Increased plasma cell frequency and accumulation of abnormal syndecan-1plus T-cells in Igµ-deficient/lpr mice

Jane Seagal1, Nira Leider1, Gizi Wildbaum1, Nathan Karin1,2 and Doron Melamed1,2

1 Department of Immunology, Bruce Rappaport Faculty of Medicine and 2 Rappaport Family Institute for Research in the Medical Sciences, Technion–Israel Institute of Technology, Haifa 31096, Israel

Correspondence to: D. Melamed; E-mail: melamedd{at}techunix.technion.ac.il
Transmitting editor: I. Pecht


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The expression of µH chain is an important checkpoint in B cell development. In mice deficient for IgM transmembrane tail exons (µMT mice) B cell development is blocked at the pro-B stage. However, we showed that Fas-deficient µMT mice (µMT/lpr) develop a very small population of isotype-switched B cells and produce high titers of self-reactive serum antibodies. In addition, µMT/lpr mice develop severe lymphoproliferation and both pathologic processes occur at young ages. This may suggest that lack of Fas–Fas ligand signaling exacerbates murine lupus in B cell lymphopenic mice. To test this we analyzed antibody and plasma cell formation, and accumulation of abnormal T cells in µMT/lpr mice. Our results show that the µMT/lpr mouse is particularly permissive for the development and accumulation of antibody-producing cells, thereby explaining the high titers of serum antibodies in these mice. In addition, we found that accumulating cells in spleen and lymph nodes of µMT/lpr mice are {alpha}ß T cells expressing the abnormal B220+/CD3+ surface markers, a phenotype also described for other Fas-deficient mouse models. Strikingly, we found that accumulating cells in µMT/lpr mice express the membrane proteoglycan syndecan-1, a known plasma cell marker. Development of these cells is blocked in mice deficient for TCRß and TCR{delta}. We also found that both antibody production and lymphoproliferation in µMT/lpr mice are Th1 regulated. Our results, therefore, suggest that in the µMT/lpr mouse model a small population of isotype-switched B cells is sufficient for the initiation and propagation of Th1-regulated murine lupus.

Keywords: B cell development, Fas signaling, lupus, self-tolerance, serum antibody


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
B cell development is a process strictly regulated by Ig gene rearrangements and assembly (1,2). The requirement of functional Ig heavy and Ig light expression and signaling in promoting B cell development has been shown in several models of mutated mice, in which B cell development in the bone marrow (BM) is blocked or altered (reviewed in 2,3). In addition, B cell development is limited by negative selection, imposed by self-tolerance mechanisms (2,4,5). Thus, B lymphocytes expressing incompetent receptors are subjected to negative selection and are eliminated by apoptosis.

Fas (CD95) is a surface receptor that transmits apoptotic signals upon binding of Fas ligand (FasL) in a wide variety of cell types (6) and is particularly important in controlling activated lymphocytes (79). However, in contrast to its known function in mature cells, the role of Fas signaling in developing lymphocytes is unclear. Several studies in Ig transgenic (Tg) mice failed to demonstrate a role for the Fas pathway in negative selection and elimination of developing B cells (1012). Mice deficient for functional Fas or FasL (lpr/lpr or gld/gld respectively) develop an autoimmune lupus-like disease, which is characterized by production of high titers of autoantibodies in serum, immune complex glomerulonephritis, vasculitis and arthritis (13). In addition, lack of functional Fas signaling in these mouse models is also characterized by massive accumulation of abnormal CD3+ T lymphocytes that aberrantly express B220, and lack expression of CD4 or CD8 (13). Many studies have utilized these mouse models to show that both T and B lymphocytes are required for the onset and propagation of the disease (1416).

In early studies we used mice deficient for the IgM transmembrane tail exon (µMT), in which B cell development is blocked at the pro-B stage (17), to probe the role of Fas in the negative selection of developing B cells (18). Strikingly, we found that Fas-deficient µMT mice (µMT/lpr) develop a very small population of isotype-switched, mature Ig+ B cells and produce high titers of self-reactive serum antibodies. In addition, µMT/lpr mice develop severe lymphoproliferation. Both production of autoantibodies and lymphadenopathy occur in young µMT/lpr mice (8–10 weeks) (18), suggesting that partial B cell lymphopenia exacerbates murine lupus in this mouse model. In the present work further steps have been taken to define the pathological and immunological symptoms that develop in the µMT/lpr model. We found that the µMT/lpr mouse is particularly permissive for the development and accumulation of antibody-producing cells, thereby explaining the high titers of serum antibodies in these mice. In addition, we found that the abnormal cells accumulating in spleen and lymph nodes (LN) of µMT/lpr mice express the membrane proteoglycan syndecan-1. The function of this molecule in these T cells is yet to be explored. Such cells did not develop in T cell-deficient µMT/lpr mice. We also found that the antibody response and lymphoproliferation in µMT/lpr mice are Th1 regulated. Collectively, our results suggest that in the µMT/lpr mouse model a small population of isotype-switched B cells is sufficient for the initiation and propagation of Th1-regulated murine lupus.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
Normal [C57/BL6 (B6)], Igµ-deficient (B6-µMT/µMT), Fas-deficient (B6-Faslpr/Faslpr) and TCRß/TCR{delta}-deficient (B6-TCRß{delta}–/–) mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Mice deficient for Igµ and Fas (µMT/lpr) were generated by crossing µMT and lpr mice. In some experiments µMT/lpr mice were crossed with TCRß{delta}–/– mice to generate µMT/lpr/TCRß{delta}–/– mice. Mouse typing for µMT and lpr was performed by PCR as described (18). Typing for TCR{delta} deficiency was performed by PCR (primer sequences as published by the Jackson Laboratory) and for TCRß by flow cytometry. Mice were maintained under specific pathogen-free conditions at the animal facility.

Analysis of serum antibody
Titers of total IgG, IgG1, IgG2a, IgM, {kappa} and {lambda} in serum samples were determined by sandwich ELISA, using specific goat anti-mouse polyclonal reagents (Southern Biotechnology Associates, Birmingham, AL) as we have described (18). Purified antibody of each isotype tested provided the standard curve for calculation of antibody concentration.

ELISPOT
Detection of antibody-forming cells (AFC) by ELISPOT assay was performed as described (19). Briefly, 5-cm nitrocellulose filters were placed in 5-ml tissue culture Petri dishes, and coated overnight with a mixture of polyclonal goat anti-mouse {kappa} and goat anti-mouse {lambda} (2.5 µg/ml) (Southern Biotechnology Associates). After an extensive wash (0.1% Tween 20/PBS), filters were blocked with 5% BSA for 2 h, and washed twice with PBS and twice with IMDM (Gibco/BRL, Gaithersburg, MD) prior to cell addition. Spleen cells, red blood cell-depleted and resuspended in IMDM, were cultured at different concentrations on top of filters for 12–16 h. After incubation, filters were extensively washed and incubated with biotinylated goat anti-mouse IgG ({gamma} chain-specific) antibody. Streptavidin conjugated with horseradish peroxidase was used as a secondary probe and signals were generated by the ECL reaction. Spots generated on film were counted and frequency was calculated.

Flow cytometry
Single-cell suspensions from spleen and LN were stained for surface marker expression using FITC-, phycoerythrin (PE)- and biotin-conjugated antibodies, visualized with streptavidin TriColor (Caltag, San Francisco, CA). Antibodies used for cell staining were: CD19 (clone 1D3), TCRß (clone H57), TCR{delta} (clone GL3), syndecan-1 [(CD138), clone 281-2] (all from PharMingen, San Diego, CA), B220 (clone RA3-6B2; Southern Biotechnology Associates) and CD3 (clone KT3; Serotec, Oxford, UK). Data for three-color analysis were collected on a FACSCalibur and analyzed using CellQuest software (Becton Dickinson, Mountain View, CA).

Intracellular cytokine staining and analysis
Intracellular staining for spleen and LN was performed as we have described (20). Briefly, spleen and LN cells were stimulated with phorbol myristate acetate (50 ng/ml), ionomycin (0.2 mM) and monensin (0.2 mM) for 5 h (all from Sigma-Aldrich, St Louis, MO). Stimulated cells were first stained for surface expression of B220 and syndecan-1. For intracellular cytokine stain, membranes were first lysed with saponin (0.5%), followed by incubation with PE-labeled rat anti-mouse IFN{gamma} mAb and allophycocyanin-labeled rat anti-mouse IL-4 mAb (all from PharMingen). Cells were analyzed using a FACSCalibur.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Abnormal serum antibody production in µMT/lpr mice
Mice bearing the µMT mutation fail to produce mature B cells in the BM and completely lack serum IgM or IgG (Fig. 1A) (17,18). However, lack of functional Fas rescues the development of some isotype-switched B cells, resulting in a significant 3- to 4-fold increase in the production of serum IgG relative to normal mice (Fig. 1A). To study the nature of this unexpected antibody response we performed isotypic analysis on serum samples collected from µMT/lpr mice, relative to serum from normal mice (Fig. 1B). Light chain analysis clearly showed that antibody responses in µMT/lpr mice are non-homogenic. We found that the Ig{lambda} proportion in µMT/lpr sera was widely distributed, ranging from 0 to 30%, whereas the proportion of Ig{lambda} antibodies in normal serum is only 2–4%. Interestingly, most µMT/lpr mice produced significantly elevated levels of Ig{lambda} (up to 10-fold) relative to control. Similarly, we found non-homogenic distribution in Ig heavy isotypic analysis in µMT/lpr serum samples relative to control (not shown). The unique pattern of serum antibody distribution in each µMT/lpr mouse may suggest a selection process of particular B cell clones.



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Fig. 1. Abnormal serum antibody response in µMT/lpr mice. (A) Serum samples from normal, µMT and µMT/lpr 8- to 12-week-old mice were analyzed for total IgM (left) and IgG (right) by ELISA. For each group, titers of six to 10 individual mice are shown as well as the group mean. (B) Light chain analysis in normal and µMT/lpr serum samples. The concentrations of {kappa} and {lambda} light chains were determined by ELISA. Shown is the {kappa}:{lambda} distribution for individual mice, plotted as {kappa} on the x-axis relative to {lambda} on the y-axis. All Ig concentrations were determined using an appropriate standard curve.

 
Accumulation of abnormal T cells in spleen and LN of µMT/lpr mice
The development of severe lymphadenopathy in µMT/lpr occurs at a young age (18). Interestingly, we did not detect enlarged spleens in µMT/lpr mice (not shown). However, FACS analysis of spleen cells revealed a massive accumulation of abnormal CD3+/B220+ T lymphocytes (Fig. 2, top). In contrast to µMT and normal mice, where the number of these cells was very low, the abnormal T cell population in the µMT/lpr mice reached 50–70% (Fig. 2, top) and severity of accumulation increased with age (not shown). Further surface analysis revealed that accumulating B220+/CD3+ T cells in µMT/lpr spleens are {alpha}ß, but not {gamma}{delta}, T cells (Fig. 2, top). As shown earlier (18), the B cell population in the µMT and µMT/lpr (B220+/CD19+) is relatively low (2.5–5%, Fig. 2). A similar distribution of lymphocyte populations was found in peripheral LN of these mice (Fig. 2, bottom). The possibility that abnormal T cell accumulation in the µMT/lpr mice results from B cell lymphopenia is unlikely since such cells are not found in the µMT spleen and LN. Thus, as described for the lpr mouse model, this accumulation exclusively depends on lack of Fas signaling.



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Fig. 2. Accumulation of an abnormal T cell population in µMT/lpr mice. Spleen (top) and LN (bottom) cells from normal µMT and µMT/lpr mice were stained for B220, CD19, CD3, TCRß and TCR{delta}, and analyzed by FACS. Shown are representative plots of individual mice.

 
Increased frequency of plasma cells in µMT/lpr mice
To resolve the apparent contradiction between the very high titers of serum antibody, on the one hand, and the low number of Ig+ cells in µMT/lpr mice (18), on the other hand, we studied the frequency of plasma cells in µMT/lpr mice. To do so, two approaches had been taken: FACS analysis and direct detection of AFC by the ELISPOT method. Since syndecan-1 is used as a plasma cell marker, spleen cells from normal, µMT and µMT/lpr mice were analyzed for the expression of syndecan-1, and the pan-B cell markers B220 and CD19. Unexpectedly, we found a severe accumulation of cells expressing both B220 and syndecan-1 in spleens of µMT/lpr mice (~50% of the cells, Fig. 3A). Similar accumulation of B220+/syndecan-1+ cells was also evident in µMT/lpr LN (not shown). Further analysis for CD19 expression clearly showed that only a small fraction of these cells were indeed B lymphocytes, while the majority were not (not shown). Hence, to quantify AFC by syndecan-1 expression, we analyzed gated B220+/CD19+ spleen cells (Fig. 3B). The results showed that 20–25% of the B cells in µMT/lpr spleens expressed syndecan-1 and were considered as plasma cells, relative to only 1–1.5% plasma cells in normal mouse. Calculating the absolute numbers revealed that µMT/lpr mice had a 2.5 ± 0.9-fold increase in AFC number. As expected, such cells were essentially not detected in µMT spleens (Fig. 3A and B). To confirm and further visualize these findings we analyzed IgG AFC in spleens of these mice by ELISPOT. The results, which are presented in Fig. 3(C), clearly show increased AFC in µMT/lpr mice. Upon counting the spots we calculated that the AFC frequency in µMT/lpr mice is 3- to 3.5-fold higher relative to normal (344 ± 70 in µMT/lpr relative to 102 ± 25 in normal), results that were not significantly different from those obtained by syndecan-1 staining and FACS analysis. For comparison, in wild-type B6/lpr mice at the early onset of the lpr disease, we obtained 2.5- to 3.5-fold increase in numbers of plasma cells as measured both by FACS (Fig. 3A and B) and by ELISPOT (Fig. 3C), and this is in agreement with earlier studies (21). We concluded that differentiation of B lymphocytes to plasma cells is augmented in the µMT/lpr mice and may be associated with the early onset of the disease.



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Fig. 3. Increased frequency of plasma cells in µMT/lpr mice. (A and B) Quantitation of plasma cells by surface staining and FACS analysis. Spleen cells from normal, µMT, µMT/lpr and B6/lpr mice were stained for B220, CD19 and the plasma cell marker syndecan-1, and analyzed by FACS. (A) Viable spleen cells were plotted for B220 and syndecan-1 expression. (B) Syndecan-1 expression in the gated B220+/CD19+ cell population. Shown are representative plots of individual mice. (C) Quantitation of plasma cells in spleens of the indicated mice by ELISPOT. The spot assay was carried out as described in Methods. Representative membrane filters are shown. Spots were counted and frequencies were determined. Mean frequencies ± SEM for each group (at least five mice per group) are presented.

 
Accumulating B220+/CD3+ abnormal T lymphocytes in µMT/lpr mice express syndecan-1
The large population of B220+ cells expressing syndecan-1, which was unexpectedly detected in µMT/lpr mice, was further studied to identify this cell subset. Because of the large fraction of these cells, we speculated that most of the syndecan-1 expression is a unique feature of the abnormal T cell population accumulating in the µMT/lpr mouse. To test this hypothesis we analyzed syndecan-1 expression in gated abnormal (CD3+/B220+) T lymphocytes relative to the normal (CD3+/B220) T cell population in µMT/lpr mice. Similar comparative analysis was performed for wild-type B6/lpr mice at the early onset of the lpr disease. The results in Fig. 4 unambiguously show that >80% of the abnormal T cell population in spleen (top) and LN (bottom) in µMT/lpr mice is expressing syndecan-1. Interestingly, while abnormal T cells accumulating in LN of B6/lpr mice also express syndecan-1, we did not detect significant expression of syndecan-1 in the abnormal T cells accumulating in spleen of the B6/lpr mice (Fig. 4). As expected, normal T cells in both µMT/lpr and B6/lpr did not express syndecan-1. Hence, we show here for the first time that abnormal T lymphocytes accumulating in spleen and LN of µMT/lpr mice and in LN of B6/lpr mice express the membrane proteoglycan syndecan-1.



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Fig. 4. Abnormal T cells accumulating in µMT/lpr mice express syndecan-1. Spleen (top) and LN (bottom) cells from µMT/lpr and B6/lpr mice were stained for B220, CD3 and syndecan-1, and analyzed by FACS. Syndecan-1 expression was determined in gated abnormal B220+/CD3+ and in normal B220/CD3+ T cells. Gates for the sorted cells are shown. The results are from individual µMT/lpr and B6/lpr mice, and are representative of three to five different mice.

 
Lack of B220+/CD3+/syndecan-1+ cell accumulation in µMT/lpr mice deficient for {alpha}ß and {gamma}{delta} T lymphocytes
To test whether accumulation of B220+/CD3+/syndecan-1+ cells in the µMT/lpr mouse depends on T lymphocytes, we crossed the µMT/lpr mice with mice genetically deficient for functional ß and {delta} TCR chains to generate µMT/lpr/TCRß{delta}–/– mice. This approach completely abolished the development of mature T lymphocytes, as evident by the lack of CD3+ cells (Fig. 5). Analysis of spleen (Fig. 5) and LN (not shown) in these mice clearly showed that accumulation of B220+/CD3+/syndecan-1+ cells is T cell dependent. The abnormal B220+/CD3+/syndecan-1+ cells were completely absent in spleen and LN of µMT/lpr/TCRß{delta}–/– mice, relative to the T cell-sufficient µMT/lpr mice.



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Fig. 5. Accumulation of B220+/CD3+ syndecan-1+ abnormal T-cells is abolished in µMT/lpr/TCRß{delta}–/– mice. Spleen cells from normal, µMT/lpr and µMT/lpr/TCRß{delta}–/– mice were stained for B220, CD3 and syndecan-1, and analyzed by FACS. Shown are representative plots of individual mice.

 
A predominant Th1 response in µMT/lpr mice
To determine the type of abnormal immune responsiveness that developed in µMT/lpr mice we analyzed cytokine and serum antibody production. Spleen and LN cells from normal, µMT and µMT/lpr mice were stained for surface syndecan-1 expression, and intracellular IFN-{gamma} (a Th1-regulated cytokine) and IL-4 (a Th2-regulated cytokine) production. FACS analysis of spleen cells clearly showed that accumulating syndecan-1+ cells also produce IFN-{gamma}, but not IL-4, suggesting a biased Th1 responsiveness (Fig. 6A). Similar results were also obtained in LN (not shown). Serum antibody analysis confirmed this observation by showing increased IgG2a (Th1-regulated antibody) and decreased IgG1 (Th2-regulated antibody) in µMT/lpr mice (Fig. 6B). This is in contrast to a reciprocal IgG2a:IgG1 ratio found in a normal mouse (Fig. 6B). Since the antibody repertoire in the µMT/lpr mice is limited and self-reactive (18), the biased IgG2a production strongly supports the fact that a predominant Th1-type responsiveness develops in these mice. Thus, both autoimmunity and lymphoproliferation in the µMT/lpr mouse model are Th1 mediated, as described in other Fas-deficient lpr models (22,23).



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Fig. 6. Th1-regulated lymphoproliferation and antibody response in µMT/lpr mice. (A) Detection of IFN{gamma} (Th1-regulated cytokine) and IL-4 (Th2-regulated cytokine) production in accumulating syndecan-1+ cells by FACS. Spleen cells from the indicated mice were stained for surface syndecan-1 expression, and for intracellular IFN{gamma} and IL-4 synthesis. Cells were analyzed by FACS and representative results of individual mice from four different experiments are shown. (B) Quantitation analysis of serum IgG2a (a Th1-regulated antibody) and IgG1 (a Th2-regulated antibody) by ELISA. The IgG2a and IgG1 titers in serum samples from 10–15 normal and µMT/lpr were determined by quantitative ELISA. Titers are plotted as IgG2a on the y-axis versus IgG1 on the x-axis for individual mice.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Lack of Fas–FasL signaling results in a severe autoimmune response in the lpr and gld mouse models, and both T and B cells are required for it (1316). It is therefore surprising to find that a small subset of isotype-switched B cells, capable of developing in µMT/lpr and in µMT/gld mice (18), gives rise to high titers of anti-DNA serum IgG. The present study shows that the autoimmune-prone µMT/lpr mice are particularly permissive for the accumulation of AFC, thereby exacerbating the lupus-like disease. These mice also develop severe lymphoproliferation of abnormal T cells expressing the membrane proteoglycan molecule syndecan-1. While this study does not rule out the requirement of both T and B cells for murine lupus, it indicates that a small subset of isotype-switched B cells is sufficient for its initiation and propagation. The development of these B cells is Fas–FasL dependent.

The generation of serum IgG in µMT/lpr mice indicates that the process of isotype switching can circumvent the developmental block imposed by the µMT mutation. Indeed, earlier studies showed that a small population of isotype-switched Ig+ B cells is present in µMT/lpr spleens (18) and switching to IgA occurs in µMT mice in response to intestinal stimulation (24). BM analysis of µMT/lpr mice does not reveal a substantial change in the developmental block imposed by the µMT mutation (18). This may suggest that isotype switching occurs at very low frequency during development and such cells accumulate in the periphery. Studies showing spontaneous isotype switching in transformed B cell lines (25) support this hypothesis. Alternatively, isotype switching in µMT/lpr mice may occur in the periphery since many pro-B cells are found in spleen and LN of these mice (17,18). Studies show that under appropriate stimulation, isotype switching can be induced in Ig B cells (26), and that sIg B cells can participate in the germinal center reaction and acquire somatic mutations (27) or undergo receptor revision and express autoimmune specificity (28). Nevertheless, this process is inefficient in the µMT/lpr mice, also resulting in a non-homogenic antibody production as reflected by differential distribution of IgG isotypes between mice (18) and in the {kappa}:{lambda} ratio (Fig. 1). It is possible, though, that such non-homogenic responses reflect a process of selection that may be different between mice. The finding that µMT/lpr mice develop a self-reactive antibody repertoire supports this hypothesis (18).

Strikingly, we found that despite the very small numbers of Ig+ cells, the µMT/lpr mice provided a permissive environment for accumulation of AFC, resulting in a 2.5- to 3.5-fold increase relative to a normal mouse. While this may explain the apparent high titers of IgG in sera of these mice, it may also indicate a major process of clonal selection and expansion. This notion is supported by the findings that µMT/lpr mice have a limited repertoire and they fail to respond to exogenous antigens (18). Thus, isotype switching may allow B cells to develop, but subsequent selection proliferation and differentiation to plasma cells are augmented in the µMT/lpr mouse. Earlier studies have shown that autoimmune prone mice, including the MRL strain, develop 3- to 6-fold more plasma cells during the onset of the autoimmune disease (21). In these mice, however, accumulation of plasma cells is due to initial polyclonal activation originating from a normal-sized B cell population (13), whereas in µMT/lpr mice the Ig+ B cell pool is ~1–5% of the normal size (18). Thus, plasma cell accumulation in the µMT/lpr lymphopenic mice is a remarkable observation that may result from enhanced processes of clonal selection and expansion. In most cases all of these processes are linked to the germinal center reaction and are mediated by T cell help (1). However, recent data suggest that isotype switching, memory formation and acquisition of somatic mutations can occur outside the germinal center (29,30). Peripheral activation is thought to be an important mechanism for B cell tolerance, as lack of T cell help promotes B cell apoptosis mediated by Fas signaling (1,31,32). This may suggest that lack of Fas allows a permissive environment for selection and activation of autoreactive clones into the B cell follicle (33,34) or at the T zone/red pulp border (30), thereby leading to breakdown of self-tolerance. Similarly, in Ig Tg models, lack of Fas results in development of a small population of variant B cells, bearing non-Tg specificity, that undergo activation and isotype switching to produce high titers of anti-DNA antibodies (10,11). In Fas-deficient mice B cell expansion and autoimmunity involves T cell-dependent [both {alpha}ß and {gamma}{delta} T cells (3537)] and T cell-independent mechanisms (11,38), resulting in a gradual increase in serum IgG with age. However, lack of secreted IgM accelerates IgG autoantibody production and autoimmune disease in MRL/lpr mice deficient for secreted IgM (39). This may explain the significant accumulation of plasma cells and high titers of autoimmune serum IgG that develop early in µMT/lpr mice.

Lack of Fas or FasL also results in accumulation of abnormal B220+/TCR+/CD4/CD8 T cells in spleen and LN of lpr mice (13). This pathologic process appears to be independent of B cells, as such cells also accumulate in lpr mice bearing a complete deletion of the JH locus, where B cell development is completely blocked (16,40). The origin of these cells is not clearly defined. Early studies propose that these abnormal T cells originate from thymic precursors (41,42). More recent work suggests that these cells are post-activated T cells surviving due to lack of functional Fas signaling (4345), as T cells undergoing superantigen-induced apoptosis in vivo express B220 and Fas (46), and Tg expression of Fas in T cells blocks lymphoproliferation in MRL/lpr mice (47). We found a severe lymphoproliferation of abnormal T cells expressing {alpha}ß, but not {gamma}{delta}, TCR in the µMT/lpr mice (Fig. 2). Accumulation of these cells is accelerated in the µMT/lpr mice and obtained even in young mice (18). This may suggest an important role for the small number of selected µMT/lpr B cells in priming T lymphocytes and induction of autoimmunity, a paradigm proposed by Shlomchik et al. (40). It is also in agreement with studies showing a central role for {alpha}ß T cells in the pathogenesis of murine lupus (35,36) and reduced lymphoproliferation obtained in TCR{alpha}–/–/lpr mice (48). Other studies in T-deficient mice implicated an important role for {gamma}{delta} T cells in regulating murine lupus (35,48).

We show here, for the first time, that abnormal T cells accumulating in Fas-deficient mice express the transmembrane proteoglycan syndecan-1 (Fig. 4). In the µMT/lpr mice syndecan-1 was significantly expressed by abnormal B220+/CD3+ T cells accumulating in both spleen and LN. It is clear that these cells arise from T lymphocytes, as they could not be found in µMT/lpr mice deficient for ß and {delta} TCR (Fig. 5). We could also detect syndecan-1 expression in abnormal T cells accumulating in LN of B6/lpr mice, but not on those accumulating in the spleen. Syndecans are widely expressed on the surfaces of most cells in the body, and play an important role in cell–matrix and cell–cell interactions, as well as modulating receptor activation (49,50). In hematopoietic cells syndecan-1 is know to be expressed not only on pre-B and plasma cells, but also on malignant B lymphoid cells, although its functionality in these cells has not been defined yet (49,50). That abnormal T cells in the µMT/lpr mouse express significant levels of syndecan-1 may imply an important role of this molecule in directing these cells into the peripheral lymphoid tissue. Several studies show that syndecans may act as co-receptors in a variety of physiological processes, including cell adhesion, migration, response to growth factors, development and tumorigenesis (reviewed in 51). Recent in vitro and in vivo data suggest the involvement of syndecans in the modulation of leukocyte–endothelial interactions and extravasation during inflammation (52), and in mediating HIV infection (53). Hence, the expression of syndecan-1 may also explain the altered responsiveness of this abnormal T cell population to a variety of stimulations (54,55) and the defective traffic patterns (56). This, however, may result from complex modifications in additional surface or intracellular regulatory genes that are yet to be defined. So far, syndecan-1 has not been shown to be expressed by T cells nor has its possible involvement in pathogenesis of murine lupus been demonstrated. As this molecule is involved in regulating cell migration (51,52) it may also act, perhaps with other molecules, in directing the abnormal T cells to accumulate in peripheral lymphoid organs. It is possible, though, that expression of syndecan-1 is associated with pathogenicity of the T cells, and the differential expression of this molecule in LN and spleen of B6/lpr mice supports this possibility. The fact that in µMT/lpr mice syndecan-1 is expressed in both spleen and LN abnormal T cells may reflect the exacerbation of the disease in these mice. This hypothesis, as well as the function of syndecan-1 on these abnormal T cells, is yet to be elucidated.

Autoimmunity in Fas-deficient mice is characterized by the production of autoantibodies and nephritis, and both require B and T lymphocytes. In murine lupus, B lymphocytes have been shown to serve as antigen-presenting cells and autoantibody producers in mediating renal lymphocytic infiltration (14,16,40). This is regulated by CD4 T cells, as lpr mice deficient for CD4 or MHC class II do not produce autoantibodies and develop only moderate nephritis (37,57). In contrast, all these mice develop B220+/CD3+ abnormal T cell lymphoproliferation, suggesting that autoimmunity and lymphoproliferation are independent processes developing in Fas-deficient mice. This is supported by showing that lymphoproliferation is reduced in lpr mice deficient for MHC class I, lacking CD8 T cells (58). Early studies suggested that murine lupus in the lpr mouse is regulated by imbalance towards Th1 predominant responses (22,23). In agreement with these are our findings showing that abnormal, syndecan+ T cells accumulating in the µMT/lpr mice produce IFN-{gamma} and not IL-4 (Fig. 6). Since the antibody repertoire of these mice is limited (18), the significant increase in Th1-regulated antibodies (IgG2a) obtained in the µMT/lpr mice, relative to the Th2-regulated antibodies (IgG1), strongly supports and further extends these observations. This may suggest that dominant Th1 responses, initiated in the absence of functional Fas, promote a permissive environment in which self-reactive B lymphocytes are produced and selected to participate in an autoimmune response (34). In the µMT mice this is particularly important since B cell development is blocked at the pro-B stage and maturation is limited by isotype-switching requirements. However, the mechanism of development and selection of these cells is still unknown.


    Acknowledgements
 
This research is supported by the Israel Science Foundation, the Colleck Research Fund and the Mars Pitsburg Foundation for Medical Research.


    Abbreviations
 
AFC—antibody-forming cell

BM—bone marrow

FasL—Fas ligand

LN—lymph node

PE—phycoerythrin

Tg—transgenic


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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