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Systemic sclerosis (SSc) is a connective tissue disease characterized by autoimmunity and tissue fibrosis. In patients with SSc, there is a close association between the presence of specific autoantibodies and the development of clinical features. Although it is known that cytokines, including transforming growth factor-β, can modulate the synthesis of extracellular matrix by fibroblasts, it is not clear how autoimmunity and tissue fibrosis are interrelated. Several recent lines of evidence indicate a potential role for B cells in the development of SSc. CD19 is a critical regulator of B-cell signaling thresholds, and B cells from SSc patients exhibit increased expression of CD19, a molecule that induces SSc-specific autoantibody production in transgenic mice. Both SSc patients and tight-skin mice, a genetic model of SSc, have intrinsic B-cell abnormalities characterized by chronic B-cell activation. Remarkably, CD19 loss or B-cell depletion using antimouse CD20 antibody suppresses the development of skin hyperplasia and autoimmunity in tight-skin mice. Additionally, a recent study revealed a possible beneficial effect of antihuman CD20 antibody (rituximab) therapy for SSc patients. As B cells have a variety of functions, further investigation into the pathogenic roles of B cells, as well as trials of B-cell-targeting therapies, may shed new light on the pathogenesis of SSc.
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Systemic sclerosis (SSc) is an autoimmune disease characterized by excessive extracellular matrix (ECM) deposition in the skin and other visceral organs. The molecular basis for SSc is unknown; however, a number of studies have focused on the pathogenic mechanisms of immune activation and tissue fibrosis in SSc.1–5 The detection of specific circulating autoantibodies including anti-topoisomerase I antibody, anti-centromere antibody and anti-RNA polymerase antibody is a common identifying feature and usually precedes disease onset. However, these antibodies are not thought to contribute directly to tissue fibrosis. Instead, it is believed that the various cytokines produced by immune-activated cells modulate the synthesis of ECM by fibroblasts. Nonetheless, it is not clear how systemic autoimmunity and tissue fibrosis are interrelated.
Previous studies had focused largely on T cells as pathogenic mediators of SSc. Recently, however, a key role for B cells and plasma cells in the development of systemic autoimmune diseases has been discovered.6–8 In fact, B-cell depletion therapy by chimeric antihuman CD20 antibody has proven to be an effective therapy for several autoimmune diseases, including systemic lupus erythematosus and rheumatoid arthritis. In this review, we will highlight how altered B-cell function may contribute to the development of SSc. Targeting of B cells as a possible future therapeutic strategy for SSc patients will also be discussed.
B-cell development and function
As B cells develop, they traverse a tightly regulated pathway, from their inception as early progenitors to their terminal differentiation into plasma cells (Fig. 1).9 Progression through this pathway depends on signals that control both negative and positive selection of B cells in the bone marrow and in the peripheral lymphoid tissues. B-cell signaling thresholds are controlled by response regulators that augment or diminish B cell signals during responses to self and foreign antigens (Fig. 2).10,11 Thus, abnormalities in the function or expression of response regulators may result in autoimmunity. Among these response regulators, the cell surface signal transduction molecule CD19 is the most potent positive regulator. In contrast, CD22 interacts with both the B-cell antigen receptor (BCR) and CD19, but plays a negative regulatory role in B-cell signaling.12
Figure 1. B-cell development. The expression of CD19, CD20, CD22 and BAFF receptors during B-cell development. BAFF, B-cell-activating factor belonging to the tumor necrosis factor family.
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Figure 2. Response regulators of B-cell signaling. B-cell responses against foreign or self-antigens are controlled in part by interactions between positive (CD19, CD21 et al.) and negative regulators (CD22, CD72 et al.). Abnormal modulation of these regulators may result in autoantibody production.
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Recent assessment of B-cell function has indicated that these cells are more than just the precursors of antibody-secreting plasma cells. It is now clear that B cells serve an essential role as regulators of immune responses.13 B-cell functions are diverse, and include antigen presentation, production of cytokines, lymphoid organogenesis, differentiation of T-effector cells and modulation of dendritic cell function (Fig. 3).13 Therefore, abnormalities in these B-cell functions could contribute to the induction of systemic autoimmunity independently of autoantibody production.
Figure 3. A variety of B-cell functions. B cells are well known as precursors of antibody-producing plasma cells. However, B cells have a variety of functions including antigen presentation, co-stimulation, cytokine production, lymphoid organogenesis and dendritic cell regulation.
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B-cell abnormalities in SSc patients
Analysis of gene expression in SSc skin lesions through DNA microarrays has revealed upregulation of genes related to B cells.14 B-cell infiltration is found in the lesional skin of SSc patients and lungs of patients with SSc-associated interstitial lung disease.14–16 B cells from SSc patients have been shown to exhibit CD19 overexpression.17 Furthermore, a polymorphism of the CD19 promoter region was detected, and was found to correlate with higher CD19 expression on B cells and susceptibility to SSc.18 Together, these findings suggest the presence of intrinsic B-cell abnormalities as well as possible mechanisms for B-cell effector function in SSc. Consistent with this, peripheral B-cell homeostasis and subsets are disturbed in SSc; naive B cells expand while memory B cells and plasmablasts/early plasma cells diminish due to increased apoptosis.19 Furthermore, memory SSc B cells are chronically activated in vivo, possibly due to CD19 overexpression.19 Therefore, it is plausible that the continuous loss of memory B cells and plasmablasts/early plasma cells leads to increased production of naive B cells in the bone marrow in order to maintain B-cell homeostasis. Although memory B-cell numbers are decreased in SSc patients, those cells remaining have an enhanced ability to produce immunoglobulin (Ig) and possibly autoantibody. Therefore, SSc patients have distinct abnormalities of blood homeostasis and B-cell compartments that are characterized by expanded naive B cells and activated, but with diminished numbers of, memory B cells. In addition, CD19 overexpression in memory SSc B cells may be related to their hyper-reactivity, because memory B cells, as well as naive B cells, from SSc patients overexpress CD19. Polymorphism of CD22, a critical negative regulator of BCR signaling, is also associated with susceptibility to limited cutaneous SSc.20 This polymorphism leads to reduced CD22 surface expression on B cells in SSc patients. Thus, expression of both positive and negative regulators of B-cell signal transduction is altered in SSc patients.
A previous report demonstrated that BAFF (B-cell-activating factor belonging to the tumor necrosis factor family, also known as BLys) signaling in B cells contributed to B-cell functional abnormalities and disease development in patients with SSc.21 Patients with SSc have elevated serum levels of soluble BAFF, a potent B-cell survival factor, and this was found to correlate positively with the severity of SSc. BAFF mRNA expression is upregulated in the affected skin of patients with active SSc.21 Furthermore, BAFF receptor expression on B cells is increased in SSc patients, and SSc B cells have an augmented ability to produce IgG and interleukin (IL)-6, reflecting the possible contribution of BAFF signaling to the development of this disease.
B-cell abnormalities in mouse models of SSc
The tight-skin (TSK/+) mouse is a model for human SSc and was originally identified as a spontaneous mutation that resulted in increased synthesis and accumulation of collagen and other ECM proteins in the skin.22 TSK/+ mice produce autoantibodies against SSc-specific target autoantigens including topoisomerase I, fibrillin 1 and RNA polymerase I.
Similar to human SSc, heightened CD19 signaling is present in TSK/+ mice, but CD19 overexpression has not been detected on TSK/+ B cells.23 However, constitutive CD19 tyrosine phosphorylation is augmented in TSK/+ B cells compared with wild-type B cells. Furthermore, intracellular Ca2+ responses generated by CD19 ligation are elevated in TSK/+ B cells. Consistent with heightened CD19 signaling, TSK/+ B cells exhibit a phenotype indicative of chronic activation. Thus, the CD19 signaling pathway appears to be constitutively activated in TSK/+ B cells, and this abnormal basal signaling likely results in the characteristic hyper-responsiveness of TSK/+ B cells.
While TSK/+ mice have significantly elevated serum levels of autoantibodies such as anti-topoisomerase I antibody, CD19 loss in TSK/+ mice completely abrogates production of these autoantibodies.23 Therefore, loss of the CD19 signaling pathway dramatically inhibits autoantibody production in TSK/+ mice. Additionally, CD19 deficiency in TSK/+ mice results in an approximately 40% reduction in skin thickness. Therefore, B cells contribute to skin fibrosis in TSK/+ mice through a CD19-dependent pathway.
B-cell hyper-responsiveness in TSK/+ mice may also result from abnormalities in the function of response regulators other than CD19. For example, function of the inhibitory signaling molecule CD22 is specifically impaired in TSK/+ B cells.24 CD22 has immunoreceptor tyrosine-based inhibitory motifs that recruit src homology 2 domain-containing tyrosine phosphatase-1 (SHP-1) when phosphorylated. Importantly, CD19 is known to be a major target of the CD22/SHP-1 inhibitory pathway.12 CD19 may also be influenced indirectly by CD22, in that disrupted negative regulation of signaling by CD22 in TSK/+ B cells may result in abnormal activation of downstream signal transduction molecules including CD19.
Recently, we have assessed the roles of B cells in the development of skin thickness and autoimmunity in TSK/+ mice by using an antibody against mouse CD20. B-cell depletion using this antibody prevents autoantibody production in TSK/+ mice and significantly reduces skin hyperplasia if administrated before disease onset.25 B-cell depletion early after disease onset has intermediate effects, while the treatment of mice with established disease is not effective. Therefore, our study indicates that B-cell depletion therapy using antihuman CD20 antibody may be useful to some extent for suppressing the development of skin fibrosis in early stage SSc patients.
Another recent study found that serum BAFF levels were significantly elevated in TSK/+ mice, similar to what has been observed in SSc patients.26 Moreover, administration of a BAFF antagonist inhibited the development of skin fibrosis and autoantibody production in TSK/+ mice. The skin from TSK/+ mice exhibited upregulated expression of fibrogenic cytokines, and the use of a BAFF antagonist significantly suppressed these while augmenting antifibrogenic cytokines. Additionally, BAFF-stimulated TSK/+ B cells had a significantly enhanced ability to produce IL-6, a cytokine that may contribute to fibrosis and autoimmunity.
In a bleomycin-induced skin and lung fibrosis mouse model of SSc,27 CD19 was found to regulate fibrogenic cytokine production and autoantibody production by B cells mainly through Toll-like receptor 4 (TLR4) signaling. TLR4 signaling is activated by hyaluronan, an endogenous TLR4 ligand that is upregulated in the skin and lung by bleomycin treatment.28 Another study demonstrated that CD19 signaling was associated with the development of bleomycin-induced pulmonary fibrosis by controlling B-cell infiltration into lung tissue, possibly through upregulation of a specific chemokine on B cells.29 Taken together, multiple murine studies have demonstrated a role for B cells in regulating the development of SSc, and augmented CD19 signaling appears to be critically involved in this process.
B-cell activation and cytokine expression
CD4+ helper T cells (Th cells) have long been divided into two subgroups, Th1 and Th2, characterized by distinct cytokine secretion patterns. A third group, the Th17 subset, has been identified, but as its role in SSc is currently unknown, it will not be discussed here. While Th1 cells secrete γ-interferon (IFN-γ), IL-2 and tumor necrosis factor (TNF)-α, thus promoting cell-mediated immunity, Th2 cells produce IL-4, IL-5, IL-6, IL-10 and IL-13, thereby facilitating humoral immunity. These Th2 cytokines enhance Ig production by B cells. Furthermore, IL-4, IL-6 and IL-13 also stimulate the synthesis of collagen by human fibroblasts.30,31 In contrast, Th1 cytokines such as IFN-γ and TNF-α suppress collagen production by fibroblasts in vitro.31 Therefore, in general, a relative shift to Th2 cytokines can induce tissue fibrosis and antibody production.
Activated B cells are known to produce IL-6 and IL-10, both of which induce Th2-dominant immune responses.32,33 Because IL-6 has been reported to affect tissue fibrosis,30,34,35 increased IL-6 production by activated B cells may directly promote the tissue fibrosis seen in SSc patients. In the TSK/+ murine model, B cells stimulated with anti-IgM plus anti-CD40 antibodies produced high amounts of IL-6 in comparison to wild-type B cells.23
Activated B cells express high levels of class II major histocompatibility complex (MHC) and co-stimulatory molecules and are nearly as effective as dendritic cells in their antigen-presenting capacity. Antigen-presenting cells play a critical role in the differentiation of Th. Dendritic cells preferentially evoke a Th1 response by producing IL-12,36 whereas B cells promote the development of Th2 cells.37,38 Alternatively, B cells might influence the Th1/Th2 balance by regulating the function of dendritic cells; IL-10 produced by activated B cells inhibits IL-12 production by dendritic cells, thus promoting Th2 differentiation.39 These findings suggest that B cells are critical for the development of Th2 responses.
Although macrophages are an important source of transforming growth factor (TGF)-β1, activated B cells also secrete significant levels of active TGF-βin vitro.40 Therefore, TGF-β released from activated B cells in SSc patients may induce excessive collagen synthesis by fibroblasts. Additionally, tissue fibrosis induced by TGF-β may be enhanced by other cytokines such as connective tissue growth factor (CTGF), IL-4, IL-13 and monocyte chemotactic protein-1 (MCP-1).41–44 IL-4 induces TGF-β gene expression, and there is an interaction between IL-4 and TGF-β in regulating collagen gene expression in vitro.45 Because TGF-β can promote the expansion of antigen-specific Th2 cells,46 and CTGF gene expression is induced by TGF-β and IL-4 in skin and lung fibroblasts,47,48 TGF-β, CTGF and Th2 cytokines may cooperatively contribute to the pathogenesis of SSc (Fig. 4).
Figure 4. T helper cell (Th)1/Th2 balance and tissue fibrosis. In general, Th2-dominant immune responses induce tissue fibrosis rather than Th1 responses. Growth factors such as transforming growth factor-β and connective tissue growth factor (CTGF) likely contribute to fibrosis through cooperation with Th2 cytokines. Chronically activated B cells may directly or indirectly enhance Th2 cytokine production during the pathogenesis of systemic sclerosis. IFN, interferon; IL, interleukin; TGF, transforming growth factor; TNF, tumor necrosis factor.
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Relationship between autoimmunity and tissue fibrosis
A possible model of the relationship between systemic autoimmunity and tissue fibrosis in SSc and TSK/+ mice is shown in Figure 5. Polymorphism or mutation of B-cell response regulators such as CD19 may increase B-cell signaling, resulting in autoantibody production through breakdown of B-cell peripheral tolerance, as observed in mice overexpressing CD19.10 B cells from human SSc and TSK/+ mice are chronically activated in vivo, possibly due to enhanced B-cell signaling, which may result in the development of skin fibrosis through augmented production of cytokines, such as IL-6 and TGF-β. Furthermore, activated B cells may directly or indirectly regulate T cells, and Th2-polarized cytokines may promote collagen synthesis. Thus, both systemic autoimmunity and tissue fibrosis in SSc may be derived from intrinsic B-cell abnormalities. Consequently, both B cells and B-cell-specific signaling molecules may prove to be efficacious therapeutic targets in SSc patients.
Figure 5. A possible model linking systemic autoimmunity and tissue fibrosis in systemic sclerosis. Polymorphism or mutation of B-cell response regulators such as CD19 induces chronic B-cell activation. This may result in autoantibody production through a breakdown of B-cell peripheral tolerance. It is also possible that chronic B-cell activation results in the development of tissue fibrosis through the continuous production of cytokines, such as interleukin (IL)-6 and transforming growth factor-β (TGF-β). Furthermore, activated B cells may regulate the activation and differentiation of T cells by antigen presentation or through the co-stimulatory effects of B cells. Studies indicate that B cells induce T helper cell (Th)2-predominant cytokine production by T cells, and these Th2 cytokines may also promote tissue fibrosis. Thus, autoimmunity and tissue fibrosis may be in part derived from B-cell abnormalities in systemic sclerosis.
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Rituximab, a chimeric IgG1 monoclonal antibody that specifically binds to CD20 on B cells, is approved for the treatment of certain non-Hodgkin’s lymphomas, although it is also increasingly being used off-label for the treatment of autoimmune diseases such as multiple sclerosis, lupus nephritis and idiopathic thrombocytopenic purpura, and is also used for rheumatoid arthritis.49 This has led investigators to speculate that B cells might play a pathogenic role in these autoimmune diseases. Furthermore, use of novel monoclonal antibodies directed against B-cell surface antigens including CD19, CD20, CD22 and CD52, that directly target B cells, have been under trial for treatment of autoimmune diseases (Fig. 6).50
Figure 6. Approaches that target B cells. B-cell-targeted therapies currently under clinical trial for treatment of autoimmune diseases. Deletion of autoreactive B cells can be induced by monoclonal antibodies directed against cell surface proteins expressed on B cells. Autoreactivity can be suppressed by blocking B-cell activation pathways, either intrinsically or extrinsically, with monoclonal antibodies, soluble receptors or ligands. BAFF, B-cell-activating factor; IL, interleukin; TNF, tumor necrosis factor.
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Regarding SSc, standard doses of rituximab depleted B cells in both peripheral blood and skin, and were well tolerated in a small, open-label study.15 However, the treatment did not show significant effects on skin sclerosis or autoantibody titers. Nonetheless, a significant reduction in the myofibroblast score was seen, and the patients remained clinically stable during the study period without developing progressive pulmonary or renal disease. In addition, a 24-week small open-label study demonstrated that rituximab treatment significantly improved the skin score and histological findings without related serious side-effects.51 Furthermore, a 1-year randomized controlled small study demonstrated that rituximab significantly improved lung function in SSc patients with interstitial lung diseases.52 There are also some additional case reports supporting the effectiveness of rituximab for treatment of SSc. For example, a case with SSc-associated interstitial lung diseases was successfully treated with rituximab.53 Although the exact mechanism by which rituximab exerts its effects in SSc remains unclear, improvement of skin sclerosis was more commonly found in patients with evidence of B-cell depletion in the skin.52 Larger, randomized multicenter studies with longer evaluation in early diffuse cutaneous SSc patients will be needed to confirm the efficacy and safety of rituximab for the treatment of SSc.
Regulatory B cells
B cells positively regulate immune responses through activation of T cells and production of antibodies. However, specific minor subsets of B cells can negatively regulate inflammation and autoimmunity.54–56 The Tedder laboratory has recently shown that IL-10-producing regulatory B cells (called B10 cells) predominantly localize within a rare CD1dhighCD5+ B-cell subset.54 Although B10 cells only represent 1–2% of splenic B cells, they dramatically inhibit the induction of antigen-specific inflammatory reactions and autoimmunity.54,55 Thereby, B cells may regulate multiple components of the immune system and development of autoimmune diseases through varied combinations of their multipurpose cellular and humoral functions.57,58
At this time, formal proof for the existence and inhibitory function of regulatory B cells remains to be found in humans. It may be possible to identify pathways leading to regulatory B-cell activation, expansion and function, which will allow this potent B-cell subset to be manipulated for therapeutic benefit. For example, the selective expansion of autoantigen-specific regulatory B cells may be sufficient to diminish autoimmunity. Alternatively, an approach that selectively depletes mature B cells while sparing regulatory B cells may offer a particularly potent therapy for autoimmune diseases including SSc. Future B-cell-directed therapies coupled with mechanistic assays to monitor B-cell subsets and their functions will enable us to clarify the mechanisms of autoreactivity and pathogenicity in SSc, and elucidate the roles of B cells in the development of this disease.
Greater understanding of B-cell involvement in the progression of SSc will require the testing of B-cell-targeted therapies in well-designed clinical trials that include mechanistic assays to monitor specific B-cell subsets and their functions. While systemic B-cell depletion using anti-CD20 antibody is one potential method for treating SSc, intrinsic or extrinsic regulation of B-cell-related signaling related CD19, BAFF, CD22 and CD40 are also a promising treatment options (Figs 1,6). Furthermore, identification of pathways that lead to induction of regulatory B cells will likely result in the ability to expand this cell population selectively, and this eventually may become the preferred option for treating SSc.