The development of biologics that target specific mediators of inflammation has led to several highly successful therapies for the treatment of RA [36, 37]. Biological therapies for RA are directed at neutralizing TNF-α, interleukin (IL)-1, IL-6 or IL-17, blocking T cell co-stimulation [cytotoxic T lymphocyte antigen-4 (CTLA-4)-Ig] or depleting B cells (Fig. 1). Among these biological therapies, B cell depletion  and neutralization of TNF-α [38-49] and IL-6 [50, 51] have been studied most successfully for their effects on ACPA levels and B cell populations. There are some limited data on the effects of oral disease-modifying anti-rheumatic drugs (DMARDs) on ACPA levels  and very few data on the effects of CTLA-4-Ig and IL-17 neutralization on ACPA levels, although there is good reason to believe that T cell co-stimulation and IL-17 also regulate ACPA levels.
Figure 1. Citrullinated peptides serve as antigens when encountered by antigen-presenting cells (APC) including macrophages within the joint. APC present citrullinated peptides via class II major histocompatibility complex (MHC II) to T cells. Macrophages secrete cytokines including interleukin (IL)-1 and IL-6. Cytokines such as IL-6 stimulate B cells via binding to IL-6R, resulting in B cell activation and differentiation of B cells into antibody-producing plasma cells. Tocilizumab is a humanized monoclonal antibody that binds to and inhibits the IL-6R. IL-6 inhibitors that bind directly to IL-6 are now in development. Anakinra is an IL1 receptor antagonist (IL-1ra). T cell activation occurs via two signals delivered by APC. The first signal occurs when an antigen is presented by MHC II to a T cell receptor (TCR). The second signal occurs via co-stimulatory molecules CD80 and CD86 binding to CD28 on the surface of the T cell. Abatacept is a fusion protein composed of the Fc region of immunoglobulin (Ig)G1 fused to the extracellular domain of cytotoxic T lymphocyte-4 (CTLA-4). Abatacept binds CD80/CD86, which blocks CD28 activation. Abatacept is a selective co-stimulation modulator, as it inhibits the co-stimulation of T cells. Activated T cells secrete several cytokines, including tumour necrosis factor (TNF)-α and IL-17. TNF inhibitors neutralize TNF-α. TNF-α is a proinflammatory cytokine that mediates apoptosis . In addition, TNF-α is a growth factor for B lymphocytes inducing the production of IL-1 and IL-6 [106, 107]. Moreover, through nuclear factor kappa B (NF-κB) activation, TNF-α up-regulates MHC molecules, interferon (IFN)-γ production and TNF receptor 2 (TNFR2). Anti-IL-17 inhibitors are under investigation for the treatment of rheumatoid arthritis (RA) and have shown promising results in early-phase studies in RA patients. Activated T cells also stimulate B cells via CD40L (CD154) binding to CD40 on B cells. Rituximab is a chimeric monoclonal antibody that binds CD20, which is found primarily on the surface of B cells. Rituximab binding to B cells results in deletion of B cells.
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B cell depletion (rituximab)
Recent studies have indicated that responsiveness to rituximab therapy (anti-CD20) is better in RA patients who are anti-CCP-positive, suggesting a possible relationship between the pathogenic capacity of ACPA and the effectiveness of rituximab . The effectiveness of B cell depletion therapy with rituximab (anti-CD20) strongly supports a role for B cells in the pathogenesis of RA [52-54]. Besides providing evidence of therapeutic efficacy for B cell depletion in RA, these studies provided evidence for the existence of CD20-negative long-lived plasma cells in humans. In these studies, it was noted that depletion of CD20+ naive and memory B cells had little effect on total serum immunoglobulin levels and no effect on anti-tetanus antibody levels . However, autoantibody levels of RF and anti-CCP decreased significantly with anti-CD20 therapy and anti-CCP levels were associated with improvement and relapse in RA patients treated with rituximab  (Table 1). These data suggested that most serum antibodies, and in particular vaccine-induced antibodies such as tetanus toxoid antibodies, are produced by CD20-negative long-lived bone marrow plasma cells. In contrast, autoantibodies in RA may be produced primarily by short-lived CD20+ memory B cells that differentiate into short-lived antibody-secreting plasmablasts [55-57]. Studies have shown that short-lived plasmablasts in synovium secrete autoantibodies and are an important source of ACPA and RF [58, 59]. Short-lived synovial plasmablasts are reduced by rituximab therapy, and plasmablast depletion during rituximab therapy correlates with responsiveness to rituximab therapy [34, 35]. The reduction of short-lived synovial plasmablasts by rituximab provides a potential pathophysiological mechanism for the ability of rituximab to reduce ACPA levels and to improve arthritis symptoms in RA patients.
Table 1. Summary of studies on the effects of B cell depletion with rituximab on anti-cyclic citrullinated peptide (CCP).
|Author (reference)||Year||Disease duration||Treatment||Study length||Effect of non-TNF biologic on ACPA levels|
|Cambridge ||2003||18 years||Rituximab with or without i.v. cyclophosphamide||33·5 months||CCP levels reduced, especially in responders|
|Kormelink ||2010||12 years||Rituximab||6 months||IgG-ACPA reduced in good–moderate responders|
|Rosengren ||2008||12·2 years||Rituximab||8 weeks||Reduced anti-CCP antibody (not in synovial tissue)|
|Toubi ||2007||NSa||Rituximab||4 months||Unchanged anti-CCP antibodies despite documented clinical response|
B cell depletion with rituximab in RA also provided important information about the role of memory B cell subsets in the pathogenesis of RA. Leandro et al.  reported that disease relapse in rituximab-treated RA patients was associated with a higher frequency of B cells, which had a memory (CD27+) phenotype at the time of B cell repopulation. In this study, patients with greater than 3 × 106/l CD27+ memory B cells at the time of B cell repopulation had an earlier relapse than patients with fewer CD27+ memory B cells [60, 61].
Other researchers have suggested that B cell depletion may have other effects on B cells that lead to indirect reductions of autoantibody levels. For example, some researchers have suggested that B cell depletion may mimic TNF-α blockade by eliminating lymphotoxin-alpha (LT-α) and TNF-α-secreting B cells .
In contrast to the data on rituximab therapy, there are less consistent data on the correlation between declines in anti-CCP levels and clinical responsiveness to TNF-antagonists in RA patients. Although TNF antagonists reduce RF levels [39-49], these same studies have not established conclusively whether oral DMARDs and/or TNF-antagonists reduce anti-CCP levels. As summarized in Table 2, some studies have identified reductions in anti-CCP levels with TNF-antagonist therapy [38, 39, 43-46, 48], while others have seen no effect of TNF-antagonists on anti-CCP levels [40-42, 47, 49]. In some of these studies TNF-antagonist therapy was compared to oral DMARD therapy [43, 45]; TNF antagonists, but not oral DMARDs, decreased anti-CCP levels, although in these studies subjects receiving only oral DMARDs did not have as great a reduction in disease activity. All these studies examined the effect of TNF antagonists on anti-CCP levels and did not examine the effect of TNF antagonists on specific ACPA levels.
Table 2. Summary of studies on the effects of tumour necrosis factor (TNF)-antagonists on anti-cyclic citrullinated peptide (CCP) levels.
|Author (reference)||Year||Disease duration||Treatment||Study length||Outcome|
|Alessandri ||2004||n.s.a||MTX + infliximab||24 weeks||Reduced anti-CCP levels|
|Bobbio-Pallavicini ||2004||9·4 years||MTX + infliximab||78 weeks||No reduction in anti-CCP levels|
|Caramaschi ||2005||12·6 years||MTX or AZA + infliximab||22 weeks||No reduction in anti-CCP levels|
|De Rycke ||2005||n.s.‡||MTX + infliximab||30 weeks||No reduction in anti-CCP levels|
|Atzeni ||2006||6–8 years||MTX versus MTX + adalimumab||48 weeks||Reduced anti-CCP levels only in group treated with adalimumab|
|Chen ||2006||8–9·5 years||MTX versus MTX + etanercept||24 weeks||Reduced anti-CCP levels|
|Cuchacovich ||2008||–||MTX + adalimumab||24 weeks||Reduced anti-CCP levels|
|Vis ||2008||10 years||MTX + infliximab||46 weeks||Reduced anti-CCP levels|
|Bacquet-Deschryver ||2008||8 years||MTX or LEF + anti-TNF||52–104 weeks||No reduction in anti-CCP levels|
|Bos ||2008||7·9–9·5 years||MTX + adalimumab||28 weeks||Reduced anti-CCP levels|
|Bruns ||2009||–||Oral DMARD + infliximab||48 weeks||No reduction in anti-CCP levels|
There are several factors that may be confounding the analysis of anti-CCP levels during TNF antagonist treatment. For example, differences in disease duration may affect the ACPA response during TNF antagonist therapy; a reduction in anti-CCP levels with anti-TNF therapy was more likely in RA patients with a disease duration of less than or equal to 1 year [38, 40]. Although all anti-CCP2 assays are derived from the same source, some have suggested that the inadequate dilution of serum samples makes the anti-CCP test too sensitive, thereby preventing the detection of variations in the antibody titre during treatment . Other confounders may also affect ACPA levels, including cigarette smoking and periodontal infections with P. gingivalis , which probably provide citrullinated antigenic sources for ACPA production. In addition, many of the published studies did not control for SE status, which may influence ACPA levels due to the potential for stronger and sustained autoimmune responses in those with the SE.
There are several potential mechanisms whereby TNF-α may regulate ACPA levels. One postulate is that TNF antagonists can down-regulate the production of inflammatory cytokines, thereby modulating autoantibody generation, particularly in the synovial compartment . In support of this hypothesis, Anolik et al.  found that lymphoid architecture was altered in patients on etanercept, an anti-TNF therapy that also blocks LT-α. RA subjects treated with etanercept had a significant decrease in follicular dendritic cell (FDC) staining and germinal centres were reduced significantly in number and size . This suggested that TNF antagonists altered B cell populations and probably impacted the ability of B cells to enter or survive a germinal centre reaction.
The changes in lymphoid architecture mediated by TNF antagonists may contribute to the reductions in memory B cells noted in RA patients treated with these therapies [62, 65, 66], which may be important, as memory B cells in RA patients express anti-CCP autoantibodies . In addition, TNF antagonists decrease the proportion of memory B cells expressing CD86 after 6 months of therapy . This is probably important because CD86 and the related molecule CD80 are co-stimulatory proteins that are up-regulated on the cell surface during B cell activation [69, 70]. RA patients have a higher proportion of naive and memory B cells expressing CD86 than healthy controls , which probably favours increased ACPA production.
Immune complexes formed from citrullinated fibrinogen and human ACPA stimulate TNF-α production from macrophages , and co-ligation of the FcγR and Toll-like receptor-4 (TLR-4) by immune complexes containing citrullinated fibrinogen also stimulate TNF-α production . In RA patients, Catalan and colleagues found reduced FcγRIIB expression on memory B cells and plasmablasts compared to healthy controls , suggesting that TNF-α or other downstream cytokines may influence the expression of FcγRIIb on B cells. In addition, a polymorphism in the FcγRIIB gene has been linked to susceptibility to systemic lupus erythematosus (SLE) in humans, and altered function of FcγRIIB could also affect the humoral response against citrullinated proteins in RA patients . For example, Chen et al.  demonstrated an association between a functional polymorphism in FcγRIIB and anti-CCP-positive RA in an Asian cohort.
Although the emphasis in this section has been on the negative effect of TNF antagonism on ACPA levels, studies have shown that therapy with TNF antagonists, but not therapy with other biologics or DMARDs, is associated with the development of anti-nuclear (ANA) and anti-dsDNA autoantibodies [40, 72]; some patients treated with TNF antagonists develop a clinical syndrome that resembles SLE . The relatively selective induction of ANA and anti-dsDNA autoantibodies during treatment with TNF antagonists may be due to the effects of TNF antagonists on apoptosis and enhanced nuclear antigen presentation on the surface of apoptotic cells [40, 72]. A recent report indicated that TNF antagonists increased levels of cytoplasmic Lyn preferentially and up-regulated B cell surface CD20 expression . Lyn plays a role in the initiation of the B cell receptor (BCR)-mediated pathway . Stimulation of B cells via the BCR results in the immediate activation of Lyn, which phosphorylates tyrosine residues of Ig-α/β rapidly and activates a number of downstream signalling proteins, including Syk . This results ultimately in the expression of antibodies.
Recent clinical trials have suggested that blocking IL-17 may be an effective treatment for RA [78, 79]. IL-17 is produced by Th17 cells, which are also important producers of TNF-α . IL-6 drives Th17 cell differentiation by activating the transcription factor STAT-3 [81, 82]. A recent study in Nature Immunology by Doreau et al.  indicated that B cell development and differentiation of B cells into immunoglobulin-secreting cells is regulated by IL-17A and BAFF. IL-17 alone or in combination with BAFF influences the survival, proliferation and differentiation of human B cells directly, and the two in combination are more efficient than either cytokine separately in promoting persistence of self-reactive B cells .
B cell depletion by rituximab followed by increased BAFF mRNA expression in human monocyte-derived macrophages of patients with RA is believed to represent a homeostatic attempt to replenish B cells . BAFF transgenic animals express high levels of BAFF and develop autoantibodies . In the study by Doreau et al. , IL-17A serum levels were elevated in SLE patients and disease severity in SLE patients was correlated directly with IL-17A levels. IL-17A serum levels are also elevated in RA patients and, like SLE patients, BAFF serum levels are elevated in RA patients [78, 79, 85-88] and correlate with disease activity [88, 89]. The differentiation of B cells into immunoglobulin-secreting B cells was regulated by nuclear factor-kappa B (NF-κB_ and the NF-κB-regulated transcription factor TWIST1, which induced the expression of TWIST2 and BCL2A1. TWIST2 expression was up-regulated in B cells from patients with SLE and was correlated directly with SLE disease severity and IL-17A levels; BCL2A1 transcript levels in B cells correlated with IL-17A levels. Doreau et al.  also found that IL-17A and BAFF induced expression of MEF2C, which is an important mediator of BCR-induced proliferation . Importantly, IL-17A and BAFF expression are regulated by TNF [91-94] and TWIST1 is over-expressed in the synovium of patients with RA .
Tofacitinib (CP-690,550) is a novel oral Janus kinase (JAK) inhibitor (Fig. 2) that was approved recently by the American Food and Drug Administration (FDA) for the treatment of rheumatoid arthritis. To date, studies in humans on the effects of tofacitinib treatment on ACPA and RF levels have not been reported. However, results from studies of animal models of arthritis indicate that IL-6 levels decrease following administration of tofacitinib [97, 98]. Furthermore, Tanaka and Yamaoka reported that tofacitinib inhibited human IL-17 expression in synovial tissue [97, 98]. Taken together, because tofacitinib suppresses IL-6 and IL-17, and as IL-6 and IL-17 are associated with ACPA and RF production, it seems likely that tofacitinib reduces RF and anti-CCP levels. Clinical studies will be needed to assess this possibility.
Although oral DMARDs reduce RF levels , these studies have not established conclusively whether oral DMARDs reduce anti-CCP levels. As noted above, in studies comparing TNF-antagonist therapy to oral DMARD therapy [43, 44] (Table 3) TNF antagonists, but not oral DMARDs, decreased anti-CCP levels, but in these studies subjects who received only oral DMARDs did not have significant reductions in disease activity. In a study by Mikuls et al. , in which subjects received methotrexate (MTX), sulfasalazine (SSZ) and hydroxychloroquine (HCQ) and had substantial reductions in disease activity, the authors found that reductions in anti-CCP levels were greatest in subjects with early disease while RF reductions were dependent upon disease activity reductions. Besides the Mikuls study, no other published studies have identified an effect of oral DMARDs on anti-CCP levels. Although oral DMARDs reduced both RF and anti-CCP levels in the study by Mikuls et al.  (Table 3), it remains unclear what mechanisms are involved in the reduction of autoantibody levels by oral DMARDs, given that less effective combinations of oral DMARDs did not reduce anti-CCP levels [43, 44].
Table 3. Summary of studies on the effects of oral disease-modifying anti-rheumatic drugs (DMARDs) on anti-cyclic citrullinated peptide (CCP) levels.
|Author (reference)||Year||Disease duration||Treatment||Study length||Effect of DMARD on ACPA level|
|Mikuls ||2004||Study 1||Study 1||13·7 ± 8·6 months||Reduced anti-CCP level in disease duration ≤ 12 months|
|<1 year||MTX versus HCQ/SSZ|
|Study 2||Study 2|
|<1 year||Minocycline versus placebo|
|Study 3||Study 3|
|52·4 ± 82·4 months||Minocycline versus HCQ|
|Atzeni ||2006||6–8 years||MTX versus MTX + adalimumab||6 months for MTX group (stable clinical course of the disease)||No effect on APCA in MTX group|
|Chen ||2006||8–9·5 years||MTX versus MTX + etanercept||24 weeks||No significant reduction in anti-CCP levels in MTX group|