Systemic sclerosis (SSc) is a connective tissue disease characterized by excessive extracellular matrix deposition in the skin and other visceral organs. Because clinical manifestations of SSc are heterogeneous, various subtypes of SSc have been proposed, such as CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasias). The most widely accepted classification system is that proposed by LeRoy et al (1), which includes limited cutaneous SSc (lcSSc), a type similar to CREST syndrome, and diffuse cutaneous SSc (dcSSc), a more rapidly advancing type with more frequent visceral involvement. The presence of autoantibodies is a central feature of SSc; antinuclear antibodies (ANAs) have been detected in >90% of patients (2). SSc patients have autoantibodies that react to various intracellular components, such as DNA topoisomerase I, centromere, RNA polymerases, U1RNP, and U3RNP (2). In addition, hyper-gammaglobulinemia and polyclonal B cell hyperactivity are detected in SSc patients (3, 4). Furthermore, a recent study demonstrated that down-regulation of B cell function leads to improved skin fibrosis in the tight-skin mouse, a genetic model of SSc (5). Although the pathogenesis of SSc remains unknown, the B cell abnormalities characterized by autoantibody production and polyclonal B cell activation play an important role. In particular, long-lived memory B cells are considered to play a crucial role as autoreactive B cells.
Although memory T cells can be distinguished from naive T cells by expression of different CD45 isoforms, individual cell surface markers that can directly identify all memory B cells were, until recently, not known. The most definitive marker of memory B cells identified to date is the presence of somatically mutated, high-affinity antigen receptors (6), but accumulating evidence has shown that cell surface CD27 is a useful marker of human memory B cells (6–12). CD27 is a type I glycoprotein expressed on some B cells and the majority of T cells, and is a member of the tumor necrosis factor receptor family (10). The interaction of CD27 with its ligand on T cells, CD70, can induce quick activation that leads to differentiation into plasma cells (9, 10). Importantly, recent studies on circulating B cells at the single-cell level confirmed that essentially all circulating CD27+ B cells display hypermutated, rearranged VH genes, while no mutations are identified in CD27− B cells (6, 7, 10, 13, 14). B cells with high levels of CD27 (CD27high) express low levels of CD19 and surface Ig, high amounts of CD38 and CD138, and no CD20, a pattern found on plasma cells (13, 15). The majority of these cells do not, however, look like mature plasma cells, but like plasma cell precursors (plasmablasts or early plasma cells); analysis by sorting and Giemsa staining revealed that they have larger, less peripheral nuclei and less abundant cytoplasm (15). Thus, CD27 is a reliable and useful marker for characterizing peripheral blood B cell subpopulations and homeostasis.
Recent studies using CD27 as a marker of memory B cells have revealed a disturbance of peripheral B cell compartments and homeostasis in systemic autoimmune disorders. Patients with systemic lupus erythematosus (SLE) exhibit an expanded population of CD27high,CD38+ plasmablasts that correlates with disease activity, while the number of CD27− naive B cells and CD27+ memory B cells is reduced due to marked B lymphocytopenia (13, 15, 16). However, patients with Sjögren's syndrome show a normal number of naive B cells, but a significantly reduced number of CD27+ memory B cells without expansion of plasmablasts (14, 17). Thus, these findings indicate distinct types of abnormal B cell homeostasis in some systemic autoimmune disorders.
Despite the potential contribution of B cell abnormalities to the pathogenesis of SSc, no studies have examined phenotypic and functional abnormalities in peripheral blood B cell compartments from SSc patients. The current study showed distinct altered B lymphocyte homeostasis characterized by an expanded naive B cell population, a diminished but highly activated memory B cell population, and a reduced plasmablast population in SSc.
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The present study revealed disturbed homeostasis of peripheral B cell subsets in SSc. Both dcSSc and lcSSc patients exhibited a pattern of expanded numbers of naive B cells and reduced numbers of plasmablasts and memory B cells, which had increased expression of CD80, CD86, and CD95. CD80 and CD86 are critical costimulatory molecules of B cells, and B cell activation is required to up-regulate expression of both molecules; thus, increased CD80 and CD86 expression on memory B cells indicates that they are constantly activated in SSc. Furthermore, CD95 expression is up-regulated after B cell activation, and increased CD95 expression coincides with the acquisition of sensitivity to CD95-mediated apoptosis (24); thus, up-regulated CD95 expression on SSc memory B cells may result in their enhanced spontaneous apoptosis and diminished number. Moreover, it is possible that the continuous loss of memory B cells and plasmablasts increases the production of naive B cells in an attempt to maintain B cell homeostasis in SSc. Remarkably, although memory B cells were decreased in SSc patients, they had a significantly enhanced ability to produce IgG, which may result in hypergammaglobulinemia.
Studies investigating phenotypic abnormalities of blood B cells are limited in SSc. Furthermore, previous studies examined only total B cells, not the naive and memory B cell compartments. Previous studies suggested that SSc B cells were activated since the frequency of B cells expressing activation markers, including HLA–DR and CD25, was increased in SSc (4, 25, 26). However, the present study showed a normal frequency of HLA–DR–positive cells and normal expression levels of HLA–DR in both naive and memory B cells (data not shown). This discrepancy may be due to the different antibodies used for assay: all naive and memory B cells had detectable HLA–DR expression in this study, while HLA–DR expression was detected in only ∼10% of total B cells in previous studies (25, 26). Due to the marked reduction in memory B cells and normal expression of activation markers on expanded naive B cells in SSc, it would be difficult to detect phenotypic abnormalities of B cells by examining total B cells as a whole. Thus, this is the first study to reveal the distinct activated phenotypic abnormalities in memory B cells in SSc.
We previously reported that B cells from SSc patients overexpress CD19 by ∼20% (21). CD19 is a critical cell surface signal transduction molecule on B cells that regulates signaling thresholds important for humoral immune responses and autoimmunity (22, 23). Remarkably, transgenic mice with a similar increase in CD19 expression exhibit hypergammaglobulinemia and produce characteristic autoantibodies with specificities similar to autoantibodies in human SSc (21). Furthermore, B cells from tight-skin mice display enhanced CD19 signaling with a constantly activated phenotype, hypergammaglobulinemia, and spontaneous autoantibody production; these phenomena are completely eliminated by the loss of CD19 expression from B cells (5). The present study showed that CD19 is overexpressed in naive B cells and, to a greater extent, in memory B cells from SSc patients. This CD19 overexpression was specific to SSc, since SLE patients exhibited significantly down-regulated CD19 expression, which is associated with decreased expression of CD21, a C3d receptor (21, 27). In SLE, this down-regulation of CD19 and CD21 expression was suggested to be secondary to interaction with immune complexes bearing fragments of C3 (27). Furthermore, up-regulation of CD19 expression on memory B cells compared with naive B cells may render memory B cells more responsive to transmembrane signals, since CD19 expression levels correlate closely with B cell function in mice (28). These results suggest that the CD19 overexpression in SSc memory B cells induces both the activated phenotype of memory B cells and augmented IgG production.
Although autoantibody production is a common feature of systemic autoimmune disorders, B cell abnormalities have been shown to differ among systemic autoimmune disorders (13–15, 17). The present study confirms that plasmablasts are the predominant blood B cell population in SLE (13, 15). However, a previous study showed a reduced frequency of naive B cells in SLE (13), while this study showed a normal frequency, although the absolute number was similarly diminished in both studies due to B lymphocytopenia. This discrepancy may be due to differences in race and/or level of disease activity in the patient groups studied.
In contrast to SLE, SSc patients had significant reductions in both memory B cells and plasmablasts. A similar reduction in memory B cells was observed in patients with Sjögren's syndrome (14, 17). In Sjögren's syndrome, preferential accumulation of memory B cells in the inflamed parotid gland may lead to their reduction in the blood (14). However, this is unlikely in SSc because few, if any, B cells infiltrated into the affected tissues (29). Instead, the continuous loss of memory B cells by activation-induced apoptosis in SSc could explain the reduction in blood memory B cells, which may result in the subsequent reduction in plasmablasts. However, the decrease in plasmablasts in SSc may also result from their preferential localization in the lungs, since an increased plasma cell number is seen within the lungs (30). Unlike SLE and Sjögren's syndrome, SSc exhibits an expansion of naive B cells, which could be explained by increased B cell production in bone marrow to compensate for the reduction in memory B cells and plasmablasts. Recent studies demonstrated an increased presence of blood germinal center precursors (CD19+,CD27−,CD38+) in juvenile SLE or Sjögren's syndrome (15, 17); however, such a B cell subset was not increased in SSc (data not shown). Thus, SSc had a pattern of disturbed B cell homeostasis that was distinct from other systemic autoimmune disorders.
Although steroid treatment did not affect the increased frequency of naive B cells in dcSSc, it reduced the number of expanded naive B cells almost to the normal level. Similarly, there was no significant difference in the memory B cell frequency between steroid-treated and untreated dcSSc patients; however, steroid treatment further decreased the absolute number of memory B cells in dcSSc to a level similar to that in SLE patients, most of whom were treated with steroids. Since steroids are potent apoptosis inducers (31), the reduction in memory B cells in SSc may be due to a higher responsiveness of memory cells to steroids. However, this possibility is unlikely, since steroid treatment did not further decrease the frequency of memory B cells in dcSSc patients. These results suggest that both naive and memory B cells are highly sensitive to steroids. Alternatively, the reduction in naive and memory B cells may result from decreased early B cell precursors, which are also highly sensitive to steroids (32). In contrast, the frequency of plasmablasts increased and their absolute number did not further decrease after steroid treatment in dcSSc, suggesting that plasmablasts have a markedly lower responsiveness to steroids compared with naive and memory B cells. Although this analysis involved only a small number of untreated dcSSc patients, collectively the results indicate that steroids may have a differential effect on various B cell subsets.
A recent study showed that memory B cells expressing CD80 are able to secrete particularly large amounts of class-switched Ig, and can efficiently and rapidly present antigen to T cells and activate them (33). Therefore, the increase in CD80+ memory B cells in SSc may be responsible for the augmented IgG production by the memory B cells. Furthermore, since autoreactive B cells may function as effective antigen-presenting cells to naive T cells, and this function depends in part on CD80 expression (34), dysregulation of the CD80+ memory B cell subset in SSc may contribute to autoimmune pathogenesis. However, autoantibodies could not be produced by stimulated SSc memory B cells (data not shown). Consistent with this, it has been demonstrated that autoantibodies are not produced by B cells cultured alone, but are produced by B cells and T cells cultured together, suggesting that collaboration between autoreactive T and B cells is essential for in vitro autoantibody production in SSc (35). Collectively, these findings suggest that the hyperreactivity of SSc memory B cells can induce more efficient and stronger activation of helper CD4+ T cells, resulting in further activation of B cells by helper CD4+ T cells. This positive feedback loop of T and B cell collaboration may finally lead to hypergammaglobulinemia, abnormal cytokine secretion, and spontaneous autoantibody production in SSc.
Critical roles of B cells in the development of autoimmunity and disease expression in animal models of systemic autoimmune disorders have been reported. Elimination of B cells in lupus-prone mice results in a complete abrogation of glomerulonephritis, vasculitis, and skin disease (36). Furthermore, lupus-prone mice with B cells that cannot secrete antibodies still develop nephritis and vasculitis (36). This finding suggests that B cells, independent of autoantibodies, are essential for lupus pathogenesis either by serving as antigen-presenting cells or by contributing directly to local inflammation through cytokine secretion. Skin fibrosis in tight-skin mice is consistently improved by the diminished B cell function induced by loss of CD19, which causes a parallel decrease in IL-6 production by B cells (5). Pathogenic autoantibodies from B cells are also important, since K/BxN mice, a model for human rheumatoid arthritis, have hyperactive B cells that cause hypergammaglobulinemia and produce arthritogenic autoantibodies (37). Recent studies have consistently shown that B cell depletion by anti-CD20 mAb is effective in treating patients with rheumatoid arthritis or SLE (38–40), suggesting that B cells are essential for disease expression of human systemic autoimmune disorders. Taken together, the current findings showing disturbed B cell homeostasis and memory B cell hyperactivity in SSc suggest that B cells are a potential target in the treatment of SSc.