb
1X
ix
2X
2X
>lXb
2X
NA
NA
2X


Akt expression
ratio (scid
compared to NOD)
2X
2X
IX
3X
4X
5X
3X
IX
2X
4X
>1X
NA
NA
IX


aThe fold increase ratio based on the akt expression detected by gel electrophoresis.
bThe amount of akt expressed is 2x or more than the scid control when compared to the adoptively-
transferred scid.










In Situ Apoptosis Detection Kit.

The presence of apoptotic cells was detected by TUNEL staining using fixed

section of smg. As seen in Fig. 3-3, apoptosis has detected in untreated scid mice at 4 wks

pos-transfer and 8 wks post-transfer. Apoptotic cells were also detected in the

scid (T and B NOD) mouse, scid (T Ig"ull and B IL-4- -) mouse and the

scid (T Ign"ull and B NOD) mouse. The average number of apoptotic cells per five

fields was determined. As seen in Table 3-6, the adoptively transferred scid mice showed

a higher number of apoptotic cells compared to the scid parental controls.

Detection of Infiltration

Immunohistochemistry

Many classification systems use the presence of infiltration in the salivary glands as

a definitive marker of Sj S. Lymphocytic infiltrates are seen in patients with Sj S and the

NOD/LtJ. After euthanization the submandibular glands of the adoptively-transferred and

control mice were removed. A fraction of those submandibular glands were fixed and set

in paraffin blocks. Afterwards, sections of paraffinized tissue were placed onto glass

slides. The tissue was then treated with immunohistochemistry reagents to visualize

infiltrates present in the submandibular gland. These slides were stained with Vector

RedTM and DAB to visualize T and B-cells, respectively. A stained T-cell appears red and

the B-cell appears as brown. A light green counterstain was used as well. As seen in Figs.

3-3 A and 3-3 A, there is no infiltrates present in the NOD-scid. Figs. 3-3 B and 3-3 B

show infiltration of NOD/LtJ submandibular glands. There is a predominance of B-cells

in the population. Figs. 3-3 C and 3-3 C show infiltration is present in the

scid (T Ign"1ul + B IL4-/-), with T-cells being the predominate cell.









Sections of paraffinized tissue were also made into hematoxylin and eosin stained

slide. The slides were visualized by brightfield microscopy in order to detect the presence

of infiltrates. Infiltrated lymphocytes were counted to determine the focus score. The

NOD at both 4 weeks and 8 weeks post-transfer had a focus score of >2 (Table 3-6). The

scid positive control did not show any infiltration at week 4 or week 8.


0 3,- lip .


r -


Figure 3-2: Immunohistochemical staining of submandibular glands for lymphocyte-
infiltrations. Staining was performed on paraffin-embedded sections of
submandibular glands. B-cells stained with DAB (brown), T-cells stained with
VectorRed (red) and counterstained with light green. A) 20 wk old NOD-scid
smg gland; focus score = 0. B) 20 wk old NOD/LtJ smg gland; focus score =
1. C) 16 wk old (4 wks post-transfer) NOD-scid (T Ig""ul + B IL4 -);
focus score = 1. Magnification = 10x.















































Figure 3-3. Hematoxylin/eosin-stained tissue sections of submandibular glands. Staining
was performed on paraffin-embedded sections of submandibular glands. A) 20
wk old NOD-scid smg gland; focus score = 0. B) 20 wk old NOD/LtJ smg
gland; focus score = 1. C) 16 wk old (4 wks post-transfer) NOD-scid (T -
Igg"ll + B IL4--); focus score = 1. Magnification = 10x.




The scid (T and B NOD) had a focus score of >2 at 4 weeks with no infiltration


seen by week 8.The scid (T Igg"ull and B IL4-/-) at 4 weeks post transfer had a focus


score of 1 (moderate infiltration), but by 8 weeks no infiltration was detected. The


scid (T Iggnull and B NOD) showed no infiltration at 4 weeks with a focus score of >2


at 8 weeks. The other adoptively transferred mouse groups did not show infiltration at 4


weeks or 8 weeks post-transfer.


* S --: .
S t I *

16*.. ;, ^
I.' -' ,* ;.* *
-. .*4 I 4
'. *? *
-. ,' a' *'* *, *u t
r -, **
C,
4 ~ 7


t '" -: *" -' '



-~ *r


S ..," -- .. ,,

, ,;t t. .
.... ._, -. .. ; ,


I.'#, .'. H. -. .,
:. : .' ,.- .. .
S -* .. : ,, o ,.
: C" -^ -*^ *:"^ '^ ^

~ ~ ~ ~ '. I :.^^l;''l L.^^/ : -_ '-.' ^











Table 3-6. Focus score derived from H+E stained slides of smg glands. A focus score of 1
is equivalent to 50 lymphocytes in a view. 50 lymphocytes in a view is given a
score of 1; 100 lymphocytes is given a score of 2; and over 100 lymphocytes
is given a score of >2.

Recipient NOD-scid (age) Donor cells Focus score


4 weeks
NOD/LtJ
8 weeks

4 weeks
NOD-scid
8 weeks

4 weeks
NOD-scid
8 weeks

4 weeks
NOD-scid
8 weeks

4 weeks
NOD-scid
8 weeks

4 weeks
NOD-scid
8 weeks

4 weeks
NOD-scid
8 weeks

4 weeks
NOD-scid
8 weeks

4 weeks
NOD-scid
8 weeks


Positive Control



Negative Control


Unfractionated NOD

Unfractionated NOD

T and B NOD

T and B NOD

T- NOD and B- I14-/

T- NOD and B- 114-/-

B IL4

B IL4

T- Ig[ nu and B I14

T- Iggull and B- I14/

T- Ig nu""

T Ig 1ull

T Iggnl and B NOD

T Iggnl and B NOD














CHAPTER 4
DISCUSSION

Studies using the NOD-scid mouse have led to advancement in the research of Sj S.

Observations on the scid mouse have provided clarification of the glandular and protein

changes that occur in the salivary gland in phase I of Sj S disease. Although the scid

mouse has these glandular changes, its lack of progression to phase II identifies the

critical role lymphocytes have in development of phase II of the disease. Providing the

scid mouse with appropriate lymphocytes was hypothesized to result in progression of

phase II. The results of this thesis provide details on the ability to adoptively transfer

various combinations of lymphocytes from the NOD/LtJ, NOD.IL-4--, and NOD.Iggn""

into the NOD-scid mouse, thereby, transferring phase II of Sj S to this non-autoimmune

host.

The NOD mouse is considered a good mouse model for Sj S research and is the

positive control used in the research of this thesis. It is expected that lymphocytes from

the NOD, placed in the scid mouse, should result in Sj S development. The data show a

46% decrease in the salivary flow, of adoptively transferred scid mice from week 4 post-

transfer to week 8, receiving T and B-cells from the NOD. However, there is no change

in the salivary flow of scid recipients of unfractionated NOD splenocytes. The stable

salivary flow observed in the scid (Unfractionated NOD) mouse implies that there is a

population of cells in the splenic lymphocytes capable of inhibiting disease transfer. The

removal of this population allows for Sj S progression, as seen in the









scid (T and B NOD) mouse. Presumably, the T-cells from the NOD activates antibody

production by the B NOD, thus resulting in the observed salivary loss. The T NOD

also show an ability to transfer disease when combined with B-cells from the NOD.IL 4-/

. The NOD.IL-4-- mouse does not develop phase II disease, even though disease

precursors are present. The absence of IL 4 cytokine, which is necessary for class

switching, prevents disease progression. However, T-cells from NOD mice provide the

IL-4 cytokine, thus allowing antibody production and the resulting decrease in salivation.

The scid (B IL-4--) mouse had little change in saliva volume, possibly due to the

absence of an appropriate T-cell. B -cells from the NOD mouse, as seen in the

scid (T and B NOD) mouse, have an ability to transfer disease when activated by an

appropriate T-cell. The NOD.Ig""ul, which lacks B-cells, has functional T-cells that

produce IL-4 and are capable of activating B-cells. The recipients of T-cells from the

Ig"u"ll mouse and B-cells from the NOD mouse did not show a decrease in salivary flow

as might be predicted, yet transfer of phase II is possible as shown by the

scid (T and B NOD) mouse and the scid (T NOD and B IL-4 -) mouse. Thus,

transferred disease may be delayed in certain combinations, such as the

scid (T Ign""ul and B NOD) mouse. In the NOD environment, antigen presentation by

B-cells to T-cells, or other APC, may occur before the splenocytes are fractionated.

Under such conditions, T-cells are already producing the cytokines required for class

switching before transfer into the scid mouse. This would allow for rapid transfer of

disease. However the Ign"ull, without B-cells capable of presenting antigens, may have

"naive" T-cells. Transfer of these "naive" T-cells may then require a longer incubation

for development of phase II. Thus, it is possible that scid recipients of T Igj"ull and









B IL-4-- did not show a decrease in salivary flow at 8 weeks post-transfer due to the

"naive" nature T-cells from the Ig""1ul mouse. In a previous study, T-cells from the

Ig"ull" were transferred into the IL-4- mouse, resulting in development of phase II (23).

However, the researchers of this study allotted more incubation time for progression of

phase II (12 weeks compared to the 8 weeks allotted in this thesis). It is also possible that

there is a loss in salivary flow in the

scid (T Ign"ull and B NOD) mouse and the scid (T Ign"ull and B IL-4- -), but this

loss is belied by the method of collection. Pooling of saliva during collection and then

quantifying may mask any loss of salivary flow that is occurring in some of the treated

mice. In the scid (T and B NOD) mouse and the scid (T NOD and B IL-4 -) mouse

the loss of salivary flow for the 4 scid mice was not simultaneous. A variation in timing

of the appearance of phase II was also observed in the T Igni"ul transfer to NOD.IL-4--

study. The scid recipients of T-cells from the Ig"ull mouse had normal salivary flow,

indicating no disease progression.

The NOD has been shown previously to be a good positive control, typically

having a 45 75% loss in salivary flow by 20 weeks of age (17,22,25). However, a loss in

saliva volume was not detected in these experiments. Nevertheless, the parental NOD-

scid mouse had stable salivary flow. Diabetes, a disease present in the NOD, appeared in

all of the scid mice that received B and T-cells from the NOD, but not unfractionated

splenocytes. It is apparent that diabetes was co-transferred with phase II. Due to the

presence of diabetes, 0.1 cc of insulin was administered to each mouse, once a day.

Despite treatment, a few of the experimental and positive controls expired early. In order









to prevent the loss of all of the treated mice, some were euthanized prior to 8 weeks post

transfer. This early euthanization could have affected the results.

Concomitant with the loss of saliva secretion, there are significant changes in the

composition and activity of salivary proteins. Probably indicating multiple mechanisms

of action. The decline in amylase may be a result of glandular changes that results from

defects in glandular homeostasis (21). Amylase, an enzyme that hydrolyzes starch, is one

of the glandular proteins that have an age-related decline in NOD and scid mice, and

therefore, a marker of phase I disease. The NOD and scid controls, as well as the

adoptively transferred scid mice, all had a decline in salivary amylase activity with the

exception of the scid (Unfractionated NOD) mouse and scid (T NOD and B IL-4 )

mouse, which had increases of 46%. The decline in amylase activity indicates that there

are glandular changes occurring, to which the adoptively transferred lymphocytes may

respond. Although the scid (T NOD and B IL-4-/-) mouse has an increase in amylase

activity, the decrease in salivary flow suggests transfer of disease. It is not clear why

there is a discrepancy in this particular adoptive transfer combination. Observation of the

results shows variability in the amylase activity of the submandibular glands amongst the

treated scid mice. The NOD positive control and scid negative control both showed a

decline in amylase activity in the submandibular gland lysate. The scid (T and B NOD)

mouse, scid (T Ignu"ll and B IL-4--) mouse and scid (T Ign"ull and B NOD) mouse

showed variable levels of declining amylase activity in the submandibular gland.

However, the scid (Unfractionated NOD) mouse, scid (T NOD and B IL-4'-) mouse,

scid (B IL-4--) mouse and scid (T Igni"l) mouse showed an increase in amylase

activity. It is not certain what causes such variability in detection of amylase activity in









the submandibular gland, when this same method is useful for detection of salivary

amylase activity. There seemed to be significantly less amylase in the submandibular

gland lysate compared to the saliva. Many samples were analyzed undiluted (a few were

diluted 1:100). From a technical point of view, though, it is possible that this assay was

not sensitive enough to detect amylase activity in the smg lysate.

Phase I of Sj S is marked by aberrant production of salivary proteins. There is

production of two neoteric forms of PSP, which enables the protein concentration to

remain constant, despite declines in other major proteins (21). Results show that there is

no change in salivary protein concentration for the adoptively transferred mice or the

controls. However, there were decreases in submandibular gland lysate protein

concentration from scid mice that received a combination of T and B-cells, irrespective of

the origin of the lymphocytes. These decreases in protein concentration from 4 weeks to

8 weeks post-transfer may be indicative of degradation of the submandibular gland by

apoptosis. The scid (T Ign""ll) mouse also exhibited a decrease in protein concentration.

Apoptosis was measured to determine the effect the transferred lymphocytes may

have on inducing cell death in the submandibular gland. Apoptosis occurs in the NOD

and the scid during phase I of Sj S. This apoptosis is lymphocyte-independent and is most

likely a result of the aberrant glandular homeostasis (27,28,29). As Sj S progresses to the

effector stage, the NOD mouse exhibits increased levels of apoptosis in the

submandibular gland, which has been shown to be lymphocyte-dependent (13). The

results of the caspase-3 activity analysis revealed an increase of activity in all of the scid

recipients receiving combinations of T and B-cells, except for the









scid (Unfractionated NOD) mouse. The increase in Caspase-3 activity in scid recipients

of T and B-cell transfer, indicate that the transferred lymphocytes have an effect on

apoptosis. The scid (T Ign"ll) mouse had a large increase (120%) in caspase activity.

This may indicate that there is T-cell dependent role in apoptosis via cytokine production

or cytotoxic action of CD8+ T-cells.

Caspase-3 activation is one of the last required events before apoptosis is

completed, thus the observed increase in activity. However, AKT, as an inhibitor of

apoptosis, would exhibit a decrease of expression. The results indicate a decrease in AKT

expression in the scid (T and B NOD) mouse and scid (T NOD and B IL-4-/-) mouse

compared to the level of AKT expression in the NOD. These groups also had a decrease

of AKT expression when compared to the scid negative control. These findings suggest

that AKT production has been decreased and may be allowing activation of apoptosis in

the submandibular gland. This correlates with the increased level of caspase-3 activity

seen in these two groups. Interestingly, the scid (Unfractionated NOD) mouse depicted

an increase of AKT expression when compared to the NOD and scid, as well as a

decrease in capase-3 activity. This is possibly due to what is a still an unidentified,

inhibitory factor in the unfractionated NOD splenocyte population. The scid (B IL-4- )

mouse did not show a decrease in AKT expression when compared to the NOD mouse

and had a slight increase in expression, when compared to the scid mouse. The increase

of AKT expression in the scid (B IL-4-/-) mouse and concurrent decrease of caspase-3

activity, would indicate a cessation in apoptosis activity. The

scid (T Ign"ull and B IL-4-/-) mouse did not show a decrease in AKT expression

compared to the NOD, but had a decrease in AKT expression compared to the scid









mouse. This group also showed an increased level of caspase-3 activity, which is

indicative of apoptosis. The scid positive control had an average 2-fold increase of AKT

expression compared to the NOD mouse.

The presence of elevated capase-3 activity and decreased AKT expression suggests

the presence of apoptotic cells. Visualization of apoptotic cells by flow cytometry was

attempted. However, apoptosis was only detected in the scid (T Ign""ll) mouse and

scid (B IL-4 -) mouse. It is possible that apoptotic cells were lost in the preparation of

the acinar cells and unable to be detected by flow cytometry. The high level of apoptosis

detected in the scid (T Ig"ull) mouse by flow cytometry may be the result of cytokine

production. It is not clear why a high level of apoptosis was detectable in the

scid (B IL-4 -) mouse. The results of the TUNEL stain show apoptotic cells in the scid

mouse, parental control and the adoptively treated scid mice. Previous studies show that

apoptotic cells are present in phase II, thereby indicating the development of phase II in

the adoptively transferred scid mice. Previous findings show that the number of apoptotic

cells increases as the scid mouse ages (17,19). However the NOD-scid parental control

had a decrease in the number of apoptotic cells present.

A definitive aspect of Sj S is the appearance of infiltrates in the submandibular

gland. The results from the stained histology slides show the presence of infiltrating

lymphocytes in the scid (T and B NOD) mouse, scid (T Ig null and B IL-4'-) mouse,

and the scid (T Igf"ull and B IL-4-/-) mouse. This localization of lymphocytes in the

submandibular gland is a precursor to the loss of salivary flow, thus indicating a transfer

of Sj S-like disease. Interestingly, the scid (T and B NOD) mouse depict a high level of

infiltration (focus score >2) at 4 weeks, and also exhibited a decrease in salivation.









However, the scid (T Ig ~"ll and B NOD) mouse did not have infiltration until 8 weeks

post-transfer, and did not lose salivary flow within that time. This supports the theory that

adoptive transfer of T-cells from the Ign""ul mouse into the scid recipient may require a

longer incubation for Sj S to develop. A similar situation is observed in the

scid (T Ign""ul and B IL-4--) mouse, which had a lesser amount of infiltration then the

scid (T and B NOD) mouse and did not have a decrease in salivation at 8 weeks post-

transfer. Infiltration in the scid (T NOD and B IL-4 -) mouse was not observed at 4 or

8 weeks post-transfer; however, there was a loss of salivary flow. This raises the

possibility of a great effect by cytokine or other soluble factors.

In conclusion, transfer of a clinical Sjogren's Syndrome-like disease into the scid

mouse by adoptive transfer is possible under certain conditions. The results of this thesis

show that a transfer of T-cells from the NOD with functional B-cells will allow transfer

of phase II to the scid recipient. Seemingly, the transfer of entire NOD splenocyte

population may allow for a delay in Sj S or even disease prevention. The results of the

different analysis show that the scid (Unfractionated NOD) mouse consistently had

results that were contradictory to the results observed in the scid (T and B NOD)

mouse. This provides more evidence of a possible inhibitory factor present in the

unfractionated NOD splenocytes. There is a population of T-cells that can inhibit

development of diabetes suggesting an important role of regulatory cells within the NOD

mouse. This population may be present in the unfractionated population, and capable of

preventing disease progression. Although the loss of salivary flow was not detected in

adoptive transfer of T-cells from the Ig"nul mouse together with functional B-cells,

adoptive transfer of phase II may be possible. It is possible that T-cells from the Ign"ull









may not be presented with the necessary antigens until interacting with a functional B-

cell. In this situation, the development of disease would require a longer period of time. It

is also probable that pooling of saliva during collection may mask individual changes in

flow rates and protein content, especially since the loss of salivation may occur at

different times in individual mice. Future directions of this research would be to use the

NOD.B10.H-2b mouse as one of the donor mice. The NOD.B10.H-2b develops SjS, but

does not develop diabetes as per the NOD (30), thus preventing diabetes complications.

Utilization of the NOD.B 10.H-2b should allow for longer observation of the treated

scid mice. In addition to the analysis performed in this study, detection of autoantibodies

in the adoptively transferred mouse sera would be beneficial. Completion of future

experiments may lead to a clarification of the etiopathology of Sj gren's Syndrome,

which may result in better treatment for human patients.















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BIOGRAPHICAL SKETCH

Vinette B. Brown was born in Brooklyn, New York, in 1979. She comes from a

very large extended Jamaican family. When she was 10 years old, she and her parents

moved to Hollywood, Florida, where she graduated from Hollywood Hills High School

in 1997. Afterwards, she received a Bachelor of Science in zoology, with a minor in

business from the University of Florida in 2001. She went on to receive a Master of

Science in molecular genetics and microbiology in August 2004 from the University of

Florida. Vinette will work towards earning a Doctor of Dental Science degree at the

School of Dental and Oral Surgery at Columbia University.