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Characterization of the B Cells in Lupus-Prone and Resistant Mouse Models


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CHARACTERIZATION OF THE B CELLS IN LUPUS-PRONE AND RESISTANT MOUSE MODELS By BIYAN DUAN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2006

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Copyright 2006 by Biyan Duan

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This document is dedicated to my parents and my sister.

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ACKNOWLEDGMENTS I would like thank my dissertation committee members, Dr. Michael J. Clare-Salzler, Dr. William L. Castleman, Byron P. Croker, Dr. Eric S. Sobel and my mentor, Dr. Laurence Morel, for their encouragement, support, and excellent suggestions. Their advice was crucial to my progress in this research. iv

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TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................iv TABLE ..............................................................................................................................vii LIST OF FIGURES .........................................................................................................viii ABSTRACT .........................................................................................................................x CHAPTER 1 INTRODUCTION........................................................................................................1 Peripheral B-Cell Populations......................................................................................2 B1 Cells.................................................................................................................2 Marginal Zone and Follicular B (B2) Cells...........................................................6 Peripheral B-Cell Development....................................................................................9 BAFF and Receptors for BAFF..................................................................................10 The NZB/W Derived Mouse Models for Lupus Prone and Resistance......................11 2 MATERIALS AND METHODS...............................................................................18 Experimental Animals................................................................................................18 Immunofluorescence Staining....................................................................................18 B-Cell Isolation and Cell Culture...............................................................................19 Flow Cytometry..........................................................................................................19 Bone Marrow Transfers..............................................................................................20 Treatment of Mice with TI Antigens..........................................................................21 Western Blotting Assay..............................................................................................21 Statistics......................................................................................................................21 3 RESULTS AND DISCUSSION: ABNORMAL MARGINAL ZONE AND MARGINAL ZONE B CELLS are found in both lupus-prone and lupus resistant mice.............................................................................................................................22 Different Marginal Zone Phenotypes Between Lupus-Prone and Resistant-Mice.....22 Reciprocal Bone Marrow Transfers Indicate MZB Translocation is Determined by Multiple Factors......................................................................................................24 Functional Studies of MZB Cells...............................................................................25 MZB Respond to TI-2 Antigen Ficoll.................................................................25 MZB Migration in Response to TI-1 Antigen LPS.............................................26 v

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Discussion...................................................................................................................27 4 RESULTS AND DISCUSSION: ROLE OF PERITONEAL CAVITY B CELLS IN LUPUS SUSCEPTIBILITY..................................................................................45 Activation, Proliferation and Apoptosis.....................................................................45 In Vivo Spontaneous Proliferation:.....................................................................45 In Vitro B-cell Apoptosis....................................................................................46 Peritoneal Cavity B-Cell Activation....................................................................46 Cytokine Production...................................................................................................46 Discussion...................................................................................................................47 5 RESULTS AND DISCUSSION: SPLEEN B CELLS IN THE LUPUS PRONE AND RESISTANT MICE..........................................................................................56 Splenic B1 and B2 Cells.............................................................................................56 Splenic Naive B-Cell Functional Properties...............................................................57 B-Cell Proliferation after Stimulation.................................................................57 Activation-Induced Cell Death............................................................................57 B-Cell Activation and Differentiation After In Vitro Stimulation......................58 Discussion...................................................................................................................59 Peripheral B-Cell Development..................................................................................65 6 SUMMARY AND CONCLUSIONS.........................................................................74 LIST OF REFERENCES...................................................................................................76 BIOGRAPHICAL SKETCH.............................................................................................93 vi

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TABLE Table page 1-1. Pathological phenotypes of mouse models.................................................................13 vii

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LIST OF FIGURES Figure page 1-1 Diagram of white pulp and red pulp............................................................................13 1-2 Peripheral B-cell development stages..........................................................................14 1-3. BAFF/APRIL and their receptors...............................................................................15 1-4. NZM models of lupus prone and resistance mice......................................................16 3-1. Reduced MZ in TC and NZM versus enlarged MZ in TAN......................................34 3-2 Splenic MZB populations............................................................................................35 3-3. Intrafollicular location of MZB cells in TC and NZM...............................................36 3-4. MZB cell phenotypes..................................................................................................37 3-5. Greatly reduced MZMs in the TC and NZM mice.....................................................38 3-6. Reduced VCAM-1 levels on TC marginal zone.........................................................39 3-7. Marginal zone of TC and B6 bone marrow chimera..................................................40 3-8. Defect of marginal zone macrophages TC.................................................................41 3-9. TNP-Ficoll up-take by splenic MZB cells..................................................................42 3-10 In vitro TNP-Ficoll binding.......................................................................................44 3-11 TAN MZB cells do not migrate after LPS treatment................................................44 4-1 Peritoneal B1 and B2 populations...............................................................................50 4-2 In vivo proliferation of peritoneal cavity B cells.........................................................51 4-3 PerC B1 cells apoptosis...............................................................................................51 4-4 PerC B2 cells apoptosis...............................................................................................52 4-5 PerC B-cell activation..................................................................................................52 viii

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4-6 Peritoneal B-cell activation after 48 hrs of stimulation...............................................53 4-7. Intracellular IL-10 level..............................................................................................54 4-8. Intracellular IL-6 level................................................................................................55 5-1. Splenic B cell populations..........................................................................................63 5-2. Splenic B cell activation.............................................................................................64 5-3. Definition of splenic B-cell developmental subpopulations.......................................65 5-4. Peripheral B-cell development...................................................................................66 5-5 Splenic B cell populations in mice after reciprocal bone marrow transfer.................67 5-6. B-cell proliferation.....................................................................................................68 5-7. B-cell apoptosis rate during of in vitro stimulation....................................................69 5-8 B-cell activation during in vitro culture......................................................................70 5-9. B-cell activation and differentiation with a low dose of anti-IgM stimulation..........71 5-10. B-cell activation under treatment with high dose of anti-IgM stimulation..............72 5-11 ERK phosphorylations...............................................................................................73 ix

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy CHARACTERIZATION OF THE B CELLS IN LUPUS-PRONE AND RESISTANT MOUSE MODELS By Biyan Duan May 2006 Chair: Laurence Morel Major Department: Pathology, Immunology and Laboratory Medicine The general hypothesis is that in the models of lupus-prone and resistant mice, B cells function differently and contribute to the phenotypes and pathogenesis. Thus, the studies of B-lymphocytes in these models may help to understand the role of B-cells and to identify involved genes.Defect splenic marginal zone (MZ) has been found in the lupus-prone NZM2410 and B6.Sle1/2/3 (TC), but not in the lupus resistant NZM.TAN (TAN) mice. I hypothesize that this defect is due to the abnormal functions of marginal zone B cells and contributes to the pathogenesis. Immuno-fluorescence study revealed that lupus MZ B cells were trans-located inside the follicle. MZ macrophages (MZM) were also found defect only in the lupus mice. Reciprocal bone marrow transfer indicated that the lack of MZM was not the reason of MZB translocation, and MZB miss-location was determined by both bone marrow and stroma-derived factors. Functional studies revealed that MZ B cells in TC and TAN mice were abnormal. The second goal of the research was to study the phenotypes and functions of the peritoneal cavity (PerC) B cells in these strains. Both lupus-prone and resistant mouse models have increased PerC B cells, especially B1 cells. Studies showed TAN PerC B1 x

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cells had lower activation and higher resistance to activation induced cell death, while B1 cells from NZM2410 and TC had higher proliferation. Finally TAN B1 cells produce little IL-6 and IL-10, while TC B1 cells make more IL-6 and a normal levels of IL-10, suggesting a regulatory role of TC B1 cells in immune response. The third goal was to study the properties of splenic B cells. Both strains have increased Transitional 1 and Transitional 2 populations and less mature follicular B cells. In vitro stimulations revealed the B cells from lupus mice had a lower activation threshold and higher proliferation/differentiation abilities, while TAN B cells showed the opposite directions. Lupus B cells also had abnormal BAFF-R/TACI expressions, indicating the mechanism behind the phenotypes. Overall these results suggest that B-cell populations in lupus-prone and resistant mice are quite different and may play an important role in the pathogenesis. xi

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CHAPTER 1 INTRODUCTION The general hypothesis of the study is that in the NZM models of lupus-prone and resistant mice, B cells function differently and contribute to the phenotypes and development of the disease. Thus, the studies of B-lymphocytes in these murine models may help to understand the role of B-cell in lupus development and to identify the genes responsible Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease that affects multiple organs and leads to end-organ damage. SLE is characterized by the production of large amounts of antibodies against a wide spectrum of auto-antigens, including ssDNA, dsDNA and RNPs. B cells have long been recognized as effectors to receive specific T-cell help and to secrete these pathogenic auto-antibodies which result in tissue damage. Recent studies suggest that B-cell may play a role other than the autoantibody producer. In the lupus-prone MRL lprmice, modification of B-cell that blocking the antibody secretion does not prevent disease development(1). In the NZB/W derived lupus model NZM2410, genetic dissection shows that the expression of Sle susceptible locus on B cells is essential for the development of autoimmunity(2). T-cell hyper-activation has been found in lupus(3-6), while in MRL lpr, T-cell activation is greatly dependent on B cells (7), suggesting the role of B-cell as (auto)antigen presenting cells. Furthermore, B-cell depleting treatments lead to remission of SLE symptoms in patients(8-11). All of these findings demonstrate the pivotal role played by B cells in pathogenesis. B cells are heterogeneous and include multiple subpopulations with distinct 1

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2 functions and properties. However, the exact role of each subpopulation in the pathogenesis of lupus is still not clear. This study is focused on the characteristics and functions of different B-cell subsets in lupus prone and resistant mouse models. Peripheral B-Cell Populations Based on their location, surface marker expression, and functions, the peripheral mature B cells are now divided into three distinct populations: B1 cells, B2 (follicular B, FoB) cells and marginal zone B (MZB) cells(12). B1 Cells Based on the expression of the pan-T cell surface glycoprotein-CD5, B cells were originally divided into two populations: B1 (CD5+) and B2 (CD5-) cells(13). Further studies defined B-1 cells as a population possessing a distinct pattern of surface markers: B220 lo IgM hi IgD lo CD9 + CD43 + CD23 lo and conventional circulating B-2 cells as B220 hi IgM hi/lo IgD + CD9 CD43 CD23 hi (14;15). B1 B cells are also larger and exhibit more side scatter than do conventional B-2 cells by flow cytometry. Subsequently, B1 cells were divided into two populations: B1a (CD5+) and B1b(CD5-), i.e. they share all other surface markers and properties except CD5. It is still not clear whether these subsets are two distinct cell types or different development stages of one population. B1b cells contribute only to a small proportion of the B1 population. Recent studies show that they have distinct functions from B1a cells. B1b cells are responsible for long-term T-independent immunity specific for B. hersmii (16) and are critical in producing adaptive pneumococcal polysaccharide antibodies (17). For practical reasons, most studies performed on B1 cells are based on results of CD5+ B1a cells. Most B1 cells resides in body cavities such as peritoneal (PerC) and pleural cavities, where they comprise the majority of the local B-cell population. While in the spleen and lymph nodes, B1 cells is

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3 the minority of the local B cells and most of B cells are CD5-. Finally, B1 cells are normally absent from peripheral blood(14). Compared to B2 cells, B1 cells are long-lived, self-renewing and non-circulating B cells with limited B-cell receptor (BCR) diversity and affinity(18;19). Adoptive transfer suggested that B1 cells originated from fetal liver and thus belong to a developmental lineage different from B2 cells (14). When transferred into irradiated mice, fetal liver cells could reconstitute both the B1 and B2 compartments, while adult bone marrow only generated B-2 cells (20;21). B1 cells are refractory to activation through B cell receptor (BCR) ligation, and do not undergo somatic hypermutation after stimulation (22;23). B1 cells mainly participate in T-cell independent responses. They respond to LPS much quicker than B2 cells (24) and uptake TI type II antigen dextran, but not ficoll (25). Recent studies also reveal that B1 cells from the spleen are phenotypically and functionally different than those obtained from peritoneal cavity (24;26): Unlike peritoneal B1 cells, splenic B1 cells do not express the myeloid marker CD11b (Mac 1) and have much lower surface IgM and CD80 levels. In addition, they make little natural IgM. Transcriptional factors Notch1 and Notch2 are expressed at high levels by splenic but not peritoneal B1 and B2 cells. Finally, peritoneal B1 cells respond to phorbol eater in vitro, while B2 and splenic B1 cells need the presence of the calcium ionophore ionomycin to proliferate. Due to the low concentration of splenic B1, most B1-cell studies are focused on peritoneal B1 cells. CD5 has been recognized as a negative regulator of BCR signals in B1 cells (27;28). Its cytoplasmic tail contains a docking site for SH2-phosphatase SHP-1, which is critical for diminishing BCR signaling after antigen ligation(29). SHP-1 deficient

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4 motheaten mice have lymphocyte over-activation and suffer from severe autoimmune symptoms(30;31). Studies on intracellular signaling pathways reveal that B1 cells have constitutive ERK and NF-AT activation, but lack of NFkapaB induction upon BCR cross-linking(32), which is similar to tolerant B cells(33;34). It has been suggested that CD5 expression by B1 cells occurs after auto-antigen exposure to down-regulate their activation status(35). Targeted deletion of CD5 leads to the activation of anergic B cells and results in the loss of tolerance (36;37), indicating the important role of CD5 expression on B1 cells. B1 cells are the major producer of IgM natural antibodies, which are poly-reactive and weakly auto-reactive(38;39). Through specific up-regulation of the transcription factor Blimp-1, which was believed to be plasma cell-specific, B1 cells spontaneously secrete a large amount of natural IgM (40). Natural antibodies recognize a broad range of antigens from many bacterial/viral pathogens prior to the exposure. Thus, they are very important for the early response to bacterial and viral infections(41). Mice lacking natural antibodies suffer from higher susceptibility to influenza infections and have increased mortality(42). On the other hand, natural IgM can also bind to constituents of self, such as phosphorylcholine (PC) (16), phosphatidyl choline (PtC) (13;43) and oxidized low-density lipoprotein (LDL)(44). The production of auto-reactive natural antibodies has implicated B1 cells a potential contributors to the development of autoimmune diseases, such as lupus. In fact, elevated B1 cells have been observed in autoimmune patients with Sjogrens syndrome, SLE and rheumatoid arthritis (RA) as well as in mouse models of lupus(14;45-47). The (NZB x NZW) F1 lupus mouse and its derivative NZM2410 have large numbers of B-1 cells which have accumulated in the peritoneal cavity and, to a

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5 lesser extent, in the spleen(48). Elimination of B1 cells by hypotonic shock with repeated water injections on NZB and (NZB x NZW) F1 mice decreased autoantibody production and reduced kidney pathology(49), suggesting that B1 cells may play a role by producing pathological autoantibodies. While some other studies do not support this notion. For example, in the well-characterized FAS-deficient MRL lpr mouse model, B2, but not B-1a cells are responsible for the autoantibody production (50). Furthermore, over-expression of IL-5 in the (NZB x NZW)F1 model greatly increased the number of B-1a cells, but significantly reduced anti-dsDNA antibody production and the incidence of nephritis (51). These results suggest that the role of B1 cells as autoantibody producer is context-dependent. In addition to antibody production, B-1a cells express high levels of co-stimulatory molecules B7-1, B7-2 and display enhanced antigen presentation capabilities (48). In aged (NZB x NZW)F1 mice, target organs such as kidney and thymus are found to contain infiltrated myeloid cells with over-expressed CXCL13 (BLC) (52;53). B1a cells are attracted to the target organs by the high local BLC levels and defective in homing to peritoneal cavity. The accumulated B1a cells thus can activate autoreactive T cells through their potent antigen presentation capability and contribute to the damage of target organs (53;54). B-1a cells are the main source of B cell-derived IL-6 and IL-10 (55), indicating their regulatory role in immune response. IL-6 promotes B-cell survival and strongly induces differentiation of B-cell to plasma cell (56). It also promotes T-cell growth through augmentation of IL-2 production and IL-2 receptor expression (57;58), and rescues T-cell from apoptosis (59;60). IL-6 has been linked to the development of

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6 autoimmune diseases, including lupus and RA (61-64). In the lupus mouse models MRL lpr and (NZB x NZW)F1, increased IL-6 levels has been found (62;65). Furthermore, administration of exogenous IL-6 to NZB/W mice increased anti-DNA autoantibody production and accelerated glomerulonephritis progression (66-68). Studies on human SLE patients have also found elevated IL-6 levels that correlate with disease activity (69;70). In the kidney, increased IL-6 can induces the proliferation of mesangial cells and is involved in the development of glomerulonephritis (71;72). IL-6 blockade treatment dampened the progression of SLE and decreased the severity (58;73). These data show a pivotal role for IL-6 in lupus pathogenesis and also suggest the role of IL-6 producing B1a cells in lupus. IL-10 is an anti-immflamatory cytokine that strongly inhibits the activation of myeloid cells including monocytes, dendritic cells and macrophages(74-76). IL-10 can promote B cell differentiation, proliferation, and antibody production (77). IL-10 is also involved in regulatory T-cell differentiation and functions(78;79). The role of IL-10 in SLE is still controversial, as it has been shown to both inhibit (80) and exacerbate (81) disease in animal models as well as in patients (82;83). The effect of IL-10 maybe follow a time-dependent fashion, since early increased levels of IL-10 leads to decreased disease severity (Morel Lab, unpublished data). Marginal Zone and Follicular B (B2) Cells Conventional B2 cells are the major B-cell participants T-dependent immune responses. It has been shown that in the SLE, B2 cells are responsible for the bulk of high affinity autoantibodies(50;84). Apart from the production of autoantibodies, the exact role of B2 cells in lupus is still not clear. The splenic CD5B cells are composed of two populations, follicular B (FoB) and

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7 marginal zone B (MZB) cells, based on their phenotypes, functions and anatomical locations. The FoB cells are IgM lo IgD hi CD1d CD21 lo CD23 hi while MZB cells are IgM hi IgD lo CD1d hi CD21 hi CD23 lo and are CD9+ (as are B1 cells) (85;86). FoB cells make up the majority of the splenic B-cell compartment, and are short lived circulating cells with a highly diverse BCR repertoire. MZB cells are the major cell population in the marginal zone (MZ), an anatomically distinctive region surrounding the follicles in the spleen. In rodents, the marginal venous sinus additionally lies between the MZ and the follicle (Figure 1-1). Most of the spleen blood flow exits the circulation through the marginal sinus, and then the marginal zone. In addition to B cells, the MZ also contains specialized macrophages that express the scavenger receptor SIGN-R1, called marginal zone macrophages (MZM), stromal cells called reticular cells, and very few T cells(87-89). The location of MZB cells makes them the first-line to encounter blood-borne pathogens, and they have evolved properties and functions to fit this position(90;91). MZ B cells are long-lived and non-circulating cells, and respond to a wide spectrum of T-dependent and T-independent antigens (88;92). After encountering with the cognate TD antigens, MZ B cells migrate into the follicle toward the T-cell area, where they can activate naive T cells more efficiently than FoB cells and quickly differentiate to plasma cells(93;94). Furthermore, the MZB population has a biased BCR repertoire which contains a large amount of auto-reactive clones(95-97). These properties also suggest that they maybe contributors to autoimmunity. Expansion of the MZB population has been found in NZB/W and estrogen-induced lupus mice (98-100). On the other hand, sequestration of autoreactive B cells into the MZ area has also been proposed to be a mechanism to prevent autoimmunity (101;102), and this process is inefficient in

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8 autoimmune MRL lpr mice (103). The location and migration of B cells are controlled by many factors including chemokines and integrins. As indicated by figure 1-1, re-circulating B cells express high levels CXCR5 and are directed to the follicle B-cell area, where CXCL13, the CXCR5 ligand, is highly expressed(104;105). T cells expressing CCR7 are attracted to the T-cell zone by CCL19/21(104). It has been shown that upon the stimulation of antigens, FoB cells up-regulate CCR7 and migrate to the T-B border of the follicle(106). The situation for MZ B cells is more complicated. MZB cells also express the B-cell zone chemokine receptor CXCR5, which by itself can direct MZB to enter into follicles. However, MZB cells are retained in the MZ by the integrin ligands ICAM-1 and VCAM-1 through their surface expression of LFA-1 and alpha-4/beta-1(107;108). The sphingosine 1-phosphate (S1P) receptors S1P1 and possible S1P3 also participate in the retention of MZB cells (109). S1P is produced by sphingosine kinase-mediated phosphorylation of sphingosine, and present abundantly in peripheral blood (110). MZB cells express much higher levels of S1P1 and S1P3 than FoB cells. Upon antigen encounter, MZ B cells quickly down-regulate the levels of S1P1 and S1P3, and tip the balance toward follicular migration(109). Marginal zone macrophages are also found to be involved in MZB cells retention through the contact of its scavenger receptor MARCO to MZB cells (111). Loss of MZM or blocking MARCO binding by antibodies allows the MZB cells to migrate to the follicle (111). There is also evidence to show that the normal development and maintenance of the marginal zone is dependent upon B-cell, especially MZB cells. Following total B cells depletion by Ig-alpha deletion or CD70 over-expression, both marginal zone macrophages and marginal metallophilic macrophages are lost and the MZ

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9 area is missing (112). Peripheral B-Cell Development B cells that are newly emerged from bone marrow contain many self-reactive clones and must undergo peripheral negative selections before the maturation(113). Based on previous studies, B-cell peripheral maturation was divided into 2 or 3 transitional (T) stages according to classification by different surface markers(114;115). In the 2-stage transitional system, the T1 stage is IgM hi IgD-, CD21 lo CD23CD24+, and the T2 stage is IgM hi IgD+, CD21 hi CD23 hi CD24+. In contrast, the MZB cells are IgM hi IgD-, CD21 hi CD23-, CD24-, and the FoB cells are IgM lo IgD+, CD21 med CD23 med CD24(116). Furthermore, T2 cells are circulating and contain the precursors of both MZB and FoB cells(116) (Figure 1-2 A). In contrast, the 3-transitional stage scheme proposed by others was based on the immature B-cell marker AA4.1(117). The T1 cells are IgM hi CD21 -/lo CD23-, AA4.1+. T2 cells are -IgM hi CD21 med CD23+, AA4.1+, and T3 cells are IgM hi CD21 hi CD23+, AA4.1+(117). In a subsequent refinement, the population which is IgM hi CD21 hi CD23+, AA4.1+ was postulated to be the precursors of MZB cells (118) (Figure 1-2 B). Regardless of the classification scheme utilized, it has been shown that the T1 stage contains large amounts of auto-reactive clones, most of which are removed during the transition from T1 to T2 by negative selection and receptor editing (119). Thus the accumulation of T1 cells, which could increase the workload of this check-point or a defect of this check-point per se could allow the maturation of auto-reactive clones. In addition to negative selection, other studies have indicated that specificity-based positive selection also occurs (120-123). All of these suggest that check points during the B-cell

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10 peripheral development are important for the formation of normal mature B-cell repertoire. BAFF and Receptors for BAFF B-lymphocyte stimulator(BLys/BAFF/THANK/TNFSF13B) is a TNF superfamily member. TNF family members are type II transmembrane proteins that form homotrimers as membrane-bond or soluble ligands to their cognate receptors(124). BAFF has been identified as a trophic factor critical for B-cell development, growth and survival(125). The major producers of BAFF are the peripheral blood mononuclear cells (126;127). Macrophages, monocytes, dendritic cells(DC), follicular dendritic cells (FDC) and even B cells can also make BAFF in the context of antigen encounter and activation(127-129). In addition to promoting the peripheral B-cell maturation from the T1 to the T2 stage, BAFF also enhances the survival of immature T1, T2 cells as well as mature B cells(130-132). BAFF and its receptors have been implicated in autoimmunity. High levels of BAFF have been observed in patients with autoimmune diseases such as systemic lupus erythematosis (SLE) and rheumatoid arthritis (RA)(133;134). In animal models, BAFF deficient mice lack the mature B cells, while BAFF transgenic mice show elevated B-cell numbers, especially T2 and MZB cells, and develop autoimmune disorders (135). Furthermore, the lupus-prone models NZB/W F1 and MRL lpr mice have high levels of BAFF in the periphery(136). Experiments on B-cell receptor transgenic mice showed that B cells compete with each other for the binding to BAFF, and that the self-reactive clones have reduced competitiveness, which results in increased susceptibility to deletion(137). In contrast, excess amounts of BAFF can rescue auto-reactive clones from this process and allows them to enter the follicle and marginal zone (138). Three receptors have been identified for BAFF and its homologue APRIL(a

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11 proliferation-inducing ligand): BAFF receptor (BAFF-R, BR3), B-cell maturation antigen (BCMA) and transmembrane activator and CAML interactor (TACI). The BAFF-R is the only receptor exclusively for BAFF(132;139), while both BAFF and APRIL can bind BCMA and TACI(136;140) (Figure 1-3). BAFF-R is predominantly expressed by all peripheral B cells, but is down-regulated when the cells are activated and become germinal center (GC) B cells(141;142). BCMA is expressed at high levels by plasma cells, plasmablasts and germinal center B cells(141;143). Research has shown that BCMA plays an important role in plasma cell survival rather than B-cell maturation(143). TACI is expressed predominately by marginal zone and activated B cells, but not GC B cells. Signals through TACI is essential for T-independent type II responses (144). However, lack of TACI also resulted into increased B-cell survival, activation, and the development of autoimmune disorders, suggesting that TACI has a role as a negative regulator of T-dependent responses (144-146). The BAFF/APRIL ligands and their receptors compose a delicate system that regulates B-cell development, activation and homeostasis. Increased BAFF has been implicated in autoimmunity, while abnormal expressions of the receptors could also tip the balance of positive and negative signals and lead to the same outcome. The NZB/W Derived Mouse Models for Lupus Prone and Resistance The NZM2410 (NZM) is one of 27 inbred strains derived from an intercross between the NZW and NZB strains(147). NZM2410 mice develop lupus nephritis spontaneously, with 80% of the animals from both sexes affected by 6 months of age and have the histological features that closely resemble those of human patients (148). Analysis of backcrosses between NZM2410 and C57/BL6 (B6) identified 4 genomic intervals containing lupus susceptible loci Sle1~4 on NZM2410 chromosomes 1, 4, 7 and

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12 17(149). Subsequently four congenic strains, B6.NZMSle1, -Sle2, -Sle3, and -Sle4 were produced by the backcross, each carrying one of the corresponding NZM2410-derived genomic intervals on the B6 genome (Figure 1-4) (149). Phenotypic analysis of these congenic strains revealed each of the loci contribute to the lupus phenotypes. Sle1 leads to the break of tolerance to nuclear antigens, and results in abnormal phenotypes in both B and T cells(150-153). Sle2 leads to B cell hyperactivity and increased B1 cells(154;155). Sle3 leads to decreased activated induced cell death in CD4+ T cells and affects immunoglobulin heavy chain diversity (156-158). SLE pathogenesis can be fully reconstituted by recombining the Sle1-3 loci on the B6 genomic background (i.e. B6.Sle1/2/3) (159). The B6.Sle1/2/3 (TC) mice display severe splenomegaly, full penetrance of SLE and lupus nephritis at an early age (159). Another strain derived from NZW and NZB, TAN, share the lupus susceptible loci Sle1, Sle2, and Sle3 common to both the NZM2410 and TC strains. However, TAN mice display a dominant resistance to SLE (table 1-1). Although they develop the comparable levels of splenomegaly, TAN mice produce less anti-nuclear antibodies, and do not develop lupus nephritis when followed to 12 month of age. In contrast, aged TAN mice develop a high incidence of marginal zone lymphoma at old age (Morel et al. unpublished data). In this study, TAN mice were used as a lupus-resistant model as compared to NZM and TC mice.

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13 MZ sinus MZ B-cell MZ macrophage Marginal zone (ICAM1+, VCAM1+ FoB-cell zone CXCL13+ T-cell zone CCL19/CCL21+ Red pulp CXCL12+ Figure 1-1 Diagram of white pulp and red pulp.

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14 NF T1 T2 FoB MZB B A T1 T2/ T3 Pre-MZB MZB FoB NF Figure 1-2 Peripheral B-cell development stages. (A) Two-transitional stage scheme. (B) Three-transitional stage scheme. NF, newly formed immature B-cell from bone marrow. T1/T2/T3, transitional 1/2/3 B cells. Pre-MZB, MZB precursors.

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15 Figure 1-3. BAFF/APRIL and their receptors. Soluble or membrane bonded BAFF and APRIL are trimers. BAFF can bind all three receptors, BAFF-R, TACI and BCMA, while APRIL does not bind to BAFF-R. After ligation, the receptor trimerizes and transduces signals into the cell.

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16 B6.Sle1 B6.Sle2 NZM2410 lupus +++ NZM TAN lupus NZB NZW NZM/Aeg strains derivation C57/BL6 (B6) Normal lupus B6.Sle3 B6.Sle1/2/3 (TC) lupus +++ Figure 1-4. NZM models of lupus prone and resistance mice

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17 Table 1-1. Pathological phenotypes of mouse models TAN NZM TC Mortality 12mo 10% 85% 100% Proliferative GN 0% 55% 72% Anti-chromatin 39% 86% 88% Anti-dsDNA 35% 85% 78%

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CHAPTER 2 MATERIALS AND METHODS Experimental Animals TAN mice were maintained in a conventional colony (after several failed attempts to re-derive this strain as specific pathogen free (SPF)). All other strains, NZM2410, TC, B6 and B6.Ly5a (purchased from Jackson Lab, Bar Harbor, ME) were kept in SPF housing as specified by the University of Florida Animal Care Services. Our studies have shown that the housing conditions (conventional versus SPF) did not switch the lupus susceptibility (data not shown). B6.Ly5a mice express a different allotype of CD45 on their leukocytes than that of B6 mice (CD45.1 on B6.Ly5a versus CD45.2 on B6), which can be distinguished by monoclonal antibodies. All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Florida. Immunofluorescence Staining Fresh tissues were embedded in OCT, then snap-frozen and stored at o C. All samples were cut at o C with a cryostat into sections of 6-7um in thickness. Sections were fixed in cold acetone for 10 minutes, briefly air-dried and kept in o C until staining. For staining, sections were first blocked with blocking buffer containing 10% rat serum in PBS for 20 min, then stained with fluorochrome-conjugated monoclonal antibodies for 30 min. Anti-mouse MOMA1-FITC was purchased from Serotec (UK), SIGN-R1 (ER-TR9)-biotin was from BMA (Switzerland), while B220 (RA3-6B2)-APC, IgM (160)-APC, CD1d-biotin, and anti-TNP-Biotin were purchased from BD Pharmingen (San Diego, CA). Biotinylated antibodies were further detected with 18

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19 streptavidin-Alexia 568 from Molecular Probes (Carlsbad, CA). Sections were finally washed, mounted with Prolong Gold media from Molecular Probes and analyzed with a Zeiss Axiom fluorescent microscope. B-Cell Isolation and Cell Culture Splenic CD43naive B cells were purified with the B-cell isolation kit (Miltenyi Biotech, Auburn, CA) according to the manufactures instructions. Purified B cells were counted with cell counter and adjusted to a concentration of 10 6 /ml in complete RPMI1640 containing 10% FCS, then cultured at 37 o C, 5% CO 2 with different stimulation conditions. Peritoneal cells were pre-incubated for 3hr in the same conditions to remove adherent macrophages. All cell culture were conducted using 6-, 12or 24-well tissue culture plate (Corning Life Sciences, Acton, MA). For B-cell stimulations, cells were treated with goat anti mouse IgM F(ab) 2 (Jackson Lab, Bar Harbor, ME) at 0, 1 and 10g/ml or LPS (Sigma, St. Louis, MO) at 1g/ml. Flow Cytometry Single cell suspensions from spleen or peritoneal lavage were treated with FcRgamma Blocker (anti-CD16/32, clone 2.4G2) in flow cytometry buffer (5% FCS in PBS) for 20 min on ice. For the in vitro TNP-Ficoll binding assay, splenocytes suspensions were incubate with TNP-ficoll (Biosearch, Novato CA ) at different concentrations for 30 minutes at 37 o C. Samples were then stained for 20 min with fluorochromeor biotinconjugated monoclonal antibodies against mouse CD1d, CD5 (53-7.3), CD11a (M17/4), CD19 (1D3), CD23 (B3B4), CD45.1 (A20), CD45.2 (104), CD80 (16-10A), CD86 (GL1), IgM (II/41) anti-TNP (G235-2356) (all from BD Pharmingen, San Diego, CA), and CD21 (7E9) (Purified from a hybridoma clone provided by Dr. Boackle, University of Colorado, Denver, CO.). Since all of the strains

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20 that carry Sle1 allele has mutation on extracellular domain of CD21, the commercially available anti-CD21 clone 7G6 binds poorly to the mutated CD21, while clone 7E9 binds well. Biotinylated antibodies were further detected with Strepavidin PerCP-Cy5.5. For intracellular cytokine stains, cells were further fixed and permeabilized with Cytofix/Cytoperm solution for 30 min on ice,. then stained with anti-IL-6, or -IL-10 antibodies according to manufacturers instructions. Samples were finally analyzed with BD FACS Calibur machine, and at least 60,000 cells were counted. Bone Marrow Transfers Three days prior to transfer, recipient mice were started on drinking water containing 40mg/L of the antibiotic Septra (Sulfamethoxazole and Trimethoprim, Hi-Tech Pharmacal CO, Amityville, NY). One day before the experiment, recipient mice were irradiated twice at 525 Rads, each dose separated by 4 hours. On the day of transfer, donor mice were euthanized and bone marrow was flushed with Hanks Solution. Single cell suspension was prepared and washed with cytotoxicity media (0.3% BSA, 0.025M HEPES, 1x Pen/Strep in RPMI1640), and adjust to a concentration of 5x10 7 cells/ml. To remove T cells, the bone marrow cell suspension was incubated with 1:100 anti-Thy1, anti-CD4 10g/ml, anti-CD8 10g/ml (Accurate Chemical, Westbury, NY) for 1 hour at 4 o C. Cells were washed and incubated twice with 1:10 Guinea Pig complement (Accurate Chemical, Westbury, NY) twice for 60 and 30 minutes respectively. Finally, the cells were washed and adjusted to a concentration of 2x10 7 cells/ml, and a volume of 0.5ml was injected into a tail vein intravenously. After bone marrow transfer, recipient mice were continued on antibiotic drinking water for three days, and maintained under SPF conditions.

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21 Treatment of Mice with TI Antigens Mice of 7~9 month old from different strains were injected i.p. with T-independent antigen LPS or Ficoll. For LPS treatment, mice were injected intra-peritoneal with 100ug LPS (Sigma, St. Louis, MO) for 3 hours. For TI-2 antigen ficoll treatment, mice were injected intra-peritoneal with 30ug TNP-Ficoll (Biosearch, Novato CA ) for 30 minutes. Mice were sacrificed after the treatment and spleen samples were snap-frozen and stored at -80 o C for future Immunofluorescent assay. Western Blotting Assay Cultured cells were collected and lysed in RIPA buffer containing protease and phosphatase inhibitor (Santa Cruz Biotechnology, Santa Cruz, CA) for about 15 minutes on ice. Protein concentration was determined by Bio-Rad Protein Assay (Bio-Rad, Hercules, CA). Equal amount of protein (10ug) was resolved on Bio-Rad 12.5% SDS-PAGE gels by electrophoresis, and then transferred to PVDF membrane (Amersham, Piscataway, NJ) by electro-blotting. Membranes were blocked overnight at 4 o C with blocking buffer (0.2% Tween 20, 5% non-fat dry milk in PBS) and then probed with primary antibody for 1 hour at room temperature. Rabbit anti-mouse ERK1/2, phosphorylated ERK1/2 (Cell Signaling, Danvers, MA) was used as primary antibody at 1:1000 dilution in blocking buffer. The goat anti-rabbit IgG HRP (Cell Signaling, Danvers, MA) was used as second antibody at 1:2000. Finally, detection was performed with the ECL plus system (Amersham, Piscataway, NJ) and exposed to X-ray film. Statistics All data were analyzed by ANOVA. Statistical significance was obtained when P < 0.05.

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CHAPTER 3 RESULTS AND DISCUSSION: ABNORMAL MARGINAL ZONE AND MARGINAL ZONE B CELLS ARE FOUND IN BOTH LUPUS-PRONE AND LUPUS RESISTANT MICE Diminished splenic marginal zones (MZ) have been found in the lupus-prone NZM2410 and B6.Sle1/2/3 (TC), while lupus resistant NZM.TAN (TAN) mice have enlarged MZ (B.P. Croker, unpublished observations). I hypothesized that these phenotypes are due to the abnormal functions of marginal zone B cells in lupus-prone and lupus-resistant mice. I also hypothesize that the different MZ phenotypes of these strains contributes to their lupus susceptibilities. Different Marginal Zone Phenotypes Between Lupus Lupus-Prone and Resistant Resistant-Mice Histological examination of the spleen revealed the agedependent changes of the marginal zone among the NZM, TC and TAN mice. From 5 or 6 months of age, significantly reduced or absent splenic marginal zone areas and IgM hi MZB cells were observed in both NZM and TC mice. In contrast, the lupusresistant strain TAN showed a markedly enlarged marginal zones with accumulation of IgM hi CD5+ cells (Figure 3-1). Flow cytometry analysis did not show a corresponding reduction or enlargement of MZB population in the spleen of lupus-prone or resistant mice (Figure 3-2). This inconsistence indicated the possibility of an altered location instead of the loss of MZB cells in the lupus-prone mice. It also suggested that the B cells present in the MZ of TAN mice could have different characteristics from normal MZB cells. Further immunofluorescent studies using another MZB cell marker, CD1d, 22

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23 revealed that the MZB cells in the lupus TC and NZM mice were in fact translocated inside the follicles (Figure 3-3). Chemokine receptor CXCR5 is expressed on mature B cells and is essential for directing B cells into the follicular B-cell area (52;161). Flow cytometry studies revealed that lupus mice MZB cells expressed a higher CXCR5 levels than that of B6, indicating thesuggesting a reason forof their translocation. The TAN MZB cells, on the other hand, did not showed increased CXCR5 levels, but had a significantly high percentage of CD5 expression (Figure 3-4). This is consistent with previous observation by histology showing that TAN MZ area was occupied by CD5+ cells. Thus the B1type MZB cells were unique for TAN mice. Since CD5 is a negative regulator of B-cell receptor signaling, the CD5+ MZB cells in TAN mice may have different phenotypes from that of other strains. Subsequently, the number of ER-TR9+ MZ macrophages (MZM) were was also found to be reduced in the lupus mice but was normal in the TAN mice, while the Moma-1 expressing metalophillic macrophages were intact in all strains (Figure3-5). MZMs has have been suggested to interact with MZB cells directly and is are important for the retention of MZB cells (111). These results indicate suggested that the loss of MZM may at least partially responsible for the diminished marginal zone in lupus mice. Interactions between integrin ligands VCAM-1, ICAM-1 and receptors LFA-1, alpha-4/beta-1 integrin on the cell surface are also critical for the retention of MZB cells (107;108). So we studied the expression levelss of these molecules on the MZB cells and on the stroma cells in of the follicles. Flow cytometry analysis for the levels of LFA-1 on TC MZB cells did not find significant changes (data not shown). Since it is hard to isolate stromal cells, we used an iImmunofluorescencet approach. Studies on the

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24 frozen sections revealed a slightly lower VCAM-1 levels on the MZ area of TC spleen, but not on that of TAN and B6 spleen (figure Figure 3-6). Since lowerLower VCAM-1 levels indicates a lowerless binding for the integrin and a weaker retention factor, which may tip the balance toward follicular migration. This suggests that the stroma cells in TC MZ area may also contribute to the phenotype of MZB cell trans-location. Reciprocal Bone Marrow Transfers Indicates MZB Translocation is Determined by Multiple Factors The defective MZ phenotype of lupus mice can be determinominated by either stroma-derived factors, i.e. stromal cell produced cytokines and, integrin ligands, or by bone marrow derived factors, i.e. MZB cell itself, or myeloid cells like MZMs. Since stromal cells are radiation-resistant, and all bone marrowderived cells are sensitive to irradiation, we conduct reciprocal bone marrow transfers on irradiated B6 and TC mice to study evaluate these possibilities. The B6 and TC recipient mice were lethally irradiated and transferred with bone marrow from untreated TC or B6 mice. The rationale is if the TC MZB cell phenotype was dominant by bone-marrow derived factors, the expected results would be a normal MZ in TC mice that received B6 bone marrow (B6 TC), and an intra-follicular MZ in B6 mice transferred with TC bone marrow (TC B6). The reverse results would be expected if stromal cells are solely responsible for the phenotype. Three months after transfer, mice were sacrificed and spleen sections were examined as mentioned before. Unexpectedly, both B6 recipients that received TC bone marrow and TC recipients transferred with B6 bone marrow showed normal MZ (Figure 3-7). We also look at the presence of ER-TR9+ marginal zone macrophages on bone marrow transferred mice. None-the-less, the TCderived bone marrow could not give rise

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25 to a normal amount of marginal zone macrophages, while in the TC recipients transferred with B6 bone marrow, the MZMs rim was present normally (Figure3-8). To verify the origin of the MZB and MZM and rule out the possibility of recipient bone marrow cell contamination, We we also did performed reciprocal bone marrow transfers between B6.Ly5a and TC (allotype Ly5b) mice and determine the cell origin by antibodies that discriminate the allotypic marker Ly5a and Ly5b. Results showed that the MZMs and MZB cells observed are both donor bone marrow derived. And recipient mice have the same manifestation on MZB cells and MZMs (data not shown). Furthermore, flow cytometry analysis on 3 months after reciprocal bone marrow transfer revealed the reconstitution of B-cell phenotypes and functions according to the derived by donor bone marrow origin, i.e. the TC B6 mice have abnormal B-cell subsets and B6 TC mice showed similar phenotype as unmanipulated B6 (data showed and discussed ion Chapter 5). This suggested first firsthat the deficiency fect of in MZM number was intrinsic tofor the TC bone marrow, although its pathological significance is still not clear. Second, these results indicate that the lack of MZMs was not the determining factor of MZB cells translocation into the follicles in TC mice, and vice versa, thus both the MZB -cells per se and the stromal cells in the marginal zone are most likely to contributed to this phenotype. Functional Studies of MZB Cells MZ B cells are the major responders to T-independent antigens (12). To study their functions in the lupus-prone and resistant strains, mice were challenged with the TI antigens TNP-ficoll and LPS. The responses of MZ B cells were assayed by immunofluorescence and flow cytometry. MZB Respond to TI-2 Antigen Ficoll. Mice were injected i.p. with 30 g TNP-Ficoll each, then sacrificed after 30

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26 minutes. Frozen spleen sections were produced, and the uptake of ficoll by MZB cells was revealed by using anti-TNP and anti-IgM antibodies. MZB cells that bind TNP-Ficoll are double positive. Results showed less ficoll binding by TC MZB cells compared to B6 and TAN MZB cells. And In addition, a small amount of ficoll-binding cells present in the follicle of TC mice (Figure 3-9). To rule out the possibility that the low binding of TC MZB cells to ficoll was due to the limited access resulting from their translocation into follicles, in vitro studies were conducted. Fresh splenic single cell suspensions were taken from mice without any treatment, then incubated with various amounts of TNP-ficoll for 30 minutes. The binding of ficoll was determined by flow cytometry with anti-TNP antibody and gated on MZB cells. We confirmed that non-MZB splenic cells were negative for TNP-binding (data not shown). The results showed that both TAN and TC mice MZ B cells had different degree of impaired binding capability to the TI-2 antigen-Ficoll. The TAN MZB cells had similar binding ability as B6 at low Ficoll concentration ( < 5 ug/ml). At higher concentrations, they seemed to be saturated and can not bind any more of ficoll as of B6 MZB. In contrast, TC MZB cells have severe defect of ficoll uptake even at lowest concentration tested (Figure 3-10). MZB Migration in Response to TI-1 Antigen LPS. It has been shown that the MZB cells migrate into the follicles when they encounter the TI-1 type antigen LPS (162). To determine MZB cell function in response to TI-1 antigen, mice at 7~9 month old were injected i.p. with 100ug LPS. After 3 hrs, the spleens were sectioned and stained with MZB cell markers to reveal their location. The results show that after 3 hrs of LPS treatment, the TAN MZB cells were still stayed located in the MZ area, while MZB cells in TC and normal B6 mice were localized inside

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27 the follicles (Figure 3-11). This suggests that the response to TI-1 type antigen for TAN MZB cells is impaired in terms of migration in response to LPS. It should be noted that most TC MZB cells were already inside the follicule before LPS exposure, and therefore it cannot be determined whether or not they responded to LPS. Discussion Both the lupus-prone NZM, TC and resistant TAN mice have abnormal lymphoid micro-structures. This is at least partially due to the fact that they all carry the lupus susceptible locus Sle3 (156). We have also shown that lupus-prone mice accumulate plasmablasts and plasma cells in the spleen at the expense of their normal migration to the bone marrow (163). The studies here show that the phenotype of the MZ cells correlate to the lupus susceptibility of the mouse strains. The lupus-prone strains MZN and TC have missingmiss the marginal zone area, with MZB cells trans-localized inside the follicles and lack of marginal zone macrophages. The lupus-resistant TAN mice, on the other hand, have an enlarged marginal zone, with a MZ arrestednon-migrating MZB cells having a large proportion of CD5 expression and normal marginal zone macrophages. The localization and migration of MZB cells is controlled by multiple factors and is a balanced outcome (164). In this study, we conducted reciprocal bone marrow transfers to dissect the reasons for the MZB cell miss-aberrant location in lupus prone mice. The results showing normal of MZ recovery in both directions of the transfers could be explained as the process was not long enough for the manifestation of MZ defect on the experiment animals. Although it can not be totally excluded at this point, This this is, although can not be totally excluded, not likely the case, since flow-cytometric analysis of bone marrow transferred mice have foundshowed the reconstitution of donor

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28 phenotypes, i.e. abnormal B-cell subsets and functions in TC B6 mice, which is were identical toreminiscent to unmanipulated adult TC mice (see Chapter 4), indicating the development of lupus phenotype. Still further Additional transfers with longer procedure time before sacrifice are to be conducted to verify this notion. The alternative explanation is that both stroma cells and lymphocyte/myeloid cells are necessary but not sufficient to cause the TC MZB-cell phenotype. Early studies have shown the interactions between integrin receptors and ligands are important for the organization of follicular structures (164;165). Specifically for the B cells, the integrins LFA-1 (CD11a) and 41alpha-4beta-1 expressed by B cells interact with their respective ligands ICAM-1 and VCAM-1 expressed by the stromal cells to direct B cells entry to the spleen white pulp (165). Marginal zone B cells express higher levelss of LFA-1 and 41alpha-4beta-1 than follicular B cells to facilitate their entering to the MZ, and this interaction is also critical for their retention in this area (107). Ablation of integrin binding by neutralizing antibodies caused the releasing of marginal zoneMZ B cells from marginal zone (107). We speculated the changes of integrin levelss on either MZB or stromal cells may be involved in the MZBcell translocation in lupus mice. Till now noNo difference on LFA-1 levels was found between MZB cells from TC and B6, while immunofluorescent study revealed that in TC mice spleen, the MZ has a slightly may have lowerless VCAM-1 levels than the surrounding area, indicating a reduced VCAM-1 expression by their MZ stromal cells. In contrast, TAN and B6 MZ showed a the similarly higher VCAM-1 levels. In addition, alterations of other integrin molecules e.g. 41 alpha-4beta-1 and ICAM-1 are not excluded and will be assayed in the future. In short, this suggests that the less

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29 diminished interactions between MZB cells and stromal cells may participate in the MZ phenotype in lupus mice. Flow cytometry studies have found increased levels of the chemokine receptor CXCR5 on TC and NZM MZB cells, which may also contribute to this phenotype. CXCR5 is the receptor for chemokine CXCL13 (BLC), and it is well known that FoB cells that express CXCR5 enter follicles following the gradient of CXCL13 established by the follicular stroma cells (166). MZB cells also express CXCR5, but other factors that counter the CXCL13 chemoattractant skew the balance to their retention in the marginal zone. One of these factors is are the integrins and their ligands just mentioned above. The receptors for the lysophospholipid sphingosine-1 phosphate (S1P) are also shown important in this process (109). S1P is found in abundant concentrations in the peripheral blood (167). MZ B cells express higher levelss of S1P1 and S1P3 than the follicular B cells, which prevent them from entering the follicles (109). Antigen exposure results to the down regulation of S1P receptors, and treatment with FTY720, an inhibitor for S1P receptor functions, can both abolish the S1P retention and allow the marginal zone B cells to migrate following the CXCL13 gradient (109;168). In the mice lacking of both CXCL13 and S1P1, the marginal zoneMZ B cells stayed in the marginal zone in spite of antigen stimulation (109), suggesting the importance of balance between S1P and CXCL13 chemoattractant. Thus in the case of MZB cells in lupus prone NZM and TC mice, higher CXCR5 levels is likely to tip the balance toward the follicular migration. However, in vitro chemotaxis assay did not show increased migration in response to CXCL13 by MZB cell from lupus mice (data not shown). It is possible that the slightly increased CXCR5 levels itself is not significant enough to cause the increased migration

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30 of MZB cells, and other factors discussed here may also contribute to the MZ phenotype. This notion is consistent with the reciprocal bone marrow transfer results mentioned above. In particular, the ligand CXCL13 may also play a role in this process, as higher CXCL13 expression has been found in peripheral organs of lupus-prone NZB/W F1 mice, which leads to the accumulation of attracted B1 cells in targeted organs (52;169;170). So in the following follow-up studies, the follicular CXCL13 levels in the lupus prone and resistance mice will be assayed. Furthermore, the involvement of S1P-mediated MZB cell retention can not be excluded. It is not known that if the expression of S1P receptors and/or the down-stream signaling are compromised in the lupus mice MZB cells. Due to the lack of commercially available antibodies for S1P receptors, we still can not assay their expression levelss in vivo. The A chemotaxis assay on S1P will be conducted in future studies. Furthermore, we still cannot exclude the role of activation in the function and location of MZB cells, since TC mice with CD86 knocked out showed less disease with little or no MZB translocations (Morel Lab, unpublished data). MZMs is constitute another major cell population in the marginal zone (111). MZMs express type A scavenger receptor MARCO and C-type lectin SIGN-R1 and are important for capture and clearance of blood-borne pathogens (171;172). Early studies have suggested that MZMs were involved in the retention MZB cells through the contact of the surface receptor MARCO to MZB cells, and MARCOblocking antibody treatment leads to MZB MZB-cell migration (111). Our studies on unmanipulated and reciprocal bone marrow transferred mice suggested a correlation between MZM defect deficiency and lupus susceptibility. However, the bone marrow transfer studies also show ed that the MZM defect is not the cause of MZB cell translocation in lupus mice. It is

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31 possible that unlike the normal MZB cells in B6, lupus mice MZB cells (gradually) lose their dependence on the contact with MARCO for their retention. Consistent with this notion, the MARCO knockout on B6 mice resulted a diminished of MZB cell population and MZ area (173), while in our lupus-prone NZM and TC mice, the MZB population is not significantly changed in spite of lacking MZM (Figure 3-2). Reciprocal MZB cell transfer between TC and B6 mice with MARCO blocking treatment may help to verify this notion. Besides, the significance of MZM defect in lupus pathogenesis is still not clear. Recent studies using conditional knockout models have shown that B cells, especially the MZB cells itself themself may also play a pivotal role in the development and maintenance of normal marginal zone (112). Lymphotoxin expressed by B cells have been found to be necessary for the differentiation and maturation of stromal cells, and further inducing their production of chemokine in the white pulp (174-178). Depletion of B cells results to thein defects of the marginal zone and the loss of marginal zone macrophages (112). Thus in the case of NZM and TC mice, the missing MZ and ER-TR9+ MZMs could be the consequence, rather than the cause of MZB MZB-cell translocation. MZB cells have been implicated in lupus autoimmunity (101;179). Specially, increased number of MZB cells has been found in NZB/W F1 mice (98). Compared to FoB cells, MZB cells respond to TD antigens more rapidly and are more potent T-cell activators (94). Besides, early studies have shown that autoreactive B cells could be positively selected into MZ, which is a postulated mechanism to prevent autoimmunity (100;101;180). In consistent consistence with this notion, the theory of follicular

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32 exclusion also states that auto-reactive B cells are excluded from follicles to prevent their activation, proliferation and differentiation, and finally the excluded B cells would undergo anergy or apoptosis (181-184). In the case of lupus prone NZM and TC mice, although we did not observed higher number of MZB cells and higher MZB MZB-cell activation indicated by CD86 levels (data not shown), their intra-follicular localization may already give them a privilege to participate in follicular responses, which does not occur for MZB cells than the counterpart in TAN and B6 mice, unless they are specifically activated by their antigen or by LPS. In addition, the lupus prone NZM and TC mice have activated auto-reactive T-cell clones and less regulatory T cells ((185) and unpublished data), which may further facilitate the trans-located MZB cells to initiate autoimmune response in the follicles. On the other hand, the CD5+ MZB cells in TAN mice may also contribute to their resistant phenotype. CD5 is a negative regulator for BCR signaling (29;36;186). The expression of CD5 thus can increase the activation threshold of TAN MZB cells, leading to their poor responsiveness to TI antigens and the impaired ability to participate TD responses. The reason for the CD5 expression on TAN MZB cells is still not clear. Besides, only TAN mice develop high instance of marginal zone lymphoma at 12 month old (Morel et al, unpublished), and the tumor cells are all CD5+ indicating the MZB cells in TAN have distinct properties. To directly test the role of MZB cells in the lupus susceptibility of these strains, we planed to conduct reciprocal MZB cell transfer and in vitro characterization on purified MZB cells. However, our attempt to isolate MZB population was unsuccessful due to the lack of high-speed cell sorting facility.

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33 The functions of marginal zone B cells in lupus mice were was also defective in terms of TI-2 antigen up-take. Together with the lack of MZMs, this indicates that the marginal zone of lupus mice is incompetent to react against blood bourn pathogens. It is possible that the ineffective clearance of bloodborne pathogens increase the chance of inflammatory damage of peripheral organs and the release of auto-antigens. The components of pathogen organelles may also activate multiple B-cell clones and elicit persistent adaptive responses, increasing the risk of auto-reactive cross-reaction and bystander activation. Further investigations are needed to verify this hypothesis. In short, this study shows that the marginal zone and MZM phenotypes correlate to the lupus susceptibility but are separate from each other. And the defect on in lupus prone mice is due to the translocation of MZB cells into the follicle, results resulting from defective of itselfMZB cells and the stroma cells.

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34 C D B A Figure 3-1. Reduced MZ in TC and NZM versus enlarged MZ in TAN mice. Representative slides from 3 mice of each strain at 7~9 month old. Spleen sections were stained with FITC conjugated Moma1 (green), CD5 PE (red) and APC-IgM (blue). MOMAMoma-1+ metallophillic macrophages mark the boundary of the follicle (A) B6. (B) TC. (C) TAN. (D) NZM. B6 follicles have a normal marginal zone. MZB cells are IgM high CD5and are located outside the green ring of Moma-1 stain. The CD5 high T-cell zone and IgM+ B-cell zone are well defined by the stain. (B) TC and (D) NZM mice showed reduced to absent marginal zone, and T-cell zone and B-cell zone are mixed together and poorly defined. (C) TAN mice have enlarged MZ area, and the cells in MZ express both IgM and CD5 (purple). Arrows indicate MZ area. (100x)

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35 TAN NZM TC B6 0 5 10 15 20 25 30 35 40 45 50 55strain% of total B cells Figure3-2 Splenic MZB populations determined by flow cytometry. MZB cells were gated as B220+, CD21 hi CD23 lo/. No significant reduction (P>0.05) of MZB was found between, TAN, TC and NZM compared to normal B6 mice.

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36 C D Moma1 CD1d IgM B A Figure 3-3. Intrafollicular location of MZB cells (IgM+, CD1d+) in the TC and NZM. Representative slides from 3 mice of each strain at 7~9 month old. (A) B6 spleen follicles with normal marginal zone. IgM+, CD1D+ MZB cells (purple color) localized outside the Moma-1 (green) positive rim. (C) TAN MZB cells localized on the both sides of Moma1 rim with increased cell layers. (100x)

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37 CD5+MZB TAN NZM TC B6 0 10 20 30 40 50 60 70 80 90***strainCD5+ % MZB CXCR5+ TAN NZM TC B6 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5**strainCXCR5+ %AB Figure 3-4. MZB cell phenotypes. Fresh splenic cells analyzed with by flow cytometryer. MZB cells are defined as IgM+, CD1d high (A) MZB cells from both NZM and TC have express significantly higher levels of chemokine receptor CXCR5. (B) Significantly high proportion of MZB cells in TAN express B1 B-cell marker CD5. P<0.05, ** P<0.01, *** P<0.001.

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38 (100x) Moma1 FITC SIGN-R1 C D B A Figure 3-5. Greatly reduced MZMs in the TC and NZM mice. Representative spleen sections are from 3 mice of each strain. Mice were 7~9 month old. (A) B6 spleen follicles have a continuous layer of MZMs (ER-TR9+, red) distributed outside the Moma-1 rim (green). (B) TC had much less MZMs. (C) TAN follicles with a continuous MZM layer similar to B6. (D) NZM mice like TC, has have a defective MZM layer. MZM, marginal zone macrophage. (100x)

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39 Figure 3-6. Reduced VCAM-1 levels on TC marginal zone. Representative spleen reen) E A D F B C sections are from 3 mice of each strain. Mice were 7~10 month old. Representative spleen sections were stained with anti-mouse Moma-1 (gand VCAM-1 (red). Arrows indicate the marginal zone area. (A) (B), B6. (C) (D), TC. (E)(F) TAN. TC spleen section show reduced VCAM-1 around marginal zone area. (100x)

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40 Figure 3-7. Marginal zone of TC and B6 reciprocal bone marrow transferred mRepresentative spleen sections are from 4 mice of each group. Mice about 3 month s old were lethally irradiated and conducted transferred with bone onths ) Moma1 CD1d IgM A B C D ice. marrow transferfrom the opposite strain. Spleen sections were taken 3 mafter transfer. Both B6 TC and TC B6 bone marrow chimeras show normal marginal zone. (A) (B) TC recipients received B6 bone marrow. (C(D) B6 recipients received TC bone marrow. B6 recipients received TC bone marrow. All representative slides show MZ comparable to that of unmanipulated B6 spleen. (100x)

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41 D C B A (100x) Moma1 FITC SIGN-R1 Figure 3-8. Defect of marginal zone macrophages in recipients receiving TC bone marrow. Representative spleen sections are from 4 mice of each group. (A) (B) TC recipients transferred with B6 bone marrow. The ER-TR9+ MZM layer was normal as in B6 mice. (C) (D) B6 recipients transferred with TC bone marrow. The MZM layer was defective as in unmanipulated TC mice. (100x)

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42 Moma1-FITC anti-TNP Biot IgM-APC C A B Figure 3-9. TNP-Ficoll up-take by splenic marginal zoneMZ B cells. Spleen sections were take produced 30minutes after TNP-Ficoll i.p. injection. TNP-Ficoll was exposed by anti-TNP (red). Representative spleen sections are from 3 mice of each strain. (A) B6 had an intense TNP+, IgM+ (purple) MZB cell layer surrounding Moma1+ circle (B) TC MZ shows much less Ficoll binding MZB cells in MZ. Also a few TNP+, IgM+ cells localized inside the follicles (red arrows). (C) TAN MZ also showed good TNP-ficoll binding in the MZ, but the presence of TNP negative MZB cells indicates less bind than in B6. (100x).

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43 MZB TNP MFI MED TNP-Ficoll 1 TNP-Ficoll 2.5 TNP-Ficoll 5 TNP-Ficoll 10 TNP-Ficoll 20 TNP-Ficoll 40 0 5 10 15 20 25 30 35 40 45TAN TC B6 Incubation dosageTNP+MFI A ********* ********* TAN TC B6 MZB gated TNP-Ficoll binding anti-TNP Biot.SAv.PerCP.Cy5.5010110210310410 Count564228140 MZB gated TNP-Ficoll binding anti-TNP Biot.SAv.PerCP.Cy5.5010110210310410 Count564228140 MZB gated TNP-Ficoll binding anti-TNP Biot.SAv.PerCP.Cy5.5010110210310410 Count564228140 MZB gated TNP-Ficoll binding anti-TNP Biot.SAv.PerCP.Cy5.5010110210310410 Count564228140 B C D E

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44 Figure 3-10 In vitro TNP-Ficoll binding capability of MZ B cells. Fresh isolated splenocytes were incubated with TNP-Ficoll at different concentration for 30 minutes. The binding of TNP-Ficoll was assayed by flow cytometry with anti-TNP monoclonal antibody. Cells were gated on MZB markers (IgM+, CD1d high). (A) Impaired TNP-ficoll up-take by TC and TAN MZB cells as compared to B6. Representative histograms of TNP-ficoll binding by MZB cells from each strain shown on (B) Medium only. (C) TNP-Ficoll 10g/ml. (D) TNP-Ficoll 20g/ml. (E) TNP-Ficoll 40 g/ml. P<0.05, ** P<0.01, *** P<0.001. B C A Moma1-FITC CD1d-Biot IgM-APC Figure 3-11 TAN MZB cells do not migrate after LPS treatment. Representative spleen sections are from 3 mice of each strain. Mice were ~7 month old. Mice were injected with 100 ug LPS i.p. for 3 hours. Spleen sections were taken and location of MZB cells (IgM+, CD1d high purple) were assayed. (A) B6 and (B) TC. MZB cells are trans-located into follicle demarcated by green Moma-1 circle. (C) TAN. MZB cells do not migrate. (100x).

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CHAPTER 4 RESULTS AND DISCUSSION: ROLE OF PERITONEAL CAVITY B CELLS IN LUPUS SUSCEPTIBILITY Both lupus-prone TC, NZM and resistant TAN mice have increased total peritoneal cavity (PerC) cells as well as B1a B-cell population. Decreased numbers of B2 cells are also found in NZM and TAN, but not TC mice (Figure 4-1). This increase in PerC B1a cell corresponds to the expression of Sle2, which is present in TAN, NZM, and TC (155;187). No difference was found in expression levels of activation markers, e.g. B7, CD69, CD40 on PerC B1 and B2 cells between these strains. Activation, Proliferation and Apoptosis Both the lupus-prone and resistant strains have increased B1 cells. I speculate the B1 cells in lupus prone mice are different from their counterpart in lupus resistant mice, and they both contribute to the strain phenotypes. It is not clear if the B1 cells in lupus mice are more prone to activate and undergo differentiation than that of TAN and B6 mice upon stimulation. If this is true, it means the B1 cells may directly participate in the pathogenesis as effectors. Furthermore, the increased cell number in both strains can result from the enhanced proliferation and/or decreased apoptosis. To test these possibilities, experiments to assay the B1 cell activation, proliferation and apoptosis properties were conducted on these strains. In Vivo Spontaneous Proliferation: Adult mice from each strain were injected with 1mg BrdU i.p. After 4 days, the spontaneous proliferation of B cells indicated by BrdU incorporation was assayed by 45

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46 flow cytometry. Results showed B1 cells from lupus-prone NZM and TC mice had more proliferation, while only B2 cells from TC mice had slightly higher proliferation (Figure 4-2). In Vitro B-cell Apoptosis Whole peritoneal cavity lavage cells from TAN, TC and B6 mice were incubated for 2 hrs at 37 0 C to remove macrophages, then cultured with medium only, anti-IgM (10 g/ml) or LPS (1 g/ml) for 16 hrs. Early apoptosis was measured by flow cytometry to detect cleaved caspase-3. Results showed that the TAN mice B1 cells had significantly lower apoptosis rate, while no difference was found on TC B1 cells (Figure 4-3). On the PerC B2 cells, only TAN mice showed lower apoptosis when treated with LPS, and the TC did not show a difference with B6 mice (Figure 4-4) Peritoneal Cavity B-Cell Activation PerC cells were cultured as mentioned above for 48 hrs. B-cell activation was evaluated by the expression of CD80 and CD86 with flow cytometry. Results showed that TAN B1 cells showed significantly less activation capability in all three conditions. The same levels of activation was observed between TC and B6 B1a cells, except with LPS stimulation, which resulted in a significantly lower activation in TC B1a cells (Figures 4-5 and 4-6). Cytokine Production Peritoneal B1 cells are the major source of B-cell derived IL-10 (55), and also produce IL-6 (188). Both cytokines are important in regulating immune responses (189). In this study, we tested the production of IL-10 and IL-6 by PerC B cells in vitro. PerC cell suspensions were cultured for 72 hrs, and the intra-cellular cytokine levels were assayed by flow cytometry. Results showed that TAN B1 cells produce significantly less

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47 IL-10 and IL-6 than either B6 and TC mice, while TC B1 cells make significantly more IL-6 when stimulated with anti-IgM (Figures 4-7, 4-8). Interestingly, B1a cells from the lupus prone TC mice do not produce more IL-10 on a per cell basis, but because TC mice have large amounts of B1 cells accumulated in the peritoneal cavity, the overall effect is an elevated IL-10 levels. This is consistent with the fact that lupus is associated with higher levels of circulating IL-10 (190). Discussion Increased levels of B1 cells has been observed in human autoimmune patient as well as some lupus mouse models(14;45-47). The role of B1 cells in the lupus pathogenesis has not been fully understood. In the NZM derived models, the lupus-prone NZM, TC and resistant TAN mice have increased peritoneal B1 cells. This is at least partially because they all carry the entire lupus susceptible locus Sle2, which leads to an age-dependent enlargement of the B1 compartment (155). Our studies here show that the peritoneal B1 cells from lupus prone and resistant mice have different functions and properties. B1 cells from lupus prone NZM and TC mice show significantly higher rate of spontaneous proliferation than that of TAN and B6 mice, indicating an active cell cycle progression. Normal B1 cells do not actively cycle in vivo (191). The increased B1 cell proliferation in NZM and TC mice reflect the dominant functions of lupus susceptible locus Sle2 carried in these strains, which have been found to cause elevated B1 cell proliferation (155). Interestingly, although TAN mice also carry Sle2, their B1 cells do not show the same high proliferation as that of lupus mice, suggesting other genes in TAN genome may suppress this function of Sle2 locus, and this correlates with the lupus susceptibility. The exact reasons for high B1 cell proliferation caused by Sle2 is still

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48 unknown (155). As B6 mice carrying Sle2 have heightened B cell responsiveness to in vitro stimuli and to in vivo antigenic challenge (154), the B1 cells from NZM and TC mice may have lower threshold in response to antigen exposure in the peritoneum, or on the other hand, they may up-regulate cell-cycle progression genes without stimulation. This hypothesis is to be tested with purified B1 cells in future studies. Both B1 and B2 cells from TAN mice peritoneum showed higher resistance to activation induced cell death when stimulated in vitro. While the lupus-prone TC mice B1 and B2 cells showed similar apoptosis rate as that of normal B6. The Sle2 locus also promote the B1 cells survival when carried by B6 genome (155) This result suggests that on the Peritoneal B1 cells of TC mice, the proliferation-promoting function of Sle2 locus is dominant while its anti-apoptotic effect is not significant. The reason for this inconsistency is not clear, probably due to the interaction with other lupus loci. On the other hand, these data also indicated that the mechanism of B1 cells accumulation in the peritoneal cavity is different among these strains: with less apoptosis more survival in TAN and high proliferation rate in the lupus mice. The responsiveness of TC peritoneal B cells to the stimulations could be the indication of their roles and functions in lupus pathogenesis. If they actively participate in the autoimmunity, the heightened activation response to stimulations is expected, and vice versa. The in vitro stimulation shows compared to that of B6, both B1 and B2 cells in the TC peritoneal cavity do not have significantly different activation in response to BCR cross-linking and to LPS. On the other hand, peritoneal B cells from TAN mice express much lower co-stimulatory molecules regardless of these stimulations. These results indicate peritoneal B1 cells in lupus TC mice may not contribute to the lupus

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49 pathogenesis through more heightened activation to antigen. And the low responsiveness and low co-stimulatory molecule expression by TAN B1 cells may contribute to the lupus resistant phenotypes. The approach to purify B1 cell is under investigation and peritoneal B1 cell transfer will be conducted to verify this notion. Peritoneal B1 cells is the major source of B-cell derived IL-10 (55), and also produce a lot of IL-6 (188), indicating their regulatory role in immune response. In vitro stimulation showed that TAN B1 cells produce much lower amount of both IL-10 and IL-6 regardless of stimulations. TC B1 cells produce similar amount of IL-10 as that of B6 B1 cell at a per cell level, and they produce more IL-6 after BCR cross-linking. Since TC mice have much more B1 cells than B6 mice, the overall effect will be a lot more total IL-10 and IL-6 production by PerC B1 cells. IL-10 is a Th2 cytokine that can promote B cell differentiation, proliferation, and antibody production (77). It has been shown to be involved in lupus pathogenesis (77). Higher levels of circulating IL-10 correlate with lupus development (190). Thus, this result suggests that B1 cells participate in lupus pathogenesis by producing IL-10. Furthermore, IL-6 is also an important cytokine that regulate immune response. IL-6 promotes B-cell survival and strongly induce differentiation of B-cell to plasma cell (56). In human SLE patients, elevated IL-6 levels correlate with lupus activity and kidney pathology (69-72). Besides, IL-6 also blocks the suppression effect of regulatory T cells (192;193), which will exacerbate status of already low Treg in lupus. Interestingly, the BCR crosslinking suggesting that upon antigen encounter, TC self-reactive B1 cells will make much more IL-6. In general, the elevated B1 cells in TC will results in more IL-6 entering the circulation and promote the disease development.

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50 Overall, these studies suggest that the peritoneal B1 cells in lupus prone NZM and TC mice are different from that of lupus resistant TAN and B6 mice. The enhanced proliferation results in the accumulation of B1 cells in lupus mice. And their B1 cells contribute to lupus pathogenesis by the mass production of IL-6 and IL-10. B1 cells in TAN mice are more resistant to apoptosis and respond poorly to stimulations in terms of activation and cytokine production. TAN NZM TC B6 0 25 50 75 100*******strain% of lymphocytes TAN NZM TC B6 0 10 20 30 40 50 60 70 80 90******strain% of lymphocytesAB Figure 4-1 Peritoneal B1 and B2 populations by flow cytometry. Mice are 5~9 month of old. (A) Peritoneal cavity B1 cells. Significantly increased B1 B cells in TC, NZM and TAN mice. (B) Peritoneal B2 cells. Both TAN and NZM mice show lower percentage of B2 cells.

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51 TAN NZM C147 B6 0 5 10 15 20***strainB1a% BrdU+ TAN NZM C147 B6 0 1 2 3 4 5 6 7 8 9 10 11 12*strainB2% BrdU+AB CD19 PE010110210310410 anti-BrdU FITC010110210310410 C Figure 4-2 In vivo proliferation of peritoneal cavity B cells. Mice were injected with 1mg BrdU i.p. and proliferation indicated by BrdU incorporation was assayed 4 days later by flow cytometry with anti-BrdU antibody. (A) B1 cells in the lupus TC and NZM PerC showed higher spontaneous proliferation than TAN and B6 mice. (B) B2 cells in TC PerC showed higher proliferation rate. (C) Representative flow cytometry plot of BrdU incorporation. Bar graph shows mean + SD P<0.05, ** P<0.01. B1 MED Casp3 TAN TC B6 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 strainB1a% Casp3+ B1 IgM Casp3 TAN TC B6 0 5 10 15 strainB1a% Casp3+ B1 LPS Casp3 TAN TC B6 0 5 10 15 20 25 30 35 strainB1a% Casp3+ABC Figure 4-3 PerC B1 cells apoptosis. Cells from peritoneal lavages were collected and cultured with (A) Medium only, (B) 10 g/ml anti-IgM, (C) 1 g/ml LPS. Intracellular activated Caspase-3 was assayed by flow cytometry. TAN B1 cells showed lower apoptosis than TC and B6 in all 3 conditions. Bar graph shows mean + SD. ** P<0.01.

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52 B2 MED Casp3 TAN TC B6 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1stainB2% Casp3+ B2 IgM Casp3 TAN TC B6 0.0 0.5 1.0 1.5 2.0 2.5*strainB2% Casp3+ B2 LPS Casp3 TAN TC B6 0 1 2 3 4 strainB2% Casp3+ABC Figure 4-4 PerC B2 cells apoptosis. (A) Medium only. (B) Stimulated with anti-IgM 10 g/ml (C) Treated with LPS at 1 g/ml. Only TAN B2 cells showed lower apoptosis in response to stimulation. TC B2 cells did not show a significant difference from B6 in the culture. Bar graph shows mean + SD. P<0.05. MED-B1 CD86 TAN TC B6 0 5 10 15 20 25 30 35**strainB1a% positive MED-B1 CD80 TAN TC B6 0 5 10 15 20 25 30 35**strainB1a% positiveAB Figure 4-5 Cells from peritoneal lavage were collected and cultured for 48 hours in medium only. Expression levels of (A) CD86 and (B) CD80 were assayed by flow cytometry. Bar graph shows mean + SD. P<0.05.

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53 IgM 10-B2 CD86 TAN TC B6 0 10 20 30 40 50 60 70 80***strainB2% positive LPS-B1 CD86 TAN TC B6 0 10 20 30 40 50 60 70***strainB1a% positive LPS-B1 CD80 TAN TC B6 0 10 20 30 40 50*strainB1a% positive LPS-B2 CD86 TAN TC B6 0 25 50 75****strainB2% positiveABCD Figure 4-6 Peritoneal B-cell activation after 48 hrs of stimulation. (A) CD86 levels of PerC B2 cells stimulated with anti-IgM at 10 g/ml. (B) (C) PerC B1 cells stimulated with LPS at 1 g/ml. (D) B2 cells stimulated with LPS 1 g/ml. Only stimulation conditions showing significant difference are shown. Both B1 and B2 cells in the TAN PerC had lower activation. Bar graph shows mean + SD. P<0.05, ** P<0.01, *** P<0.001.

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54 B1 medium TAN-MED TC-MED B6-MED 0 5 10 15 20 25***B1a% IL-10+ B1 IgM TAN-IgM TC-IgM B6-IgM 0 5 10 15 20 25 30 35***B1a% IL-10+ B1 LPS TAN-LPS TC-LPS B6-LPS 0 5 10 15 20 25 30 35 40 45***B1a% IL-10+ABC PerC B1 gated IL-10 PE010110210310410 CD86 Biot.SAv.PerCP.Cy5.5010110210310410 R5 D Figure 4-7. Intracellular IL-10 after 48 hrs of culture. Cells from peritoneal lavages were cultured for 48 hrs with (A) medium only. (B) anti-IgM 10 g/ml. (C). LPS 1 g/ml. (D) Representative plot of flow cytometry analysis for intracellular IL-10 levels. Cells were gated on B1a B-cell population. TAN PerC B1a cells produce significantly less IL-10 than TC and B6. P<0.05, ** P<0.01, *** P<0.001.

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55 MED-B1 TAN TC B6 0 5 10 15 20***strainB1a% IL-6+ IgM10-B1 TAN TC B6 0 10 20 30 40*****strainB1a% IL-6+ LPS-B1 TAN TC B6 0 10 20 30 40***strainB1a% IL-6+ABC PerC B1 IL-6 IL-6 PE010110210310410 CD80 Biot.SAv.PerCP.Cy5.5010110210310 4 D 10 R5 Figure 4-8. Intracellular IL-6 after 48 hrs of culture. Cells from peritoneal lavage were cultured for 48 hrs with (A) medium only, (B) 10 g/ml anti-IgM. (C) 1 g/ml LPS. (D) Representative plot of flow cytometry analysis for intracellular IL-6 levels. Cells were gated on B1a B-cell population. TAN PerC B1 cells make significantly less IL-6 in all 3 conditions, while TC B1 cells produce more IL-6 when treated with anti-IgM. P<0.05, ** P<0.01, *** P<0.001.

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CHAPTER 5 RESULTS AND DISCUSSION: SPLEEN B CELLS IN THE LUPUS PRONE AND RESISTANT MICE The hypothesis is that lupus prone mice have different splenic B cell functions and/or development stages and these differences reflect their roles in the pathogenesis. Splenic B1 and B2 Cells Flow cytometry studies on adult spleen showed increased B1 cells in both TAN and NZM, while proportionally less B2 cells were found in NZM, TC and TAN mice (Figure 5-1). Activation marker levels measurement found no significant differences of B7-1 and B7-2 expression on either TAN or lupus mice B-cell (data not shown). TC B2 cells showed however an increased of early activation marker CD69 and NZM B2 cells showed higher CD40 expression. In addition B-cells from the 3 strains express reduced levels of IgM on average as compared to B6, indicating that that these B cells have encountered antigens, presumably self-antigen (Figure 5-2). Newly formed B cells that are just released from bone marrow undergo a series of development stages in the periphery (113). Based on CD21 and CD23 expression, the B-cell population in the spleen has been divided into 4 sub-groups: T1 (Transitional 1), T2 (Transitional 2), follicular (FoB) and marginal zone B (MZB) cells(116) (Figure 5-3). Flow cytometry revealed that NZM, TC and TAN mice have accumulated T1 and less T2 and FoB cells than B6, suggesting a developmental arrest on T1 to T2 stage (Figure 5-4). No difference was observed for marginal zone B cells, as mentioned earlier. B-cell development can be determined by bone marrow derived cells, including the 56

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57 B cells themselves, or by non-bone marrow derived stromal cells. To identify whether abnormal B cell development in TC mice was determined by bone marrow derived cells, we performed reciprocal bone-marrow transfers between B6 and TC mice. The results of this experiment showed that the abnormal B cell subset distribution can be completely accounted for by TC bone marrow (Figure5-5). Splenic Naive B-Cell Functional Properties To study B-cell functional properties without the confounding factor of differential sub-population distribution, we analyzed the response of nave CD43B cells to stimulation. Splenic naive CD43B cells were isolated with a B-cell isolation kit from Miltenyi Biotech. The output was >96% pure. Then purified B-cells were cultured with medium only, anti-IgM (low dose: 1 g/ml or high dose: 10 g/ml), or LPS (1 g/ml). B-Cell Proliferation after Stimulation During the 72 hrs of culture in medium only, B cells from TAN and NZM, TC mice showed a higher spontaneous proliferation as compared to B6 (Figure 5-6). When stimulated with low dose of anti-IgM, B cells in lupus-prone mice showed an increased proliferation index but TAN had a decreased proliferation index. In the situation of high dose of anti-IgM stimulation, TAN mice B cells had a similar proliferation index as B6, while TC showed an even higher proliferation. Interestingly, no difference was observed with LPS treated B cells from either strain. Activation-Induced Cell Death To assess activation-induced cell death in B cells, CD43B cells were treated as mentioned before for 16 hrs, and intracellular cleaved caspase-3 levels was assayed by flow cytometry. The results showed significantly less apoptosis in lupus-prone NZM and TC B-cells when treated with 10g/ml anti-IgM. No differences were observed in B-cell

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58 treated with medium only, low dose of anti-IgM and LPS (Figure 5-7). B-Cell Activation and Differentiation After In Vitro Stimulation. To test the activation and differentiation of cultured B cells, cells were treated as mentioned before for 48 hrs, and the expression of surface activation/differentiation markers was assayed by flow cytometry as the read-out. The results showed that when left in the medium only, all TAN, NZM and TC mice showed significant higher differentiation into germinal center (GC) B cells as shown by the high levels of GL-7 expression. Besides, a higher percentage of CD19 hi population also present in the TAN and TC B cells (Figure 5-8). Since GL-7 is a germinal center marker, and activated B cells up-regulate CD19(194-196), a BCR co-receptor, these results suggested a higher spontaneous B-cell activation and differentiation among these strains. No difference was observed for the expression of early activation marker CD69 and co-stimulatory molecule B7-2 (CD86). When treated with low dose of anti-IgM at 1g/ml, the TAN B cells showed decreased CD86 and GL-7 levels. Expression of CD80 and CD19 were not significantly different (Figure-5-9). In the situation of high dose of anti-IgM treatment (10g/ml), the TAN B cells still showed significantly lower levels of CD86, although their GL-7 levels was now similar to B6, and their CD19hi B-cells were significantly less than B6. On the other hand, the B cells from lupus prone mice stimulated with high doses of IgM showed high levels of GL-7 and CD19 up-regulation (Figure 5-10). In contrast to these differences, Western blot assay shows that both TAN and TC B cells have similar elevated ERK phosphorylation compared to B6 B cells (Figure 5-11). For the B cells stimulated with LPS, no significant difference of

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59 activation/differentiation were found between the strains (data not shown), indicating that the differences in activation and differentiation reported above were specific for BCR stimulation. Discussion Although both TAN, NZM and TC carry the three lupus susceptible loci, the increased splenic B1 cells was not observed in TC mice, indicating the gene(s) responsible for high SP B1 cells map outside the three Sle loci. In addition, the levels of activation markers on splenic B1 cells from TAN and NZM were not different, suggesting that splenic B1 cell is not necessary for the development of lupus, either by their number of activation status. Both the lupus-prone and resistant mice have abnormal splenic B cell development, displayed by the accumulated T1 cells as well as decreased T2 and follicular B cells, suggesting a development arrest between the T1 and T2 stages. Reciprocal bone marrow transfer suggests that this defect can be completely accounted for by the bone marrow i.e. either B cell itself and/or interaction between B and T/myeloid cells. The immature B cells that just emerged from bone marrow contain autoreactive clones and must undergo maturation in the periphery, during which these clones are removed, and checkpoints of negative selection during T1 and T2 stages has been suggested (113;119). Thus the accumulation of T1 cells may overload this checkpoint and increase the possibility of autoreactive clones maturation in periphery. Besides, recent studies have shown that transitional B cells can present antigens to CD4+ T cells and activate T cells if exogenous costimulation is provided (197). On the other hand, interactions with activated T cells can also protect immature B cells from negative selection (197). Since the lupus prone NZM and TC, but not the resistant TAN mice, have hyper-activated T cells (unpublished), the

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60 accumulated immature B cells in these strains thus may have different outcome, and play an important role in disease development. An age-dependent splenomegaly appears in TAN, NZM and TC mice. Thus although they have a lower B2 proportion of splenic lymphocytes, the actual B2 cell number is still higher. Consistent with this, naive B cells from all the strains show a significantly higher spontaneous proliferation in vitro. Besides, both strains also have higher proportion of B-cells expressing GL7 and up-regulating CD19 without stimulation. GL7 is an activation marker that is expressed only on germinal center B cells in the periphery (198). CD19 forms complex with CD21 and CD81 and is an essential co-receptor for BCR that functions to enhance BCR signaling (199). The cytoplasmic domain of CD19 recruits vav, PI-3 kinase and Src-family kinase Lyn after BCR ligation and activates down-stream signaling (195;200). Over-expression of CD19 leads to B cell hyper-activation and development of autoimmunity (201). These results indicate a higher spontaneous B-cell activation and differentiation in these strains. Further studies were conducted using different dosage of anti-IgM F(ab) 2 to mimic different strengths of BCR ligation in vivo. Results showed that B cells from lupus-prone and resistant mice have different activation threshold. The TAN B cells responded poorly to low dose anti-IgM stimulation, even less than the medium only condition, while at this low levels, the lupus TC and NZM B cells showed significant activation and differentiation. At the high dose of anti-IgM stimulation, still, the lupus mice B cells exhibited higher proliferation and activation potential, while TAN B cells were similar as the B6 B cells. Especially TAN B cells did not show a significant up-regulation of CD19 upon stimulation. Furthermore, the lupus B cells have less activation induced cell death.

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61 One interesting observation is that both TAN and TC B cells have a similarly elevated ERK phosphorylation upon low and high dose anti-IgM, suggesting the effects of the Sle loci they share. In fact, hyper-activation of ras-ERK has been mapped to Sle1ab sub-locus recently (202). This also indicate that signaling differences between TAN and TC B cell may exist downstream of ERK. Finally, flow cytometry study showed that both TAN and lupus mice B cells have similar levels of surface IgM, which is significantly lower than that of B6, thus the amount of surface IgM did not account for the different signaling phenomenon. In short, these results indicate that compared to normal B cells, lupus mice B-cells have lower threshold on BCR signaling pathway, while TAN B cell has elevated threshold, and their TLR4 signaling pathway is not changed. It has been show that B cells are indispensable to prime T cells and to initiate lupus pathogenesis (1;7). The B-cell phenotypes and BCR signaling properties thus may contribute to the phenotypes of the strains. Both TAN and lupus mice B cells have high spontaneous activation, proliferation and activation in vitro. Accordingly, all these mice develop an age-dependent anti-chromatin and anti-dsDNA IgM autoantibody production. This can be due to the effects of 3 Sle loci they share (154;203-205). Breaking of the tolerance is not sufficient, however, for the fully development of autoimmune disease (152). The disease phenotypes of the strains correlate to and affected by their B-cell properties. As B cells from lupus prone mice have more potency to activation, proliferation and differentiation as well as resistance to apoptosis, the lupus mice generate large amount of germinal centers, long-lived plasma cells (unpublished data), class-switched high affinity IgG autoantibodies (206) and finally full development of the disease. TAN mice B cell respond poorly to low dose BCR cross-linking, and they do not

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62 up-regulate CD19 expression even with strong BCR stimulation. Accordingly, their autoantibody production is significantly lower, and most of the isotype is IgM (unpublished data). Most importantly, TAN mice do not develop lupus nephritis. These facts also indicate the impacts of lupus suppressing genes carried by the TAN genome. In summary, this study shows that in lupus prone mice, splenic B cells actively participate in the pathogenesis by the enhanced activation and differentiation to plasma cells in response to antigen stimulation. In lupus resistant TAN mice, the limited B cell activation likely contributes to the protection the host from lupus.

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63 SP B1 TAN NZM TC B6 0 10 20 30 40 50 60 70******% lymphocytes SP B2 TAN NZM TC B6 0 25 50 75********% lymphocytesAB Figure 5-1. Splenic B cell populations. Freshly isolated splenocytes were assayed by flow cytometry for (A) B1 cells and (B) B2 cells. Both TAN and NZM have increased percentages of spleen B1 cells and decreased B2 cells, while TC has only a decreased percentage of B2 cells. P<0.05, ** P<0.01, *** P<0.001.

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64 B2 CD69 TAN NZM TC B6 0 5 10 15 20***strain% of B2 B2 CD40 TAN NZM TC B6 0 10 20 30 40***strain% of B2 SP B2 IgM MFI TAN NZM TC B6 0 50 100 150 200*******strainMFIABC Figure 5-2. Activation marker expression on splenic B2 cells assayed by flow cytometry. (A) Significantly higher percentages of TC B2 cells express CD69. (B). Significantly higher percentages of NZM B2 cells express CD40. (C) Lower IgM mean fluorescence intensity on TAN, TC and NZM B2 cells. P<0.05, ** P<0.01, *** P<0.001.

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65 Peripheral B-Cell Development CD23 PE0101210310410 10 T1 CD21 Biot.SAv.PerCP.Cy5.5010110210310410 Gated on B cells FoB MZB T2 Figure 5-3. Definition of splenic B-cell developmental subpopulations with flow cytometry. Figure shows typical B-cell subpopulations from normal B6 mice spleen. Cells are gated on B220+. T1, transitional 1; T2, transitional 2; FoB, follicular B cells; MZB, marginal zone B cells.

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66 T1 TAN NZM TC B6 0 10 20 30 40 50 60 70 80 90*********strain% of B-cells FoB TAN NZM TC B6 0 10 20 30 40 50 60 70 80 90*****strain% of B-cells T2 TAN NZM TC B6 0.0 2.5 5.0 7.5*****strain% of B-cells MZ B TAN NZM TC B6 0 5 10 15 20 25 30 35 40 45 50 55strain% of B-cellsABCD Figure 5-4. Peripheral B-cell development. Freshly isolated splenocytes were assayed by flow cytometry for (A) T1. (B) FoB. (C) T2 and (D) MZB cells. Both TAN and lupus-prone NZM and TC have accumulated T1, less T2 and follicular B cells. Populations were gated on B220+ B cells. P<0.05, ** P<0.01, *** P<0.001.

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67 T1 B6 donor TC donor B6 TC NZM 0 10 20 30***% of B-cells T2 B6 donor TC donor B6 TC NZM 0 1 2 3 4 5***% of B-cells FoB B6 donor TC donor B6 TC NZM 50 55 60 65 70 75 80 85**% of B-cells MZB B6 donor TC donor B6 TC NZM 0 2 4 6 8 10 12***% of B-cellsABCD Figure 5-5 Splenic B cell populations in mice after reciprocal bone marrow transfer. Three month old B6 and TC mice were lethally irradiated and TC or B6 bone marrow was transferred. All mice were sacrificed after 3 months and splenic B cells were assayed for (A) T1, (B) T2, (C) FoB, and (D) MZB populations. Unmanipulated mice were used as controls. Splenic B-cell populations from bone marrow transferred mice were similar as the non-operated donor strains. Statistical comparisons were conducted between B6 donor and TC donor groups. P<0.05, ** P<0.01, *** P<0.001.

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68 medium TAN NZM TC B6 0 1000 2000 3000 4000 5000 6000 7000 8000*******straincpm IgM 1 index TAN NZM TC B6 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5******strainproliferation indexAB IgM 10 index TAN NZM TC B6 0 10 20 30 40 50 60***strainproliferation index LPS index TAN NZM TC B6 0 25 50 75strainproliferation indexCD Figure 5-6. B-cell proliferation indicated by 3H-Thymidine incorporation. Splenic naive B cells were isolated and cultured for 72 hrs with (A) medium only, (B) anti-IgM at 1 g/ml; (C) anti-IgM at 10 g/ml (D) LPS at 1 g/ml. Proliferation index was calculated by the relative ratio of stimulation cpm to the corresponding medium only cpm value. Without stimulation, both TAN and NZM, TC mice showed increased spontaneous proliferation. When stimulated with lose doses of anti-IgM, lupus-prone mice had significantly higher proliferation, while TAN mice show decreased proliferation as compared to B6. At higher doses of anti-IgM, TAN and NZM showed similar proliferation as B6, while TC had an even higher proliferation. Finally, no significant difference was observed in LPS-treated B cells. P<0.05, ** P<0.01, *** P<0.001.

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69 medium TAN NZM TC B6 0 10 20 30 40 50 60strainB% Casp3+ IgM 1ug/ml TAN NZM TC B6 0 10 20 30 40 50 60strainB% Casp3+ IgM 10ug/ml TAN NZM TC B6 0 5 10 15 20 25 30 35 40 45 50 55***strainB% Casp3+ LPS TAN NZM TC B6 0 5 10 15 20 25 30 35 40 45strainB% Casp3+ABCD Figure 5-7. Apoptosis rate during of in vitro stimulation. Splenic naive B cells were isolated and cultured for 16 hrs with (A) medium only, (B) anti-IgM at 1 g/ml, (C) anti-IgM at 10 g/ml, (D) LPS at 1 g/ml. Only when stimulated with high doses of anti-IgM, lupus prone TC and NZM B cells showed less cell death than TAN and B6. P<0.05, ** P<0.01, *** P<0.001.

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70 MED-B CD86 TAN NZM TC B6 0 5 10 15 20 25 30 35 40 45 50 55strainB% positive MED-B CD69 TAN NZM TC B6 0 5 10 15 20strainB% positive MED-B GL-7 TAN NZM TC B6 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5****strainB% positive MED-B CD19 hi TAN TC B6 25.0 27.5 30.0 32.5 35.0 37.5 40.0******strainB% CD19himediumABCD Figure 5-8 B-cell activation during in vitro culture. Splenic naive B cells were isolated and cultured for 48 hrs with medium only. Activation markers were assayed by flow cytometry for (A) CD86, (B) CD69, (C) GL-7 and (D) CD19. No significant difference was observed for CD86 and CD69. (C) TAN, NZM and TC showed higher GL-7 levels. (D) Both TC and TAN had much more B cells with up-regulated CD19. P<0.05, ** P<0.01, *** P<0.001.

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71 IgM 1-B CD86 TAN NZM TC B6 0 5 10 15 20 25 30 35 40 45 50 55 60 65***strainB% positive IgM 1-B CD80 TAN NZM TC B6 0 10 20 30 40strainB% positive IgM 1-B GL-7 TAN NZM TC B6 0 10 20 30***strainB% positive IgM 1-B CD19 hi TAN TC B6 15 20 25 30 35strainB% CD19 hiABCD Figure 5-9. B-cell activation and differentiation with a low dose of anti-IgM stimulation. Splenic naive B cells were isolated and cultured for 48hrs with anti-IgM at 1g/ml. Activation markers were assayed by flow cytometry for (A) CD86, (B) CD69, (C) GL-7 (D) CD19. TAN B-cell showed lower CD86 and GL-7 levels. No significant differences were observed among TC, NZM and B6 mice for all of these markers.* P<0.05, ** P<0.01, *** P<0.001.

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72 IgM 10-B CD86 TAN NZM TC B6 0 10 20 30 40 50 60 70 80**strainB% positive IgM 10-B CD80 TAN NZM TC B6 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5strainB% positive IgM 10-B GL-7 TAN NZM TC B6 0 10 20 30 40 50 60 70 80****strainB% positive IgM 10-B CD19 hi TAN TC B6 30 40 50 60 70******strainB% CD19 hiABCD Figure 5-10. B-cell activation under treatment with high dose of BCR stimulation. Splenic naive B cells were isolated and cultured for 48 hrs with anti-IgM at 10g/ml. Activation markers were assayed by flow cytometry for (A) CD86, (B) CD69, (C) GL-7 (D) CD19. P<0.05, ** P<0.01, *** P<0.001.

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73 anti-IgM 1ug/ml anti-IgM 10ug/ml p-ERK1 p-ERK2 ERK1 ERK2 A B C A B C Figure 5-11 ERK phosphorylations in isolated naive B cell after 5 minutes of stimulation. (A) TAN (B) TC (C) B6. Both TAN and TC B cell have elevated ERK phosphorylation

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CHAPTER 6 SUMMARY AND CONCLUSIONS In this study, we have characterized the properties of B-cell populations in lupus prone and resistant mice. We found that the splenic MZB cell phenotype correlated with the lupus susceptibility. In the lupus-prone TC and NZM mice, we found a loss of marginal zones, missing marginal zone macrophages, and marginal zone B cells trans-located inside the follicles. In contrast, lupus-resistant TAN mice had enlarged MZ with CD5+ marginal zone B cells retained despite of stimulation. Besides, MZB cells of both strains have defective TI-2 antigen up-take. As the MZB cells have potent antigen presenting and rapid differentiation capabilities, and also contain auto-reactive clones, the trans-localized MZB cells acquire the privilege to present antigen to and to activate CD4 T cells and ultimately differentiate into (auto)antibody-producing plasma cells. In contrast, the expression of CD5 and MZ arrest of MZB cells in TAN mice may raise the activation threshold and prevent them from developing overt autoimmunity. Furthermore, the lack of marginal zone macrophages and defects in TI-2 antigen uptake by MZB cells suggests that lupus mice are ineffective in the clearance of blood-borne pathogens. Secondly, B-cell responsiveness to BCR ligation was also correlated to the disease phenotype of these strains. The naive splenic B cells in lupus mice generally have lower activation thresholds, and higher rates of differentiation, while TAN B cells showed the opposite. All of our data strongly suggest that B cells in lupus mice actively participate in lupus pathogenesis by over-activation to antigen stimulation and differentiation to plasma cells. 74

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75 In the peritoneal cavity, TC B1 cells produced overall more IL-6 and IL-10 upon BCR cross-linking. In contrast, peritoneal B cells from TAN mice still show the decreased responsiveness, and little IL-6 and IL-10 production with stimulations. Both IL-6 and IL-10 can promote B-cell proliferation, differentiation and antibody production, and they also play a positive role in lupus progression. These results suggest that peritoneal B1-cells in lupus mice participate in the pathogenesis of lupus by producing disease promoting cytokine IL-6 and IL-10. Taken together, these studies suggest that B-cell populations contribute to the development of lupus both through effector and regulatory mechanisms.

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BIOGRAPHICAL SKETCH Biyan Duan was born in P. R. China on Sep. 23, 1971. He graduated from Tianjin Medical University and received his Bachelor of Medicine in 1995. Then he became a clinical resident of Tianjin No.3 Hospital, at the Department of Pediatrics for 3 years. In Sep. 1998 he started study for his masters degree in clinical immunology at Tianjin Medical University. In fall, 2000, he was accepted by the Interdisciplinary Program in Biomedical Sciences at the University of Florida. He did his dissertation research under the supervision of Dr. Laurence Morel and received his PhD in May 2006.


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Permanent Link: http://ufdc.ufl.edu/UFE0013828/00001

Material Information

Title: Characterization of the B Cells in Lupus-Prone and Resistant Mouse Models
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0013828:00001

Permanent Link: http://ufdc.ufl.edu/UFE0013828/00001

Material Information

Title: Characterization of the B Cells in Lupus-Prone and Resistant Mouse Models
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0013828:00001


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CHARACTERIZATION OF THE B CELLS INT LUPUS-PRONE AND RESISTANT


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2006



























Copyright 2006


































This document is dedicated to my parents and my sister.















ACKNOWLEDGMENTS

I would like thank my dissertation committee members, Dr. Michael J. Clare-

Salzler, Dr. William L. Castleman, Byron P. Croaker, Dr. Eric S. Sobel and my mentor, Dr.

Laurence Morel, for their encouragement, support, and excellent suggestions. Their

advice was crucial to my progress in this research.




















TABLE OF CONTENTS

IM Le

ACKNOWLEDGMENT S .............. .............. iv


T ABLE ............... .............. vii


LI ST OF FIGURE S .............. .............. viii


AB ST RAC T ............... ...............x


CHAPTER


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


Peripheral B-Cell Populations .............. ...............2
Bl Cell s ................ ........ .. ........ ...... ...............2

Marginal Zone and Follicular B (B2) Cells............... ...............6
Peripheral B-Cell Development............... ...............9
BAFF and Receptors for BAFF .............. .. ... .. ... .. ...............10
The NZB/W Derived Mouse Models for Lupus Prone and Resistance......................11


2 MATERIALS AND METHODS .............. ...............18


Experimental Animals .............. ...............18
Immunofluorescence Staining .............. ...............18
B-Cell Isolation and Cell Culture .............. ...............19
Flow Cytometry .............. ...............19
Bone Marrow Transfers............... ...............20
Treatment of Mice with TI Antigens............... ...............21
Western Blotting Assay .............. ...............21
Statistics............... ...............21


3 RESULTS AND DISCUSSION: ABNORMAL MARGINAL ZONE AND
MARGINAL ZONE B CELLS are found in both lupus-prone and lupus resistant
mice............... ...............22


Different Marginal Zone Phenotypes Between Lupus-Prone and Resistant-Mice.....22

Reciprocal Bone Marrow Transfers Indicate MZB Translocation is Determined by
Multiple Factors ..................... ...............24
Functional Studies of MZB Cells .............. ...............25
MZB Respond to TI-2 Antigen Ficoll................. ...............25
MZB Migration in Response to TI-1 Antigen LPS.............. ...............26












Discussion............... ...............27


4 RESULTS AND DISCUSSION: ROLE OF PERITONEAL CAVITY B CELLS
IN LUPUS SUSCEPTIBILITY............... ...............45


Activation, Proliferation and Apoptosis .............. ...............45
In Vivo Spontaneous Proliferation:. ...45
In Vitro B-cell Apoptosis .............. ...............46
Peritoneal Cavity B-Cell Activation............... ...............46

Cytokine Production.............. ...............46
Discussion............... ...............47


5 RESULTS AND DISCUSSION: SPLEEN B CELLS IN THE LUPUS -PRONE
AND RESISTANT MICE .............. ...............56


S pl eni cB 1 and B 2 Cell s ................. ... ............... 56
Splenic Naive B-Cell Functional Properties............... ...............57
B-Cell Proliferation after Stimulation .............. ...............57
Activation-Induced Cell Death................. ... ..... .... ....... ...............57
B-Cell Activation and Differentiation After In Vitro Stimulation......................58
Discussion............... ...............59

PeripheralB-CellDevelopment............... ...............65


6 SUMMARY AND CONCLUSIONS.............. ...............74


LI ST OF REFERENCE S ............... ...............76


BIOGRAPHICAL SKETCH .............. ...............93
















TABLE




















LIST OF FIGURES


Figure page


1-1 Diagram of white pulp and red pulp............... ...............13


1-2 Peripheral B-cell development stages............... ...............14


1-3. BAFF/APRIL and their receptors............... ...............15


1-4. NZM models of lupus prone and resistance mice .............. ...............16


3-1. Reduced MZ in TC and NZM versus enlarged MZ in TAN............... ...............34


3-2 Splenic MZB populations............... ...............35


3-3. Intrafollicular location of MZB cells in TC and NZM............... ...............36


3-4. MZB cell phenotypes............... ...............37


3-5. Greatly reduced MZMs in the TC and NZM mice............... ...............38


3-6. Reduced VCAM-1 levels on TC marginal zone............... ...............39


3-7. Marginal zone of TC and B6 bone marrow chimera............... ...............40


3-8. Defect of marginal zone macrophages TC .............. ...............41


3-9. TNP-Ficoll up-take by splenic MZB cells............... ...............42


3-10 In vitro TNP-Ficoll binding............... ...............44


3-11 TAN MZB cells do not migrate after LPS treatment .............. ...............44


4-1 Peritoneal Bl and B2 populations .............. ...............50


4-2 In vivo proliferation of peritoneal cavity B cells............... ...............51


4-3 PerC B 1 cell s apoptosi s .............. ...............5 1


4-4 PerC B2 cells apoptosis.............. ...............52


4-5 PerC B-cell activation............... ...............52




V111












4-6 Peritoneal B-cell activation after 48 hrs of stimulation............... ...............53


4-7. Intracellular IL-10 level............... ...............54


4-8. Intracellular IL-6 level............... ...............55


5-1. Splenic B cell populations.............. ...............63


5-2. Splenic B cell activation............... ...............64


5-3. Definition of splenic B-cell developmental subpopulations............... ...............65


5-4. Peripheral B-cell development .............. ...............66


5-5 Splenic B cell populations in mice after reciprocal bone marrow transfer .................67


5-6. B-cell proliferation. ............. ...............68


5-7. B-cell apoptosis rate during of in vitro stimulation............... ...............69


5-8 B-cell activation during in vitro culture .............. ...............70


5-9. B-cell activation and differentiation with a low dose of anti-IgM stimulation ..........71


5-10. B-cell activation under treatment with high dose of anti-IgM stimulation ..............72


5-11 ERK phosphorylations............... ...............73











Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
CHARACTERIZATION OF THE B CELLS IN LUPUS-PRONE AND RESISTANT
MOUSE MODELS

By

Biyan Duan

May 2006
Chair: Laurence Morel
Major Department: Pathology, Immunology and Laboratory Medicine

The general hypothesis is that in the models of lupus-prone and resistant mice, B

cells function differently and contribute to the phenotypes and pathogenesis. Thus, the

studies of B-lymphocytes in these models may help to understand the role of B-cells and

to identify involved genes.Defect splenic marginal zone (MZ) has been found in the

lupus-prone NZM2410 and B6.Slel/2/3 (TC), but not in the lupus resistant NZM.TAN

(TAN) mice. I hypothesize that this defect is due to the abnormal functions of marginal

zone B cells and contributes to the pathogenesis. Immuno-fluorescence study revealed

that lupus MZ B cells were trans-located inside the follicle. MZ macrophages (MZM)

were also found defect only in the lupus mice. Reciprocal bone marrow transfer indicated

that the lack of MZM was not the reason of MZB translocation, and MZB miss-location

was determined by both bone marrow and stroma-derived factors. Functional studies

revealed that MZ B cells in TC and TAN mice were abnormal.

The second goal of the research was to study the phenotypes and functions of the

peritoneal cavity (PerC) B cells in these strains. Both lupus-prone and resistant mouse

models have increased PerC B cells, especially Bl cells. Studies showed TANPerC Bl









cells had lower activation and higher resistance to activation induced cell death, while Bl

cells from NZM2410 and TC had higher proliferation. Finally TAN Bl cells produce

little IL-6 and IL-10, while TC Bl cells make more 1-6 and a normal levels of IL-10,

suggesting a regulatory role of TC Bl cells in immune response.

The third goal was to study the properties of splenic B cells. Both strains have

increased Transitional 1 and Transitional 2 populations and less mature follicular B cells.

In vitro stimulations revealed the B cells from lupus mice had a lower activation

threshold and higher proliferation/differentiation abilities, while TAN B cells showed the

opposite directions. Lupus B cells also had abnormal BAFF-R/TACI expressions,

indicating the mechanism behind the phenotypes. Overall these results suggest that B-cell

populations in lupus-prone and resistant mice are quite different and may play an

important role in the pathogenesis.














CHAPTER 1
INTRODUCTION

The general hypothesis of the study is that in the NZM models of lupus-prone and

resistant mice, B cells function differently and contribute to the phenotypes and

development of the disease. Thus, the studies of B-lymphocytes in these marine models

may help to understand the role of B-cell in lupus development and to identify the genes

responsible

Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease that affects

multiple organs and leads to end-organ damage. SLE is characterized by the production

of large amounts of antibodies against a wide spectrum of auto-antigens, including

ssDNA, dsDNA and RNPs. B cells have long been recognized as effectors to receive

specific T-cell help and to secrete these pathogenic auto-antibodies which result in tissue

damage. Recent studies suggest that B-cell may play a role other than the autoantibody

producer. In the lupus-prone MRLipr- mice, modification ofB-cell that blocking the

antibody secretion does not prevent disease development(. In the NZB/W derived lupus

model NZM2410, genetic dissection shows that the expression of Sle susceptible locus

on B cells is essential for the development of autoimmunity(2). T-cell hyper-activation

has been found in lupus(3-6), while in MRLipr-, T-cell activation is greatly dependent on

B cells (7), suggesting the role of B-cell as (auto)antigen presenting cells. Furthermore,

B-cell depleting treatments lead to remission of SLE symptoms in patients(8-11). All of

these findings demonstrate the pivotal role played by B cells in pathogenesis.

B cells are heterogeneous and include multiple subpopulations with distinct









functions and properties. However, the exact role of each subpopulation in the

pathogenesis of lupus is still not clear. This study is focused on the characteristics and

functions of different B-cell subsets in lupus prone and resistant mouse models.

Peripheral B-Cell Populations

Based on their location, surface marker expression, and functions, the peripheral

mature B cells are now divided into three distinct populations: Bl cells, B2 (follicular B,

FoB) cells and marginal zone B (MZB) cells(12).

B1 Cells

Based on the expression of the pan-T cell surface glycoprotein-CD5, B cells were

originally divided into two populations: Bl (CD5+) and B2 (CD5-) cells(13). Further

studies defined B-1 cells as a population possessing a distinct pattern of surface markers:

B2201o, IgMhi, IgDio, CD9 CD43 CD23io, and conventional circulating B-2 cells as

B220hi, IgMhi/lo, IgD CD9-, CD43-, CD23hi (14;15). Bl B cells are also larger and

exhibit more side scatter than do conventional B-2 cells by flow cytometry. Subsequently,

Bl cells were divided into two populations: Bla (CD5+) and B1b(CD5-), i.e. they share

all other surface markers and properties except CD5. It is still not clear whether these

subsets are two distinct cell types or different development stages of one population. B1b

cells contribute only to a small proportion of the Bl population. Recent studies show that

they have distinct functions from Bla cells. B1b cells are responsible for long-term T-

independent immunity specific for B. hersmii (16) and are critical in producing adaptive

pneumococcal polysaccharide antibodies (17). For practical reasons, most studies

performed on Bl cells are based on results of CD5+ Bla cells. Most Bl cells resides in

body cavities such as peritoneal (PerC) and plural cavities, where they comprise the

majority of the local B-cell population. While in the spleen and lymph nodes, Bl cells is









the minority of the local B cells and most of B cells are CD5-. Finally, Bl cells are

normally absent from peripheral blood(14).

Compared to B2 cells, Bl cells are long-lived, self-renewing and non-circulating B

cells with limited B-cell receptor (BCR) diversity and affinity(18;19). Adoptive transfer

suggested that Bl cells originated from fetal liver and thus belong to a developmental

lineage different from B2 cells (14). When transferred into irradiated mice, fetal liver

cells could reconstitute both the Bl and B2 compartments, while adult bone marrow only

generated B-2 cells (20;21).

Bl cells are refractory to activation through B cell receptor (BCR) ligation, and do

not undergo somatic hypermutation after stimulation (22;23). Bl cells mainly participate

in T-cell independent responses. They respond to LPS much quicker than B2 cells (24)

and uptake TI type II antigen dextran, but not ficoll (25). Recent studies also reveal that

Bl cells from the spleen are phenotypically and functionally different than those obtained

from peritoneal cavity (24;26): Unlike peritoneal Bl cells, splenic Bl cells do not express

the myeloid marker CD11b (Mac 1) and have much lower surface IgM and CD80 levels.

In addition, they make little natural IgM. Transcriptional factors Notchl and Notch2 are

expressed at high levels by splenic but not peritoneal Bl and B2 cells. Finally, peritoneal

Bl cells respond to phorbol eater in vitro, while B2 and splenic Bl cells need the

presence of the calcium ionophore ionomycin to proliferate. Due to the low concentration

of splenic Bl, most Bl-cell studies are focused on peritoneal Bl cells.

CD5 has been recognized as a negative regulator of BCR signals in Bl cells

(27;28). Its cytoplasmic tail contains a docking site for SH2-phosphatase SHP-1, which is

critical for diminishing BCR signaling after antigen ligation(29). SHP-1 deficient









"motheaten" mice have lymphocyte over-activation and suffer from severe autoimmune

symptoms(30;31). Studies on intracellular signaling pathways reveal that Bl cells have

constitutive ERK and NF-AT activation, but lack of NFkapaB induction upon BCR

cross-linking(32), which is similar to tolerant B cells(33;34). It has been suggested that

CD5 expression by Bl cells occurs after auto-antigen exposure to down-regulate their

activation status(35). Targeted deletion of CD5 leads to the activation of anergic B cells

and results in the loss of tolerance (36;37), indicating the important role of CD5

expression on Bl cells.

Bl cells are the major producer of IgM natural antibodies, which are poly-reactive

and weakly auto-reactive(38;39). Through specific up-regulation of the transcription

factor Blimp-1, which was believed to be plasma cell-specific, Bl cells spontaneously

secrete a large amount of natural IgM (40). Natural antibodies recognize a broad range of

antigens from many bacterial/viral pathogens prior to the exposure. Thus, they are very

important for the early response to bacterial and viral infections(41). Mice lacking natural

antibodies suffer from higher susceptibility to influenza infections and have increased

mortality(42). On the other hand, natural IgM can also bind to constituents of self, such

as phosphorylcholine (PC) (16), phosphatidyl choline (PtC) (13;43) and oxidized low-

density lipoprotein (LDL)(44). The production of auto-reactive natural antibodies has

implicated Bl cells a potential contributors to the development of autoimmune diseases,

such as lupus. In fact, elevated Bl cells have been observed in autoimmune patients with

Sjogren's syndrome, SLE and rheumatoid arthritis (RA) as well as in mouse models of

lupus(14;45-47). The (NZB x NZW) Fl lupus mouse and its derivative NZM2410 have

large numbers of B-1 cells which have accumulated in the peritoneal cavity and, to a









lesser extent, in the spleen(48). Elimination of Bl cells by hypotonic shock with repeated

water injections on NZB and (NZB x NZW) Fl mice decreased autoantibody production

and reduced kidney pathology(49), suggesting that Bl cells may play a role by producing

pathological autoantibodies. While some other studies do not support this notion. For

example, in the well-characterized FAS-deficient MRLipr mouse model, B2, but not B-la

cells are responsible for the autoantibody production (50). Furthermore, over-expression

of IL-5 in the (NZB x NZW)F1 model greatly increased the number of B-la cells, but

significantly reduced anti-dsDNA antibody production and the incidence of nephritis (51).

These results suggest that the role of Bl cells as autoantibody producer is context-

dependent.

In addition to antibody production, B-la cells express high levels of co-stimulatory

molecules B7-1, B7-2 and display enhanced antigen presentation capabilities (48). In

aged (NZB x NZW)F1 mice, target organs such as kidney and thymus are found to

contain infiltrated myeloid cells with over-expressed CXCL13 (BLC) (52;53). Bla cells

are attracted to the target organs by the high local BLC levels and defective in homing to

peritoneal cavity. The accumulated Bla cells thus can activate autoreactive T cells

through their potent antigen presentation capability and contribute to the damage of target

organs (53;54).

B-la cells are the main source of B cell-derived IL-6 and IL-10 (55), indicating

their regulatory role in immune response. IL-6 promotes B-cell survival and strongly

induces differentiation of B-cell to plasma cell (56). It also promotes T-cell growth

through augmentation of 1-2 production and IL-2 receptor expression (57;58), and

rescues T-cell from apoptosis (59;60). IL-6 has been linked to the development of









autoimmune diseases, including lupus and RA (61-64). In the lupus mouse models

MRLipr and (NZB x NZW)Fl, increased IL-6 levels has been found (62;65). Furthermore,

administration of exogenous 1-6 to NZB/W mice increased anti-DNA autoantibody

production and accelerated glomerulonephritis progression (66-68). Studies on human

SLE patients have also found elevated IL-6 levels that correlate with disease activity

(69;70). In the kidney, increased 1-6 can induces the proliferation of mesangial cells and

is involved in the development of glomerulonephritis (71;72). IL-6 blockade treatment

dampened the progression of SLE and decreased the severity (58;73). These data show a

pivotal role for IL-6 in lupus pathogenesis and also suggest the role of IL-6 producing

Bla cells in lupus.

IL-10 is an anti-immflamatory cytosine that strongly inhibits the activation of

myeloid cells including monocytes, dendritic cells and macrophages(74-76). IL-10 can

promote B cell differentiation, proliferation, and antibody production (77). 1-10 is also

involved in regulatory T-cell differentiation and functions(78;79). The role of IL-10 in

SLE is still controversial, as it has been shown to both inhibit (80) and exacerbate (81)

disease in animal models as well as in patients (82;83). The effect of IL-10 maybe follow

a time-dependent fashion, since early increased levels of IL-10 leads to decreased disease

severity (Morel Lab, unpublished data).

Marginal Zone and Follicular B (B2) Cells

Conventional B2 cells are the major B-cell participants T-dependent immune

responses. It has been shown that in the SLE, B2 cells are responsible for the bulk of high

affinity autoantibodies(50;84). Apart from the production of autoantibodies, the exact

role of B2 cells in lupus is still not clear.

The splenic CD5- B cells are composed of two populations, follicular B (FoB) and









marginal zone B (MZB) cells, based on their phenotypes, functions and anatomical

locations. The FoB cells are IgMio, IgDhi, CD1d, CD211o, CD23hi, while MZB cells are

IgMhi, IgDio, CD1dhi, CD21hi, CD23io, and are CD9+ (as are Bl cells) (85;86). FoB cells

make up the majority of the splenic B-cell compartment, and are short lived circulating

cells with a highly diverse BCR repertoire. MZB cells are the major cell population in the

marginal zone (MZ), an anatomically distinctive region surrounding the follicles in the

spleen. In rodents, the marginal venous sinus additionally lies between the MZ and the

follicle (Figure 1-1). Most of the spleen blood flow exits the circulation through the

marginal sinus, and then the marginal zone. In addition to B cells, the MZ also contains

specialized macrophages that express the scavenger receptor SIGN-R1, called marginal

zone macrophages (MZM), stromal cells called reticular cells, and very few T cells(87-

89). The location ofMZB cells makes them the first-line to encounter blood-borne

pathogens, and they have evolved properties and functions to fit this position(90;91). MZ

B cells are long-lived and non-circulating cells, and respond to a wide spectrum of T-

dependent and T-independent antigens (88;92). After encountering with the cognate TD

antigens, MZ B cells migrate into the follicle toward the T-cell area, where they can

activate naive T cells more efficiently than FoB cells and quickly differentiate to plasma

cells(93;94). Furthermore, the MZB population has a biased BCR repertoire which

contains a large amount of auto-reactive clones(95-97). These properties also suggest that

they maybe contributors to autoimmunity. Expansion of the MZB population has been

found in NZB/W and estrogen-induced lupus mice (98-100). On the other hand,

sequestration of autoreactive B cells into the MZ area has also been proposed to be a

mechanism to prevent autoimmunity (101;102), and this process is inefficient in









autoimmune MRLipr mice (103).

The location and migration of B cells are controlled by many factors including

chemokines and integrins. As indicated by figure 1-1, re-circulating B cells express high

levels CXCR5 and are directed to the follicle B-cell area, where CXCL13, the CXCR5

ligand, is highly expressed(104;105). T cells expressing CCR7 are attracted to the T-cell

zone by CCL19/21(104). It has been shown that upon the stimulation of antigens, FoB

cells up-regulate CCR7 and migrate to the T-B border of the follicle(106). The situation

for MZ B cells is more complicated. MZB cells also express the B-cell zone chemokine

receptor CXCR5, which by itself can direct MZB to enter into follicles. However, MZB

cells are retained in the MZ by the integrin ligands ICAM-1 and VCAM-1 through their

surface expression of LFA-1 and alpha-4/beta-1(107;108). The sphingosine 1-phosphate

(S1P) receptors S1PI and possible S1P3 also participate in the retention of MZB cells

(109). S1P is produced by sphingosine kinase-mediated phosphorylation of sphingosine,

and present abundantly in peripheral blood (110). MZB cells express much higher levels

of S1PI and S1P3 than FoB cells. Upon antigen encounter, MZ B cells quickly down-

regulate the levels of S1PI and S1P3, and tip the balance toward follicular

migration(109). Marginal zone macrophages are also found to be involved in MZB cells

retention through the contact of its scavenger receptor MARCO to MZB cells (111). Loss

of MZM or blocking MARCO binding by antibodies allows the MZB cells to migrate to

the follicle (111). There is also evidence to show that the normal development and

maintenance of the marginal zone is dependent upon B-cell, especially MZB cells.

Following total B cells depletion by Ig-alpha deletion or CD70 over-expression, both

marginal zone macrophages and marginal metallophilic macrophages are lost and the MZ









area is missing (112).

Peripheral B-Cell Development

B cells that are newly emerged from bone marrow contain many self-reactive

clones and must undergo peripheral negative selections before the maturation(113).

Based on previous studies, B-cell peripheral maturation was divided into 2 or 3

transitional (T) stages according to classification by different surface markers(114;115).

In the 2-stage transitional system, the Tl stage is IgMhi, IgD-, CD211o, CD23- CD24+,

and the T2 stage is IgMhi, IgD+, CD21hi, CD23hi, CD24+. In contrast, the MZB cells are

IgMhi, IgD-, CD21hi, CD23-, CD24-, and the FoB cells are IgMio, IgD+, CD21med

CD23med, CD24- (116). Furthermore, T2 cells are circulating and contain the precursors

of both MZB and FoB cells(116) (Figure 1-2 A). In contrast, the 3-transitional stage

scheme proposed by others was based on the immature B-cell marker AA4.1(117). The

Tl cells are IgMhi, CD21-no, CD23-, AA4.1+. T2 cells are -IgMhi, CD21med, CD23+,

AA4.1+, and T3 cells are IgMhi, CD21hi, CD23+, AA4.1+(117). In a subsequent

refinement, the population which is IgMhi, CD21hi, CD23+, AA4.1+ was postulated to be

the precursors of MZB cells (118) (Figure 1-2 B).

Regardless of the classification scheme utilized, it has been shown that the Tl stage

contains large amounts of auto-reactive clones, most of which are removed during the

transition from Tl to T2 by negative selection and receptor editing (119). Thus the

accumulation of Tl cells, which could increase the workload of this check-point or a

defect of this check-point per se could allow the maturation of auto-reactive clones. In

addition to negative selection, other studies have indicated that specificity-based positive

selection also occurs (120-123). All of these suggest that check points during the B-cell









peripheral development are important for the formation of normal mature B-cell

repertoire.

BAFF and Receptors for BAFF

B-lymphocyte stimulator(BLys/BAFF/THANK/TNFSFl3B) is a TNF superfamily

member. TNF family members are type II transmembrane proteins that form homotrimers

as membrane-bond or soluble ligands to their cognate receptors(124). BAFF has been

identified as a trophic factor critical for B-cell development, growth and survival(125).

The major producers of BAFF are the peripheral blood mononuclear cells (126;127).

Macrophages, monocytes, dendritic cells(DC), follicular dendritic cells (FDC) and even

B cells can also make BAFF in the context of antigen encounter and activation(127-129).

In addition to promoting the peripheral B-cell maturation from the Tl to the T2 stage,

BAFF also enhances the survival of immature Tl, T2 cells as well as mature B cells(130-

132). BAFF and its receptors have been implicated in autoimmunity. High levels of

BAFF have been observed in patients with autoimmune diseases such as systemic lupus

erythematosis (SLE) and rheumatoid arthritis (RA)(133;134). In animal models, BAFF

deficient mice lack the mature B cells, while BAFF transgenic mice show elevated B-cell

numbers, especially T2 and MZB cells, and develop autoimmune disorders (135).

Furthermore, the lupus-prone models NZB/W Fl and MRLipr mice have high levels of

BAFF in the periphery(136). Experiments on B-cell receptor transgenic mice showed that

B cells compete with each other for the binding to BAFF, and that the self-reactive clones

have reduced competitiveness, which results in increased susceptibility to deletion(137).

In contrast, excess amounts of BAFF can rescue auto-reactive clones from this process

and allows them to enter the follicle and marginal zone (138).

Three receptors have been identified for BAFF and its homologue APRIL(a









proliferation-inducing ligand): BAFF receptor (BAFF-R, BR3), B-cell maturation antigen

(BCMA) and transmembrane activator and CAML interactor (TACI). The BAFF-R is the

only receptor exclusively for BAFF(132;139), while both BAFF and APRIL can bind

BCMA and TACI(136;140) (Figure 1-3). BAFF-R is predominantly expressed by all

peripheral B cells, but is down-regulated when the cells are activated and become

germinal center (GC) B cells(141;142). BCMA is expressed at high levels by plasma

cells, plasmablasts and germinal center B cells(141;143). Research has shown that

BCMA plays an important role in plasma cell survival rather than B-cell maturation(143).

TACI is expressed predominately by marginal zone and activated B cells, but not GC B

cells. Signals through TACI is essential for T-independent type II responses (144).

However, lack of TACI also resulted into increased B-cell survival, activation, and the

development of autoimmune disorders, suggesting that TACI has a role as a negative

regulator of T-dependent responses (144-146).

The BAFF/APRIL ligands and their receptors compose a delicate system that

regulates B-cell development, activation and homeostasis. Increased BAFF has been

implicated in autoimmunity, while abnormal expressions of the receptors could also tip

the balance of positive and negative signals and lead to the same outcome.

The NZB/W Derived Mouse Models for Lupus Prone and Resistance

The NZM2410 (NZM) is one of 27 inbred strains derived from an intercross

between the NZW and NZB strains(147). NZM2410 mice develop lupus nephritis

spontaneously, with 80% of the animals from both sexes affected by 6 months of age and

have the histological features that closely resemble those of human patients (148).

Analysis ofbackcrosses between NZM2410 and C57/BL6 (B6) identified 4 genomic

intervals containing lupus susceptible loci Slel~4 on NZM2410 chromosomes 1, 4, 7 and









17(149). Subsequently four congenic strains, B6.NZMSlel, -Sle2, -Sle3, and -Sle4 were

produced by the backcross, each carrying one of the corresponding NZM2410-derived

genomic intervals on the B6 genome (Figure 1-4) (149). Phenotypic analysis of these

congenic strains revealed each of the loci contribute to the lupus phenotypes. Slel leads

to the break of tolerance to nuclear antigens, and results in abnormal phenotypes in both

B and T cells(150-153). Sle2 leads to B cell hyperactivity and increased Bl

cells(154;155). Sle3 leads to decreased activated induced cell death in CD4+ T cells and

affects immunoglobulin heavy chain diversity (156-158). SLE pathogenesis can be fully

reconstituted by recombining the Slel-3 loci on the B6 genomic background (i.e.

B6.Slel/2/3) (159). The B6.Slel/2/3 (TC) mice display severe splenomegaly, full

penetrance of SLE and lupus nephritis at an early age (159). Another strain derived from

NZW and NZB, TAN, share the lupus susceptible loci Slel, Sle2, and Sle3 common to

both the NZM2410 and TC strains. However, TAN mice display a dominant resistance to

SLE (table 1-1). Although they develop the comparable levels of splenomegaly, TAN

mice produce less anti-nuclear antibodies, and do not develop lupus nephritis when

followed to 12 month of age. In contrast, aged TAN mice develop a high incidence of

marginal zone lymphoma at old age (Morel et al. unpublished data). In this study, TAN

mice were used as a lupus-resistant model as compared to NZM and TC mice.










VIarginal zone
CAM1+, VCAM14


MZ sinus


MZ B-cell


MZ macrophage


Figure 1-1 Diagram of white pulp and red pulp.





Figure 1-2 Peripheral B-cell development stages. (A) Two-transitional stage scheme. (B)







































Figure 1-3. BAFF/APRIL and their receptors. Soluble or membrane bonded BAFF and











NZW NZB


NZM/Aeg strains derivation


9 9
NZM2410 NZM TAN
lupus+++ lupus-


C57/BL6 (B6)
Normal
lupus -






B6.Slel B6.Sle2
B6. le3







B6.Slel/2/3 (TC)
lupus +++


Figure 1-4. NZM models of lupus prone and resistance mice






17

Table 1-1. Pathological phenotypes of mouse models
TAN NZM TC

Mortality 12mo 10% 85% 100%

Proliferative GN 0% 55% 72%

Anti-chromatin 39% 86% 88%

Anti-dsDNA 35% 85% 78%














CHAPTER 2
MATERIALS AND METHODS

Experimental Animals
TAN mice were maintained in a conventional colony (after several failed attempts

to re-derive this strain as specific pathogen free (SPF)). All other strains, NZM2410, TC,

B6 and B6.Ly5a (purchased from Jackson Lab, Bar Harbor, ME) were kept in SPF

housing as specified by the University of Florida Animal Care Services. Our studies have

shown that the housing conditions (conventional versus SPF) did not switch the lupus

susceptibility (data not shown). B6.Ly5a mice express a different allotype of CD45 on

their leukocytes than that of B6 mice (CD45.1 on B6.Ly5a versus CD45.2 on B6), which

can be distinguished by monoclonal antibodies. All animal protocols were approved by

the Institutional Animal Care and Use Committee (IACUC) at the University of Florida.

Immunofluorescence Staining

Fresh tissues were embedded in OCT, then snap-frozen and stored at -80oC. All

samples were cut at -20oC with a cryostat into sections of 6-7um in thickness. Sections

were fixed in cold acetone for 10 minutes, briefly air-dried and kept in -80oC until

staining. For staining, sections were first blocked with blocking buffer containing 10% rat

serum in PBS for 20 min, then stained with fluorochrome-conjugated monoclonal

antibodies for 30 min. Anti-mouse MOMAl-FITC was purchased from Serotec (UK),

SIGN-R1 (ER-TR9)-biotin was from BMA (Switzerland), while B220 (RA3-6B2)-APC,

IgM (160)-APC, CD1d-biotin, and anti-TNP-Biotin were purchased from BD

Pharmingen (San Diego, CA). Biotinylated antibodies were further detected with









streptavidin-Alexia 568 from Molecular Probes (Carlsbad, CA). Sections were finally

washed, mounted with Prolong Gold media from Molecular Probes and analyzed with a

Zeiss Axiom fluorescent microscope.

B-Cell Isolation and Cell Culture

Splenic CD43- naive B cells were purified with the B-cell isolation kit (Miltenyi

Biotech, Auburn, CA) according to the manufacture's instructions. Purified B cells were

counted with cell counter and adjusted to a concentration of 106/mi in COmplete

RPMI1640 containing 10% FCS, then cultured at 37oC, 5% CO2 with different

stimulation conditions. Peritoneal cells were pre-incubated for 3hr in the same conditions

to remove adherent macrophages. All cell culture were conducted using 6-, 12- or 24-

well tissue culture plate (Corning Life Sciences, Acton, MA). For B-cell stimulations,

cells were treated with goat anti mouse IgM F(ab)2 (Jackson Lab, Bar Harbor, ME) at 0, 1

and 10pg/ml or LPS (Sigma, St. Louis, MO) at lyg/ml.

Flow Cytometry

Single cell suspensions from spleen or peritoneal lavage were treated with

FcRgamma Blocker (anti-CD16/32, clone 2.4G2) in flow cytometry buffer (5% FCS in

PBS) for 20 min on ice. For the in vitro TNP-Ficoll binding assay, splenocytes

suspensions were incubate with TNP-ficoll (Biosearch, Novato CA) at different

concentrations for 30 minutes at 37oC. Samples were then stained for 20 min with

fluorochrome- or biotin- conjugated monoclonal antibodies against mouse CD1d, CD5

(53-7.3), CD11a (M17/4), CD19 (1D3), CD23 (B3B4), CD45.1 (A20), CD45.2 (104),

CD80 (16-10A), CD86 (GL1), IgM (II/41) anti-TNP (G235-2356) (all from BD

Pharmingen, San Diego, CA), and CD21 (7E9) (Purified from a hybridoma clone

provided by Dr. Boackle, University of Colorado, Denver, CO.). Since all of the strains









that carry Slel allele has mutation on extracellular domain of CD21, the commercially

available anti-CD21 clone 7G6 binds poorly to the mutated CD21, while clone 7E9 binds

well. Biotinylated antibodies were further detected with Strepavidin PerCP-Cy5.5. For

intracellular cytosine stains, cells were further fixed and permeabilized with

Cytofix/Cytoperm solution for 30 min on ice,. then stained with anti-L-6, or -IL-10

antibodies according to manufacturer's instructions. Samples were finally analyzed with

BD FACS Calibur machine, and at least 60,000 cells were counted.

Bone Marrow Transfers

Three days prior to transfer, recipient mice were started on drinking water

containing 40mg/L of the antibiotic Septra (Sulfamethoxazole and Trimethoprim, Hi-

Tech Pharmacal CO, Amityville, NY). One day before the experiment, recipient mice

were irradiated twice at 525 Rads, each dose separated by 4 hours. On the day of transfer,

donor mice were euthanized and bone marrow was flushed with Hank's Solution. Single

cell suspension was prepared and washed with cytotoxicity media (0.3% BSA, 0.025M

HEPES, lx Pen/Strep in RPMI1640), and adjust to a concentration of 5x10 cells/ml. To

remove T cells, the bone marrow cell suspension was incubated with 1:100 anti-Thyl,

anti-CD4 10pg/ml, anti-CD8 10pg/ml (Accurate Chemical, Westbury, NY) for 1 hour at

4oC. Cells were washed and incubated twice with 1:10 Guinea Pig complement (Accurate

Chemical, Westbury, NY) twice for 60 and 30 minutes respectively. Finally, the cells

were washed and adjusted to a concentration of 2x10 cells/ml, and a volume of 0.5ml

was injected into a tail vein intravenously.

After bone marrow transfer, recipient mice were continued on antibiotic drinking

water for three days, and maintained under SPF conditions.









Treatment of Mice with TI Antigens

Mice of 7~9 month old from different strains were injected i.p. with T-independent

antigen LPS or Ficoll. For LPS treatment, mice were injected intra-peritoneal with 100ug

LPS (Sigma, St. Louis, MO) for 3 hours. For TI-2 antigen ficoll treatment, mice were

injected intra-peritoneal with 30ug TNP-Ficoll (Biosearch, Novato CA) for 30 minutes.

Mice were sacrificed after the treatment and spleen samples were snap-frozen and stored

at -80oC for future Immunofluorescent assay.

Western Blotting Assay

Cultured cells were collected and lysed in RIPA buffer containing protease and

phosphatase inhibitor (Santa Cruz Biotechnology, Santa Cruz, CA) for about 15 minutes

on ice. Protein concentration was determined by Bio-Rad Protein Assay (Bio-Rad,

Hercules, CA). Equal amount of protein (10ug) was resolved on Bio-Rad 12.5% SDS-

PAGE gels by electrophoresis, and then transferred to PVDF membrane (Amersham,

Piscataway, NJ) by electro-blotting. Membranes were blocked overnight at 4oC with

blocking buffer (0.2% Tween 20, 5% non-fat dry milk in PBS) and then probed with

primary antibody for 1 hour at room temperature. Rabbit anti-mouse ERKl/2,

phosphorylated ERKl/2 (Cell Signaling, Danvers, MA) was used as primary antibody at

1:1000 dilution in blocking buffer. The goat anti-rabbit IgG HRP (Cell Signaling,

Dancers, MA) was used as second antibody at 1:2000. Finally, detection was performed

with the ECL plus system (Amersham, Piscataway, NJ) and exposed to X-ray film.

Statistics

All data were analyzed by ANOVA. Statistical significance was obtained when

P$0.05.













CHAPTER 3
RESULTS AND DISCUSSION: ABNORMAL MARGINAL ZONE AND MARGINAL
ZONE B CELLS ARE FOUND IN BOTH LUPUS-PRONE AND LUPUS RESISTANT
MICE

Diminished splenic marginal zones (MZ) have been found in the lupus-prone

NZM2410 and B6.Slel/2/3 (TC), while lupus resistant NZM.TAN (TAN) mice have

enlarged MZ (B.P. Croaker, unpublished observations). I hypothesized that these

phenotypes are due to the abnormal functions of marginal zone B cells in lupus-prone and

lupus-resistant mice. I also hypothesize that the different MZ phenotypes of these strains

contributes to their lupus susceptibilities.

Different Marginal Zone Phenotypes Between Lupus Lupus-Prone and Resistant
Resistant-Mice

Histological examination of the spleen revealed the age- dependent changes of the

marginal zone among the NZM, TC and TAN mice. From 5 or 6 months of age,

significantly reduced or absent splenic marginal zone areas and IgMhi MZB cells were

observed in both NZM and TC mice. In contrast, the lupus- resistant strain TAN showed

a markedly enlarged marginal zones with accumulation of IgMhi, CD5+ cells (Figure 3-1).

Flow cytometry analysis did not show a corresponding reduction or enlargement of MZB

population in the spleen of lupus-prone or resistant mice (Figure 3-2). This inconsistency

indicated the possibility of an altered location instead of the loss of MZB cells in the

lupus-prone mice. It also suggested that the B cells present in the MZ of TAN mice could

have different characteristics from normal MZB cells.

Further immunofluorescent studies using another MZB cell marker, CD1d,









revealed that the MZB cells in the lupus TC and NZM mice were in fact translocated

inside the follicles (Figure 3-3). Chemokine receptor CXCR5 is expressed on mature B

cells and is essential for directing B cells into the follicular B-cell area (52;161). Flow

cytometry studies revealed that lupus mice MZB cells expressed a higher CXCR5 levels

than that of B6, indicating thesuggesting a reason forof their translocation. The TAN

MZB cells, on the other hand, did not showed increased CXCR5 levels, but had a

significantly high percentage ofCD5 expression (Figure 3-4). This is consistent with

previous observation by histology showing that TAN MZ area was occupied by CD5+

cells. Thus the Bl- type MZB cells were unique for TANmice. Since CD5 is a negative

regulator of B-cell receptor signaling, the CD5+ MZB cells in TAN mice may have

different phenotypes from that of other strains.

Subsequently, the number of ER-TR9+ MZ macrophages (MZM) were was also

found to be reduced in the lupus mice but was normal in the TAN mice, while the Moma-

1 expressing metalophillic macrophages were intact in all strains (Figure3-5). MZMs has

have been suggested to interact with MZB cells directly and is are important for the

retention of MZB cells (111). These results indicate suggested that the loss of MZM may

at least partially responsible for the diminished marginal zone in lupus mice.

Interactions between integrin ligands VCAM-1, ICAM-1 and receptors LFA-1,

aalpha-4/pbeta-1 integrin on the cell surface are also critical for the retention of MZB

cells (107;108). So we studied the expression levels of these molecules on the MZB

cells and on the stroma cells in of the follicles. Flow cytometry analysis for the levels of

LFA-1 on TC MZB cells did not find significant changes (data not shown). Since it is

hard to isolate stromal cells, we used an ilmmunofluorescencet approach. Studies on the









frozen sections revealed a slightly lower VCAM-1 levels on the MZ area of TC spleen,

but not on that of TAN and B6 spleen (figure Figure 3-6). Since lowerLower VCAM-1

levels indicates a powerless binding for the integrin and a weaker retention factor, which

may tip the balance toward follicular migration. This suggests that the stroma cells in TC

MZ area may also contribute to the phenotype of MZB cell trans-location.

Reciprocal Bone Marrow Transfers Indicates MZB Translocation is Determined by
Multiple Factors

The defective MZ phenotype of lupus mice can be determinominated by either

stroma-derived factors, i.e. stromal cell produced cytokines and, integrin ligands, or by

bone marrow derived factors, i.e. MZB cell itself, or myeloid cells like MZMs. Since

stromal cells are radiation-resistant, and all bone marrow- derived cells are sensitive to

irradiation, we conduct reciprocal bone marrow transfers on irradiated B6 and TC mice to

study evaluate these possibilities. The B6 and TC recipient mice were lethally irradiated

and transferred with bone marrow from untreated TC or B6 mice. The rationale is if the

TC MZB cell phenotype was dominant by bone-marrow derived factors, the expected

results would be a normal MZ in TC mice that received B6 bone marrow (B6 4 TC), and

an intra-follicular MZ in B6 mice transferred with TC bone marrow (TC & B6). The

reverse results would be expected if stromal cells are solely responsible for the phenotype.

Three months after transfer, mice were sacrificed and spleen sections were

examined as mentioned before. Unexpectedly, both B6 recipients that received TC bone

marrow and TC recipients transferred with B6 bone marrow showed normal MZ (Figure

3-7).

We also look at the presence of ER-TR9+ marginal zone macrophages on bone

marrow transferred mice. None-the-less, the TC- derived bone marrow could not give rise









to a normal amount of marginal zone macrophages, while in the TC recipients transferred

with B6 bone marrow, the MZMs rim was present normally (Figure3-8). To verify the

origin of the MZB and MZM and rule out the possibility of recipient bone marrow cell

contamination, We we also did performed reciprocal bone marrow transfers between

B6.Ly5a and TC (allotype Ly5b) mice and determine the cell origin by antibodies that

discriminate the allotypic marker Ly5a and Ly5b. Results showed that the MZMs and

MZB cells observed are both donor bone marrow derived. And recipient mice have the

same manifestation on MZB cells and MZMs (data not shown). Furthermore, flow

cytometry analysis on 3 months after reciprocal bone marrow transfer revealed the

reconstitution of B-cell phenotypes and functions according to the derived by donor bone

marrow origin, i.e. the TC & B6 mice have abnormal B-cell subsets and B6 4 TC mice

showed similar phenotype as manipulated B6 (data showed and discussed ion Chapter

5). This suggested first firsthat the deficiency fect ofin MZM number was intrinsic tofor

the TC bone marrow, although its pathological significance is still not clear. Second, ,

these results indicate that the lack of MZMs was not the determining factor of MZB cells'

translocation into the follicles in TC mice, and vice versa, thus both the MZB -cells per se

and the stromal cells in the marginal zone are most likely to contributed to this phenotype.

Functional Studies of MZB Cells

MZ B cells are the major responders to T-independent antigens (12). To study their

functions in the lupus-prone and resistant strains, mice were challenged with the TI

antigens TNP-ficoll and LPS. The responses ofMZ B cells were assayed by

immunofluorescence and flow cytometry.

MZB Respond to TI-2 Antigen Ficoll.

Mice were injected i.p. with 30 pg TNP-Ficoll each, then sacrificed after 30









minutes. Frozen spleen sections were produced, and the uptake officoll by MZB cells

was revealed by using anti-TNP and anti-IgM antibodies. MZB cells that bind TNP-

Ficoll are double positive. Results showed less ficoll binding by TC MZB cells compared

to B6 and TAN MZB cells. And In addition, a small amount officoll-binding cells

present in the follicle of TC mice (Figure 3-9).

To rule out the possibility that the low binding of TC MZB cells to ficoll was due

to the limited access resulting from their translocation into follicles, in vitro studies were

conducted. Fresh splenic single cell suspensions were taken from mice without any

treatment, then incubated with various amounts of TNP-ficoll for 30 minutes. The

binding of ficoll was determined by flow cytometry with anti-TNP antibody and gated on

MZB cells. We confirmed that non-MZB splenic cells were negative for TNP-binding

(data not shown). The results showed that both TAN and TC mice MZ B cells had

different degree of impaired binding capability to the TI-2 antigen-Ficoll. The TAN MZB

cells had similar binding ability as B6 at low Ficoll concentration (<_5 pug/ml). At higher

concentrations, they seemed to be saturated and can not bind any more of ficoll as of B6

MZB. In contrast, TC MZB cells have severe defect of ficoll uptake even at lowest

concentration tested (Figure 3-10).

MZB Migration in Response to TI-1 Antigen LPS.

It has been shown that the MZB cells migrate into the follicles when they encounter

the TI-1 type antigen LPS (162). To determine MZB cell function in response to TI-1

antigen, mice at 7~9 month old were injected i.p. with 100ug LPS. After 3 hrs, the

spleens were sectioned and stained with MZB cell markers to reveal their location. The

results show that after 3 hrs of LPS treatment, the TAN MZB cells were still stayed

located in the MZ area, while MZB cells in TC and normal B6 mice were localized inside









the follicles (Figure 3-11). This suggests that the response to TI-1 type antigen for TAN

MZB cells is impaired in terms of migration in response to LPS. It should be noted that

most TC MZB cells were already inside the follicle before LPS exposure, and therefore

it cannot be determined whether or not they responded to LPS.

Discussion

Both the lupus-prone NZM, TC and resistant TAN mice have abnormal lymphoid

micro-structures. This is at least partially due to the fact that they all carry the lupus

susceptible locus Sle3 (156). We have also shown that lupus-prone mice accumulate

plasmablasts and plasma cells in the spleen at the expense of their normal migration to

the bone marrow (163). The studies here show that the phenotype of the MZ cells

correlate to the lupus susceptibility of the mouse strains. The lupus-prone strains MZN

and TC have missingmiss the marginal zone area, with MZB cells trans-localized inside

the follicles and lack of marginal zone macrophages. The lupus-resistant TAN mice, on

the other hand, have an enlarged marginal zone, with a MZ arrestednon-migrating MZB

cells having a large proportion of CD5 expression and normal marginal zone

macrophages.

The localization and migration of MZB cells is controlled by multiple factors and is

a balanced outcome (164). In this study, we conducted reciprocal bone marrow transfers

to dissect the reasons for the MZB cell miss-aberrant location in lupus prone mice. The

results showing normal of MZ recovery in both directions of the transfers could be

explained as the process was not long enough for the manifestation of MZ defect on the

experiment animals. Although it can not be totally excluded at this point, This this is,

although can not be totally excluded, not likely the case, since flow-cytometric analysis

of bone marrow transferred mice have foundshowed the reconstitution of donor









phenotypes, i.e. abnormal B-cell subsets and functions in TC+ B6 mice, which is were

identical reminiscent to manipulated adult TC mice (see Chapter 4), indicating the

development of lupus phenotype. Still further Additional transfers with longer procedure

time before sacrifice are to be conducted to verify this notion. The alternative explanation

is that both stroma cells and lymphocyte/myeloid cells are necessary but not sufficient to

cause the TC MZB-cell phenotype. Early studies have shown the interactions between

integrin receptors and ligands are important for the organization of follicular structures

(164;165). Specifically for the B cells, the integrins LFA-1 (CD11a) and a4plalpha-

4beta-1 expressed by B cells interact with their respective ligands ICAM-1 and VCAM-1

expressed by the stromal cells to direct B cells' entry to the spleen white pulp (165).

Marginal zone B cells express higher levels of LFA-1 and a4plalpha-4beta-1 than

follicular B cells to facilitate their entering to the MZ, and this interaction is also critical

for their retention in this area (107). Ablation of integrin binding by neutralizing

antibodies caused the releasing of marginal zoneMZ B cells from marginal zone (107).

We speculated the changes of integrin levels on either MZB or stromal cells may be

involved in the MZB- cell translocation in lupus mice. Till now noNo difference on LFA-

1 levels was found between MZB cells from TC and B6, while immunofluorescent study

revealed that in TC mice spleen, the MZ has a slightly may have powerless VCAM-1

levels than the surrounding area, indicating a reduced VCAM-1 expression by their MZ

stromal cells. In contrast, TAN and B6 MZ showed a the similarly higher VCAM-1 levels.

In addition, alterations of other integrin molecules e.g. a4p l alpha-4beta-1 and ICAM-1

are not excluded and will be assayed in the future. In short, this suggests that the less









diminished interactions between MZB cells and stromal cells may participate in the MZ

phenotype in lupus mice.

Flow cytometry studies have found increased levels of the chemokine receptor

CXCR5 on TC and NZM MZB cells, which may also contribute to this phenotype.

CXCR5 is the receptor for chemokine CXCL13 (BLC), and it is well known that FoB

cells that express CXCR5 enter follicles following the gradient of CXCL13 established

by the follicular stroma cells (166). MZB cells also express CXCR5, but other factors that

counter the CXCL13 chemoattractant skew the balance to their retention in the marginal

zone. One of these factors is are the integrins and their ligands just mentioned above. The

receptors for the lysophospholipid sphingosine-1 phosphate (S1P) are also shown

important in this process (109). S1P is found in abundant concentrations in the peripheral

blood (167). MZ B cells express higher levels of S1PI and S1P3 than the follicular B

cells, which prevent them from entering the follicles (109). Antigen exposure results to

the down regulation of S1P receptors, and treatment with FTY720, an inhibitor for S1P

receptor functions, can both abolish the S1P retention and allow the marginal zone B

cells to migrate following the CXCL13 gradient (109;168). In the mice lacking of both

CXCL13 and S1P1, the marginal zoneMZ B cells stayed in the marginal zone in spite of

antigen stimulation (109), suggesting the importance of balance between S1P and

CXCL13 chemoattractant. Thus in the case of MZB cells in lupus prone NZM and TC

mice, higher CXCR5 levels is likely to tip the balance toward the follicular migration.

However, in vitro chemotaxis assay did not show increased migration in response to

CXCL13 by MZB cell from lupus mice (data not shown). It is possible that the slightly

increased CXCR5 levels itselfis not significant enough to cause the increased migration









of MZB cells, and other factors discussed here may also contribute to the MZ phenotype.

This notion is consistent with the reciprocal bone marrow transfer results mentioned

above. In particular, the ligand CXCL13 may also play a role in this process, as higher

CXCL13 expression has been found in peripheral organs of lupus-prone NZB/W Fl mice,

which leads to the accumulation of attracted Bl cells in targeted organs (52;169;170). So

in the following follow-up studies, the follicular CXCL13 levels in the lupus prone and

resistance mice will be assayed. Furthermore, the involvement of S1P-mediated MZB

cell retention can not be excluded. It is not known that if the expression of S1P receptors

and/or the down-stream signaling are compromised in the lupus mice MZB cells. Due to

the lack of commercially available antibodies for S1P receptors, we still can not assay

their expression levels in vivo. The A chemotaxis assay on S1P will be conducted in

future studies. Furthermore, we still cannot exclude the role of activation in the function

and location of MZB cells, since TC mice with CD86 knocked out showed less disease

with little or no MZB translocation (Morel Lab, unpublished data).

MZMs is constitute another major cell population in the marginal zone (111).

MZMs express type A scavenger receptor MARCO and C-type lectin SIGN-R1 and are

important for capture and clearance of blood-borne pathogens (171;172). Early studies

have suggested that MZMs were involved in the retention MZB cells through the contact

of the surface receptor MARCO to MZB cells, and MARCO- blocking antibody

treatment leads to MZB MZB-cell migration (111). Our studies on manipulated and

reciprocal bone marrow transferred mice suggested a correlation between MZM defect

deficiency and lupus susceptibility. However, the bone marrow transfer studies also show

ed that the MZM defect is not the cause of MZB cell translocation in lupus mice. It is









possible that unlike the normal MZB cells in B6, lupus mice MZB cells (gradually) lose

their dependence on the contact with MARCO for their retention. Consistent with this

notion, the MARCO knockout on B6 mice resulted a diminished of MZB cell population

and MZ area (173), while in our lupus-prone NZM and TC mice, the MZB population is

not significantly changed in spite of lacking MZM (Figure 3-2). Reciprocal MZB cell

transfer between TC and B6 mice with MARCO blocking treatment may help to verify

this notion. Besides, the significance of MZM defect in lupus pathogenesis is still not

clear.

Recent studies using conditional knockout models have shown that B cells,

especially the MZB cells itself themself may also play a pivotal role in the development

and maintenance of normal marginal zone (112). Lymphotoxin expressed by B cells have

been found to be necessary for the differentiation and maturation of stromal cells, and

further inducing their production of chemokine in the white pulp (174-178). Depletion of

B cells results to thein defects of the marginal zone and the loss of marginal zone

macrophages (112). Thus in the case of NZM and TC mice, the missing MZ and ER-

TR9+ MZMs could be the consequence, rather than the cause of MZB MZB-cell

translocation.

MZB cells have been implicated in lupus autoimmunity (101;179). Specially,

increased number of MZB cells has been found in NZB/W Fl mice (98). Compared to

FoB cells, MZB cells respond to TD antigens more rapidly and are more potent T-cell

activators (94). Besides, early studies have shown that autoreactive B cells could be

positively selected into MZ, which is a postulated mechanism to prevent autoimmunity

(100;101;180). In consistent consistence with this notion, the theory of follicular









exclusion also states that auto-reactive B cells are excluded from follicles to prevent their

activation, proliferation and differentiation, and finally the excluded B cells would

undergo energy or apoptosis (181-184). In the case of lupus prone NZM and TC mice,

although we did not observed higher number of MZB cells and higher MZB MZB-cell

activation indicated by CD86 levels (data not shown), their intra-follicular localization

may already give them a privilege to participate in follicular responses, which does not

occur for MZB cells than the counterpart in TAN and B6 mice, unless they are

specifically activated by their antigen or by LPS. In addition, the lupus prone NZM and

TC mice have activated auto-reactive T-cell clones and less regulatory T cells ((185) and

unpublished data), which may further facilitate the trans-located MZB cells to initiate

autoimmune response in the follicles.

On the other hand, the CD5+ MZB cells in TAN mice may also contribute to their

resistant phenotype. CD5 is a negative regulator for BCR signaling (29;36;186). The

expression of CD5 thus can increase the activation threshold of TAN MZB cells, leading

to their poor responsiveness to TI antigens and the impaired ability to participate TD

responses. The reason for the CD5 expression on TAN MZB cells is still not clear.

Besides, only TAN mice develop high instance of marginal zone lymphoma at 12 month

old (Morel et al, unpublished), and the tumor cells are all CD5+, indicating the MZB

cells in TAN have distinct properties. To directly test the role of MZB cells in the lupus

susceptibility of these strains, we planed to conduct reciprocal MZB cell transfer and in

vitro characterization on purified MZB cells. However, our attempt to isolate MZB

population was unsuccessful due to the lack of high-speed cell sorting facility.









The functions of marginal zone B cells in lupus mice were was also defective in

terms of TI-2 antigen up-take. Together with the lack of MZMs, this indicates that the

marginal zone of lupus mice is incompetent to react against blood bourn pathogens. It is

possible that the ineffective clearance of blood-borne pathogens increase the chance of

inflammatory damage of peripheral organs and the release of auto-antigens. The

components of pathogen organelles may also activate multiple B-cell clones and elicit

persistent adaptive responses, increasing the risk of auto-reactive cross-reaction and

bystander activation. Further investigations are needed to verify this hypothesis.

In short, this study shows that the marginal zone and MZM phenotypes correlate to

the lupus susceptibility but are separate from each other. And the defect on in lupus prone

mice is due to the translocation of MZB cells into the follicle, results resulting from

defective of itselfMZB cells and the stroma cells.



































Figure 3-1. Reduced MZ in TC and NZM versus enlarged MZ in TAN mice.
Representative slides from 3 mice of each strain at 7~9 month old. Spleen
sections were stained with FITC conjugated Momal (green), CD5 PE (red)
and APC-IgM (blue). MOMAMoma-1+ metallophillic macrophages mark the
boundary of the follicle (A) B6. (B) TC. (C) TAN. (D) NZM. B6 follicles
have a normal marginal zone. MZB cells are IgM high, CD5- and are located
outside the green ring of Moma-1 stain. The CD5 high T-cell zone and IgM+ B
-
cell zone are well defined by the stain. (B) TC and (D) NZM mice showed
reduced to absent marginal zone, and T-cell zone and B-cell zone are mixed
together and poorly defined. (C) TAN mice have enlarged MZ area, and the
cells in MZ express both IgM and CD5 (purple). Arrows indicate MZ area.
(100x)










55-
*
50-
a 45.
Til 40.
35. *

25- *
20. *
*
o 15. e



TAN NZM TC B6
strain


Figure3-2 Splenic MZB populations determined by flow cytometry. MZB cells were
gated as B220+, CD21hi, CD231ot-. No significant reduction (P>0.05) ofMZB
was found between, TAN, TC and NZM compared to normal B6 mice.



















Momal
CD1d
Ig M














Figure 3-3. Intrafollicular location of MZB cells (IgM+, CD1d+) in the TC and NZM.
Representative slides from 3 mice of each strain at 7~9 month old. (A) B6
spleen follicles with normal marginal zone. IgM+, CDID+ MZB cells (purple
color) localized outside the Moma-1 (green) positive rim. (C) TAN MZB cells
localized on the both sides of Momal rim with increased cell layers. (100x)
























-IIII


-Till
TAN NZM TC B6
strain


IVIB CXCRS+


S CD5+1VZB


3.*
.*
..

*


..
* *
*


,*,
W


.* *._
,


.*,
**


Figure 3-4. MZB cell phenotypes. Fresh splenic cells analyzed with by flow cytometryer.
MZB cells are defined as IgM+, CD1d high. (A) MZB cells from both NZM
and TC have express significantly higher levels of chemokine receptor
CXCR5. (B) Significantly high proportion ofMZB cells in TAN express Bl
B-cell marker CD5. P<0.05, ** P<0.01, *** P<0.001.



















(100x)
Momal FITC
SIGN-R1















Figure 3-5. Greatly reduced MZMs in the TC and NZM mice. Representative spleen
sections are from 3 mice of each strain. Mice were 7~9 month old. (A) B6
spleen follicles have a continuous layer of MZMs (ER-TR9+, red) distributed
outside the Moma-1 rim (green). (B) TC had much less MZMs. (C) TAN
follicles with a continuous MZM layer similar to B6. (D) NZM mice like TC,
has have a defective MZM layer. MZM, marginal zone macrophage. (100x)





















































Figure 3-6. Reduced VCAM-1 levels on TC marginal zone. Representative spleen
sections are from 3 mice of each strain. Mice were 7~10 month old.
Representative spleen sections were stained with anti-mouse Moma-1 (green)
and VCAM-1 (red). Arrows indicate the marginal zone area. (A) (B), B6. (C)
(D), TC. (E)(F) TAN. TC spleen section show reduced VCAM-1 around
marginal zone area. (100x)



















Momal
CD1d
IgM


















Figure 3-7. Marginal zone of TC and B6 reciprocal bone marrow transferred mice.
Representative spleen sections are from 4 mice of each group. Mice about 3
month s old were lethally irradiated and conducted transferred with bone
marrow transferfrom the opposite strain. Spleen sections were taken 3 months
after transfer. Both B6 4 TC and TC & B6 bone marrow chimeras show
normal marginal zone. (A) (B) TC recipients received B6 bone marrow. (C)
(D) B6 recipients received TC bone marrow. B6 recipients received TC bone
marrow. All representative slides show MZ comparable to that of
manipulated B6 spleen. (100x)




















(100x)
Momal FITC
SIGN-R1















Figure 3-8. Defect of marginal zone macrophages in recipients receiving TC bone
marrow. Representative spleen sections are from 4 mice of each group. (A) (B)
TC recipients transferred with B6 bone marrow. The ER-TR9+ MZM layer
was normal as in B6 mice. (C) (D) B6 recipients transferred with TC bone
marrow. The MZM layer was defective as in manipulated TC mice. (100x)









































Figure 3-9. TNP-Ficoll up-take by splenic marginal zoneMZ B cells. Spleen sections
were take produced 30minutes after TNP-Ficoll i.p. injection. TNP-Ficoll was
exposed by anti-TNP (red). Representative spleen sections are from 3 mice of
each strain. (A) B6 had an intense TNP+, IgM+ (purple) MZB cell layer
surrounding Momal+ circle (B) TC MZ shows much less Ficoll binding MZB
cells in MZ. Also a few TNP+, IgM+ cells localized inside the follicles (red
arrows). (C) TAN MZ also showed good TNP-ficoll binding in the MZ, but
the presence of TNP negative MZB cells indicates less bind than in B6.
(100x).

























0

/ / / /


A

45-
40-
35-
- 30-
LI..
2 25-
+
1 20-
1- 1 5-
10-
5
-


-m- TAN
-A- TC
-*- B 6


- TAN
- TC
- B6


1101


1110111
anti-TNP Blot SAvPeoP Cy5 5

E MZB gated TNP-Flcoll binding


42-

28-


anti-TNP Blot SAv PerCP Cy5 5


anti-TNP Blot SAv PerCP Cy5 5


MZB TNP MFI


Incubation dosage









Figure 3-10 In vitro TNP-Ficoll binding capability of MZ B cells. Fresh isolated
splenocytes were incubated with TNP-Ficoll at different concentration for 30
minutes. The binding of TNP-Ficoll was assayed by flow cytometry with anti-
TNP monoclonal antibody. Cells were gated on MZB markers (IgM+, CD1d
high). (A) Impaired TNP-ficoll up-take by TC and TAN MZB cells as
compared to B6. Representative histograms ofTNP-ficoll binding by MZB
cells from each strain shown on (B) Medium only. (C) TNP-Ficoll 10pg/ml.
(D) TNP-Ficoll 20pg/ml. (E) TNP-Ficoll 40 pg/ml. P<0.05, ** P<0.01, ***
P<0.001.


Figure 3-11 TAN MZB cells do not migrate after LPS treatment. Representative spleen
sections are from 3 mice of each strain. Mice were ~7 month old. Mice were
injected with 100 pug LPS i.p. for 3 hours. Spleen sections were taken and
location of MZB cells (IgM+, CD1d high, purple) were assayed. (A) B6 and (B)
TC. MZB cells are trans-located into follicle demarcated by green Moma-1
circle. (C) TAN. MZB cells do not migrate. (100x).














CHAPTER 4
RESULTS AND DISCUSSION: ROLE OF PERITONEAL CAVITY B CELLS IN
LUPUS SUSCEPTBLITY

Both lupus-prone TC, NZM and resistant TAN mice have increased total peritoneal

cavity (PerC) cells as well as Bla B-cell population. Decreased numbers of B2 cells are

also found in NZM and TAN, but not TC mice (Figure 4-1). This increase in PerC Bla

cell corresponds to the expression of Sle2, which is present in TAN, NZM, and TC

(155;187). No difference was found in expression levels of activation markers, e.g. B7,

CD69, CD40 on PerC Bl and B2 cells between these strains.

Activation, Proliferation and Apoptosis

Both the lupus-prone and resistant strains have increased Bl cells. I speculate the

Bl cells in lupus prone mice are different from their counterpart in lupus resistant mice,

and they both contribute to the strain phenotypes. It is not clear if the Bl cells in lupus

mice are more prone to activate and undergo differentiation than that of TAN and B6

mice upon stimulation. If this is true, it means the Bl cells may directly participate in the

pathogenesis as effectors. Furthermore, the increased cell number in both strains can

result from the enhanced proliferation and/or decreased apoptosis. To test these

possibilities, experiments to assay the Bl cell activation, proliferation and apoptosis

properties were conducted on these strains.

In Vivo Spontaneous Proliferation:

Adult mice from each strain were injected with 1mg BrdUi.p. After 4 days, the

spontaneous proliferation of B cells indicated by BrdU incorporation was assayed by









flow cytometry. Results showed Bl cells from lupus-prone NZM and TC mice had more

proliferation, while only B2 cells from TC mice had slightly higher proliferation (Figure

4-2).

In Vitro B-cell Apoptosis

Whole peritoneal cavity lavage cells from TAN, TC and B6 mice were incubated

for 2 hrs at 370C to remove macrophages, then cultured with medium only, anti-IgM (10

pg/ml) or LPS (1 pg/ml) for 16 hrs. Early apoptosis was measured by flow cytometry to

detect cleaved caspase-3. Results showed that the TAN mice Bl cells had significantly

lower apoptosis rate, while no difference was found on TC Bl cells (Figure 4-3). On the

PerC B2 cells, only TAN mice showed lower apoptosis when treated with LPS, and the

TC did not show a difference with B6 mice (Figure 4-4)

Peritoneal Cavity B-Cell Activation

PerC cells were cultured as mentioned above for 48 hrs. B-cell activation was

evaluated by the expression of CD80 and CD86 with flow cytometry. Results showed

that TAN Bl cells showed significantly less activation capability in all three conditions.

The same levels of activation was observed between TC and B6 Bla cells, except with

LPS stimulation, which resulted in a significantly lower activation in TC Bla cells

(Figures 4-5 and 4-6).

Cytokine Production

Peritoneal Bl cells are the major source of B-cell derived IL-10 (55), and also

produce 1-6 (188). Both cytokines are important in regulating immune responses (189).

In this study, we tested the production of IL-10 and IL-6 by PerC B cells in vitro. PerC

cell suspensions were cultured for 72 hrs, and the intra-cellular cytosine levels were

assayed by flow cytometry. Results showed that TAN Bl cells produce significantly less









IL-10 and IL-6 than either B6 and TC mice, while TC Bl cells make significantly more

IL-6 when stimulated with anti-IgM (Figures 4-7, 4-8). Interestingly, Bla cells from the

lupus prone TC mice do not produce more IL-10 on a per cell basis, but because TC mice

have large amounts of Bl cells accumulated in the peritoneal cavity, the overall effect is

an elevated IL-10 levels. This is consistent with the fact that lupus is associated with

higher levels of circulating IL-10 (190).

Discussion

Increased levels of Bl cells has been observed in human autoimmune patient as

well as some lupus mouse models(14;45-47). The role of Bl cells in the lupus

pathogenesis has not been fully understood. In the NZM derived models, the lupus-prone

NZM, TC and resistant TAN mice have increased peritoneal Bl cells. This is at least

partially because they all carry the entire lupus susceptible locus Sle2, which leads to an

age-dependent enlargement of the Bl compartment (155). Our studies here show that the

peritoneal Bl cells from lupus prone and resistant mice have different functions and

properties.

Bl cells from lupus prone NZM and TC mice show significantly higher rate of

spontaneous proliferation than that of TAN and B6 mice, indicating an active cell cycle

progression. Normal Bl cells do not actively cycle in vivo (191). The increased Bl cell

proliferation in NZM and TC mice reflect the dominant functions of lupus susceptible

locus Sle2 carried in these strains, which have been found to cause elevated Bl cell

proliferation (155). Interestingly, although TAN mice also carry Sle2, their Bl cells do

not show the same high proliferation as that of lupus mice, suggesting other genes in

TAN genome may suppress this function of Sle2 locus, and this correlates with the lupus

susceptibility. The exact reasons for high Bl cell proliferation caused by Sle2 is still









unknown (155). As B6 mice carrying Sle2 have heightened B cell responsiveness to in

vitro stimuli and to in vivo antigenic challenge (154), the Bl cells from NZM and TC

mice may have lower threshold in response to antigen exposure in the peritoneum, or on

the other hand, they may up-regulate cell-cycle progression genes without stimulation.

This hypothesis is to be tested with purified Bl cells in future studies.

Both Bl and B2 cells from TAN mice peritoneum showed higher resistance to

activation induced cell death when stimulated in vitro. While the lupus-prone TC mice

Bl and B2 cells showed similar apoptosis rate as that of normal B6. The Sle2 locus also

promote the Bl cells survival when carried by B6 genome (155) This result suggests that

on the Peritoneal Bl cells of TC mice, the proliferation-promoting function of Sle2 locus

is dominant while its anti-apoptotic effect is not significant. The reason for this

inconsistency is not clear, probably due to the interaction with other lupus loci. On the

other hand, these data also indicated that the mechanism of Bl cells accumulation in the

peritoneal cavity is different among these strains: with less apoptosis more survival in

TAN and high proliferation rate in the lupus mice.

The responsiveness of TC peritoneal B cells to the stimulations could be the

indication of their roles and functions in lupus pathogenesis. If they actively participate in

the autoimmunity, the heightened activation response to stimulations is expected, and

vice versa. The in vitro stimulation shows compared to that of B6, both Bl and B2 cells

in the TC peritoneal cavity do not have significantly different activation in response to

BCR cross-linking and to LPS. On the other hand, peritoneal B cells from TAN mice

express much lower co-stimulatory molecules regardless of these stimulations. These

results indicate peritoneal Bl cells in lupus TC mice may not contribute to the lupus









pathogenesis through more heightened activation to antigen. And the low responsiveness

and low co-stimulatory molecule expression by TAN Bl cells may contribute to the lupus

resistant phenotypes. The approach to purify Bl cell is under investigation and peritoneal

Bl cell transfer will be conducted to verify this notion.

Peritoneal Bl cells is the major source of B-cell derived IL-10 (55), and also

produce a lot of 1-6 (188), indicating their regulatory role in immune response. In vitro

stimulation showed that TAN Bl cells produce much lower amount of both IL-10 and IL-

6 regardless of stimulations. TC Bl cells produce similar amount of IL-10 as that of B6

Bl cell at a per cell level, and they produce more IL-6 after BCR cross-linking. Since TC

mice have much more Bl cells than B6 mice, the overall effect will be a lot more total

IL-10 and IL-6 production by PerC Bl cells. IL-10 is a Th2 cytosine that can promote B

cell differentiation, proliferation, and antibody production (77). It has been shown to be

involved in lupus pathogenesis (77). Higher levels of circulating IL-10 correlate with

lupus development (190). Thus, this result suggests that Bl cells participate in lupus

pathogenesis by producing 1-10.

Furthermore, IL-6 is also an important cytosine that regulate immune response. IL-

6 promotes B-cell survival and strongly induce differentiation of B-cell to plasma cell

(56). In human SLE patients, elevated 1-6 levels correlate with lupus activity and kidney

pathology (69-72). Besides, IL-6 also blocks the suppression effect of regulatory T cells

(192;193), which will exacerbate status of already low Treg in lupus.

Interestingly, the BCR crosslinking suggesting that upon antigen encounter, TC

self-reactive Bl cells will make much more IL-6. In general, the elevated Bl cells in TC

will results in more IL-6 entering the circulation and promote the disease development.







50












Ovrltee tde ugetta h ertna lcel nlps rn Z n

TC' mic are difrn rmta flpsrsstn A n 6mc.Teehne

prlfrto eut nteacuuaino lclsi upsmc.AdterB el

contibut to luu pahgeei bytems rdcto f16ad1-0 clsi


actiatio an cytkin proucton

A B
100 ** *** p
45 80*
I ~ ~ ~ ~ 7 **,e 0

25 egaL t oo 0
























1@


strain


Figure 4-2 In vivo proliferation of peritoneal cavity B cells. Mice were injected with 1mg
BrdU i.p. and proliferation indicated by BrdU incorporation was assayed 4
days later by flow cytometry with anti-BrdU antibody. (A) Bl cells in the
lupus TC and NZM PerC showed higher spontaneous proliferation than TAN
and B6 mice. (B) B2 cells in TC PerC showed higher proliferation rate. (C)
Representative flow cytometry plot of BrdU incorporation. Bar graph shows
mean + SD P<0.05, ** P<0.01.


B1 gM Casp3


B1 LPSCasp3


81 MrDCasp3


strain sri


Figure 4-3 PerC Bl cells apoptosis. Cells from peritoneal ravages were collected and
cultured with (A) Medium only, (B) 10 pg/ml anti-IgM, (C) 1 pg/ml LPS.
Intracellular activated Caspase-3 was assayed by flow cytometry. TAN Bl
cells showed lower apoptosis than TC and B6 in all 3 conditions. Bar graph
shows mean + SD. ** P<0.01.


TAN NZM C147 B6
strain





















TAN TC B6
strain


B2 LPS Casp3


B2 MIED Casp3


B2 gIgMCasp3


TAN


stain


Figure 4-4 PerC B2 cells apoptosis. (A) Medium only. (B) Stimulated with anti-IgM 10


MIED-B1 CD86


MIED-B1 CD80


TAN


TAN


IC


ICI


Figure 4-5 Cells from peritoneal lavage were collected and cultured for 48 hours in


B6
















A IgM
80
70
60
50
. 40
30


01
TAN


10-B2 CD86


B LPS-B1 CD86
70-
60-
50-
40-
30-


-1
TAN TC
strain


TC


LPS-B1 CD80


LPS-B2 CD86


TAN


strain


strain


Figure 4-6 Peritoneal B-cell activation after 48 hrs of stimulation. (A) CD86 levels of
PerC B2 cells stimulated with anti-IgM at 10 pg/ml. (B) (C) PerC Bl cells
stimulated with LPS at 1 pg/ml. (D) B2 cells stimulated with LPS 1 pg/ml.
Only stimulation conditions showing significant difference are shown. Both
Bl and B2 cells in the TAN PerC had lower activation. Bar graph shows
mean+ SD. P<0.05, ** P<0.01, *** P<0.001.


TA

























I I I


TII-"


Ill


B1 IgM


35-


+ 25
a
3 20
-
15
m 14


*
e

*
4
*


+
o
1
-
10-
m


e

**


*

*


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e
-
*


D PeCBgae


0



+
o 30.
2 25
20.
15
0
10.
5*


7
e
*


*
*
7
**g
*


**
*
**
** *
go


01


Figure 4-7. Intracellular IL-10 after 48 hrs of culture. Cells from peritoneal ravages were
cultured for 48 hrs with (A) medium only. (B) anti-IgM 10 pg/ml. (C). LPS 1
pg/ml. (D) Representative plot of flow cytometry analysis for intracellular 1-
10 levels. Cells were gated on Bla B-cell population. TAN PerC Bla cells
produce significantly less IL-10 than TC and B6. P<0.05, ** P<0.01, ***
P<0.001.


B1 medium


B1 LPS








55




A MED-B1 B igu10-si
20* *
..
15* *** +
20 *
so. .* .
10
5

TAN TC B6
Ill
TAN TC B6
strain strain


C D | PerC B1 IL-6
LPS-B1
10
40
103
30

20 e 102 __r"

10 101 R5

I I I
TAN TC B6 10"
strain 1 10 1& 1 1
IL-6 PE



Figure 4-8. Intracellular IL-6 after 48 hrs of culture. Cells from peritoneal lavage were
cultured for 48 hrs with (A) medium only, (B) 10 pg/ml anti-IgM. (C) 1 pg/ml
LPS. (D) Representative plot of flow cytometry analysis for intracellular IL-6
levels. Cells were gated on Bla B-cell population. TAN PerC Bl cells make
significantly less IL-6 in all 3 conditions, while TC Bl cells produce more IL-
6 when treated with anti-IgM. P<0.05, ** P<0.01, *** P<0.001.













CHAPTER 5
RESULTS AND DISCUSSION: SPLEEN B CELLS IN THE LUPUS -PRONE AND
RESISTANT MICE

The hypothesis is that lupus prone mice have different splenic B cell functions

and/or development stages and these differences reflect their roles in the pathogenesis.

Splenic B1 and B2 Cells
Flow cytometry studies on adult spleen showed increased Bl cells in both TAN and

NZM, while proportionally less B2 cells were found in NZM, TC and TAN mice (Figure

5-1). Activation marker levels measurement found no significant differences of B7-1 and

B7-2 expression on either TAN or lupus mice B-cell (data not shown). TC B2 cells

showed however an increased of early activation marker CD69 and NZM B2 cells

showed higher CD40 expression. In addition B-cells from the 3 strains express reduced

levels of IgM on average as compared to B6, indicating that that these B cells have

encountered antigens, presumably self-antigen (Figure 5-2).

Newly formed B cells that are just released from bone marrow undergo a series of

development stages in the periphery (113). Based on CD21 and CD23 expression, the B-

cell population in the spleen has been divided into 4 sub-groups: Tl (Transitional 1), T2

(Transitional 2), follicular (FoB) and marginal zone B (MZB) cells(116) (Figure 5-3).

Flow cytometry revealed that NZM, TC and TAN mice have accumulated Tl and

less T2 and FoB cells than B6, suggesting a developmental arrest on Tl to T2 stage

(Figure 5-4). No difference was observed for marginal zone B cells, as mentioned earlier.

B-cell development can be determined by bone marrow derived cells, including the









B cells themselves, or by non-bone marrow derived stromal cells. To identify whether

abnormal B cell development in TC mice was determined by bone marrow derived cells,

we performed reciprocal bone-marrow transfers between B6 and TC mice. The results of

this experiment showed that the abnormal B cell subset distribution can be completely

accounted for by TC bone marrow (Figure5-5).

Splenic Naive B-Cell Functional Properties

To study B-cell functional properties without the confounding factor of differential

sub-population distribution, we analyzed the response of naive CD43- B cells to

stimulation. Splenic naive CD43- B cells were isolated with a B-cell isolation kit from

Miltenyi Biotech. The output was >96% pure. Then purified B-cells were cultured with

medium only, anti-IgM (low dose: 1 pg/ml or high dose: 10 pg/ml), or LPS (1 pg/ml).

B-Cell Proliferation after Stimulation

During the 72 hrs of culture in medium only, B cells from TAN and NZM, TC

mice showed a higher spontaneous proliferation as compared to B6 (Figure 5-6). When

stimulated with low dose of anti-IgM, B cells in lupus-prone mice showed an increased

proliferation index but TAN had a decreased proliferation index. In the situation of high

dose of anti-IgM stimulation, TAN mice B cells had a similar proliferation index as B6,

while TC showed an even higher proliferation. Interestingly, no difference was observed

with LPS treated B cells from either strain.

Activation-Induced Cell Death

To assess activation-induced cell death in B cells, CD43- B cells were treated as

mentioned before for 16 hrs, and intracellular cleaved caspase-3 levels was assayed by

flow cytometry. The results showed significantly less apoptosis in lupus-prone NZM and

TC B-cells when treated with 10pg/ml anti-IgM. No differences were observed in B-cell









treated with medium only, low dose of anti-IgM and LPS (Figure 5-7).

B-Cell Activation and Differentiation After In Vitro Stimulation.

To test the activation and differentiation of cultured B cells, cells were treated as

mentioned before for 48 hrs, and the expression of surface activation/differentiation

markers was assayed by flow cytometry as the read-out. The results showed that when

left in the medium only, all TAN, NZM and TC mice showed signiHeant higher

differentiation into germinal center (GC) B cells as shown by the high levels of GL-7

expression. Besides, a higher percentage of CD19hi population also present in the TAN

and TC B cells (Figure 5-8). Since GL-7 is a germinal center marker, and activated B

cells up-regulate CD19(194-196), a BCR co-receptor, these results suggested a higher

spontaneous B-cell activation and differentiation among these strains. No difference was

observed for the expression of early activation marker CD69 and co-stimulatory molecule

B7-2 (CD86).

When treated with low dose of anti-IgM at 1pg/ml, the TAN B cells showed

decreased CD86 and GL-7 levels. Expression of CD80 and CD19 were not signiHeantly

different (Figure-5-9).

In the situation of high dose of anti-IgM treatment (10pg/ml), the TAN B cells still

showed signiHeantly lower levels of CD86, although their GL-7 levels was now similar

to B6, and their CD19hi B-cells were signiHeantly less than B6. On the other hand, the B

cells from lupus prone mice stimulated with high doses of IgM showed high levels of

GL-7 and CD19 up-regulation (Figure 5-10). In contrast to these differences, Western

blot assay shows that both TAN and TC B cells have similar elevated ERK

phosphorylation compared to B6 B cells (Figure 5-11).

For the B cells stimulated with LPS, no signiHeant difference of









activation/differentiation were found between the strains (data not shown), indicating that

the differences in activation and differentiation reported above were specific for BCR

stimulation.

Discussion

Although both TAN, NZM and TC carry the three lupus susceptible loci, the

increased splenic Bl cells was not observed in TC mice, indicating the genes)

responsible for high SP Bl cells map outside the three Sle loci. In addition, the levels of

activation markers on splenic Bl cells from TAN and NZM were not different,

suggesting that splenic Bl cell is not necessary for the development of lupus, either by

their number of activation status.

Both the lupus-prone and resistant mice have abnormal splenic B cell development,

displayed by the accumulated Tl cells as well as decreased T2 and follicular B cells,

suggesting a development arrest between the Tl and T2 stages. Reciprocal bone marrow

transfer suggests that this defect can be completely accounted for by the bone marrow i.e.

either B cell itself and/or interaction between B and T/myeloid cells. The immature B

cells that just emerged from bone marrow contain autoreactive clones and must undergo

maturation in the periphery, during which these clones are removed, and checkpoints of

negative selection during Tl and T2 stages has been suggested (113;119). Thus the

accumulation of Tl cells may overload this checkpoint and increase the possibility of

autoreactive clones maturation in periphery. Besides, recent studies have shown that

transitional B cells can present antigens to CD4+ T cells and activate T cells if exogenous

costimulation is provided (197). On the other hand, interactions with activated T cells can

also protect immature B cells from negative selection (197). Since the lupus prone NZM

and TC, but not the resistant TAN mice, have hyper-activated T cells (unpublished), the









accumulated immature B cells in these strains thus may have different outcome, and play

an important role in disease development.

An age-dependent splenomegaly appears in TAN, NZM and TC mice. Thus

although they have a lower B2 proportion of splenic lymphocytes, the actual B2 cell

number is still higher. Consistent with this, naive B cells from all the strains show a

significantly higher spontaneous proliferation in vitro. Besides, both strains also have

higher proportion of B-cells expressing GL7 and up-regulating CD19 without stimulation.

GL7 is an activation marker that is expressed only on germinal center B cells in the

periphery (198). CD19 forms complex with CD21 and CD81 and is an essential co-

receptor for BCR that functions to enhance BCR signaling (199). The cytoplasmic

domain of CD19 recruits vav, PI-3 kinase and Src-family kinase Lyn after BCR ligation

and activates down-stream signaling (195;200). Over-expression of CD19 leads to B cell

hyper-activation and development of autoimmunity (201). These results indicate a higher

spontaneous B-cell activation and differentiation in these strains.

Further studies were conducted using different dosage of anti-IgM F(ab)2 to mimic

different strengths of BCR ligation in vivo. Results showed that B cells from lupus-prone

and resistant mice have different activation threshold. The TAN B cells responded poorly

to low dose anti-IgM stimulation, even less than the medium only condition, while at this

low levels, the lupus TC and NZM B cells showed significant activation and

differentiation. At the high dose of anti-IgM stimulation, still, the lupus mice B cells

exhibited higher proliferation and activation potential, while TAN B cells were similar as

the B6 B cells. Especially TAN B cells did not show a significant up-regulation of CD19

upon stimulation. Furthermore, the lupus B cells have less activation induced cell death.









One interesting observation is that both TAN and TC B cells have a similarly elevated

ERK phosphorylation upon low and high dose anti-IgM, suggesting the effects of the Sle

loci they share. In fact, hyper-activation of ras-ERK has been mapped to Slelab sub-

locus recently (202). This also indicate that signaling differences between TAN and TC B

cell may exist downstream of ERK. Finally, flow cytometry study showed that both TAN

and lupus mice B cells have similar levels of surface IgM, which is significantly lower

than that of B6, thus the amount of surface IgM did not account for the different signaling

phenomenon. In short, these results indicate that compared to normal B cells, lupus mice

B-cells have lower threshold on BCR signaling pathway, while TAN B cell has elevated

threshold, and their TLR4 signaling pathway is not changed.

It has been show that B cells are indispensable to prime T cells and to initiate lupus

pathogenesis (1;7). The B-cell phenotypes and BCR signaling properties thus may

contribute to the phenotypes of the strains. Both TAN and lupus mice B cells have high

spontaneous activation, proliferation and activation in vitro. Accordingly, all these mice

develop an age-dependent anti-chromatin and anti-dsDNA IgM autoantibody production.

This can be due to the effects of 3 Sle loci they share (154;203-205). Breaking of the

tolerance is not sufficient, however, for the fully development of autoimmune disease

(152). The disease phenotypes of the strains correlate to and affected by their B-cell

properties. As B cells from lupus prone mice have more potency to activation,

proliferation and differentiation as well as resistance to apoptosis, the lupus mice

generate large amount of germinal centers, long-lived plasma cells (unpublished data),

class-switched high affinity IgG autoantibodies (206) and finally full development of the

disease. TAN mice B cell respond poorly to low dose BCR cross-linking, and they do not









up-regulate CD19 expression even with strong BCR stimulation. Accordingly, their

autoantibody production is significantly lower, and most of the isotope is IgM

(unpublished data). Most importantly, TAN mice do not develop lupus nephritis. These

facts also indicate the impacts of lupus suppressing genes carried by the TAN genome.

In summary, this study shows that in lupus prone mice, splenic B cells actively

participate in the pathogenesis by the enhanced activation and differentiation to plasma

cells in response to antigen stimulation. In lupus resistant TAN mice, the limited B cell

activation likely contributes to the protection the host from lupus.








63







A SPB1 B SPB2
70. g* 75- *** *** **
60. *
**
50' $ 50. *$ g*
40, o a
30 *
& 25- **
20 *
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TAN NZM TC B6 TAN NZM TC B6





Figure 5-1. Splenic B cell populations. Freshly isolated splenocytes were assayed by flow
cytometry for (A) Bl cells and (B) B2 cells. Both TAN and NZM have
increased percentages of spleen Bl cells and decreased B2 cells, while TC has
only a decreased percentage of B2 cells. P<0.05, ** P<0.01, *** P<0.001.







64




A B2 CD69 B B2 CD40
2@ 40
*
*
15 30

4 10 ** 4 20
g 4 ** *
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TAN NZM TC B6 TAN NZM TC B6
strain strain


C
SP B2 IgM MFI
200 *
**
*
eg *
go **
u.. 100 *
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1111
TAN NZM TC B6
strain




Figure 5-2. Activation marker expression on splenic B2 cells assayed by flow cytometry.
(A) Significantly higher percentages of TC B2 cells express CD69. (B).
Significantly higher percentages ofNZM B2 cells express CD40. (C) Lower
IgM mean fluorescence intensity on TAN, TC and NZM B2 cells. P<0.05,
** P<0.01, *** P<0.001.









Peripheral B-Cell Development


10

3
-


2


1
-
10


MZB T2


I


1 01 1
CD23 PE


Figure 5-3. Definition of splenic B-cell developmental subpopulations with flow


10


























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55
50
45
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TC B6


TAN NZ TC B


Figure 5-4. Peripheral B-cell development. Freshly isolated splenocytes were assayed by
flow cytometry for (A) Tl. (B) FoB. (C) T2 and (D) MZB cells. Both TAN
and lupus-prone NZM and TC have accumulated Tl, less T2 and follicular B
cells. Populations were gated on B220+ B cells. P<0.05, ** P<0.01, ***
P<0.001.






















n


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Figure 5-5 Splenic B cell populations in mice after reciprocal bone marrow transfer. Three
month old B6 and TC mice were lethally irradiated and TC or B6 bone marrow was
transferred. All mice were sacrificed after 3 months and splenic B cells were assayed for
(A) Tl, (B) T2, (C) FoB, and (D) MZB populations. Unmanipulated mice were used as
controls. Splenic B-cell populations from bone marrow transferred mice were similar as
the non-operated donor strains. Statistical comparisons were conducted between B6
donor and TC donor groups. P<0.05, ** P<0.01, *** P<0.001.







68









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TAN NM


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5


TAN NM T 6


TAN NZ TC B


Figure 5-7. Apoptosis rate during of in vitro stimulation. Splenic naive B cells were
isolated and cultured for 16 hrs with (A) medium only, (B) anti-IgM at 1
pg/ml, (C) anti-IgM at 10 pg/ml, (D) LPS at 1 pg/ml. Only when stimulated
with high doses of anti-IgM, lupus prone TC and NZM B cells showed less
cell death than TAN and B6. P<0.05, ** P<0.01, *** P<0.001.




























IIll
TAN NZM TC B6
strain


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TAN NZM TC B6
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Figure 5-8 B-cell activation during in vitro culture. Splenic naive B cells were isolated
and cultured for 48 hrs with medium only. Activation markers were assayed

by flow cytometry for (A) CD86, (B) CD69, (C) GL-7 and (D) CD19. No
significant difference was observed for CD86 and CD69. (C) TAN, NZM and
TC showed higher GL-7 levels. (D) Both TC and TAN had much more B cells
with up-regulated CD19. P<0.05, ** P<0.01, *** P<0.001.


med iu m
























IIll
TAN NZM TC B6
strain


lill
TAN NZM TC B6
strain


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Figure 5-9. B-cell activation and differentiation with a low dose of anti-IgM stimulation.
Splenic naive B cells were isolated and cultured for 48hrs with anti-IgM at
l yg/ml. Activation markers were assayed by flow cytometry for (A) CD86, (B)
CD69, (C) GL-7 (D) CD19. TAN B-cell showed lower CD86 and GL-7 levels.
No significant differences were observed among TC, NZM and B6 mice for
all of these markers.* P<0.05, ** P<0.01, *** P<0.001.


IgM 1-B CD19 hi


























TAN NZM TC B6
strain


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Figure 5-10. B-cell activation under treatment with high dose of BCR stimulation.












anti-IgM lug/ml


anti-IgM 10ug/ml


p-ERK 1


A BAB


Figure 5-11 ERK phosphorylations in isolated naive B cell after 5 minutes of stimulation.














CHAPTER 6
SUMMARY AND CONCLUSIONS

In this study, we have characterized the properties of B-cell populations in lupus

prone and resistant mice. We found that the splenic MZB cell phenotype correlated with

the lupus susceptibility. In the lupus-prone TC and NZM mice, we found a loss of

marginal zones, missing marginal zone macrophages, and marginal zone B cells trans-

located inside the follicles. In contrast, lupus-resistant TAN mice had enlarged MZ with

CD5+ marginal zone B cells retained despite of stimulation. Besides, MZB cells of both

strains have defective TI-2 antigen up-take. As the MZB cells have potent antigen

presenting and rapid differentiation capabilities, and also contain auto-reactive clones, the

trans-localized MZB cells acquire the privilege to present antigen to and to activate CD4

T cells and ultimately differentiate into (auto)antibody-producing plasma cells. In

contrast, the expression of CD5 and MZ arrest of MZB cells in TAN mice may raise the

activation threshold and prevent them from developing overt autoimmunity. Furthermore,

the lack of marginal zone macrophages and defects in TI-2 antigen uptake by MZB cells

suggests that lupus mice are ineffective in the clearance of blood-borne pathogens.

Secondly, B-cell responsiveness to BCR ligation was also correlated to the disease

phenotype of these strains. The naive splenic B cells in lupus mice generally have lower

activation thresholds, and higher rates of differentiation, while TAN B cells showed the

opposite. All of our data strongly suggest that B cells in lupus mice actively participate in

lupus pathogenesis by over-activation to antigen stimulation and differentiation to plasma

cells.









In the peritoneal cavity, TC Bl cells produced overall more IL-6 and 1-10 upon

BCR cross-linking. In contrast, peritoneal B cells from TAN mice still show the

decreased responsiveness, and little 1-6 and 1-10 production with stimulations. Both

IL-6 and IL-10 can promote B-cell proliferation, differentiation and antibody production,

and they also play a positive role in lupus progression. These results suggest that

peritoneal Bl-cells in lupus mice participate in the pathogenesis of lupus by producing

disease -promoting cytosine IL-6 and IL-10.

Taken together, these studies suggest that B-cell populations contribute to the

development of lupus both through effector and regulatory mechanisms.















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