Markers of B-lymphocyte activation are elevated in patients with early rheumatoid arthritis and correlated with disease activity in the ESPOIR cohort

For decades – ever since the discovery of rheumatoid factor (RF) – B cells have been known to play a pathogenic role in established rheumatoid arthritis (RA) [1–4]. More recently, the secretion of RF and antibodies against cyclic citrullinated peptide (anti-CCP) was demonstrated to precede RA clinical onset by many years [5, 6], which suggests that activation of autoreactive B cells might be an early pathogenic event. However, very little is known about the activation of alloreactive B cells in patients with early RA. Markers of B-cell activation, such as beta2-microglobulin, immunoglobulin levels, free light chains (FLCs) of immunoglobulins, and BAFF (B-cell activating factor of the tumor necrosis factor [TNF] family) – which are all elevated in established RA [7–10] – could be useful in determining the extent of B-cell activation in early RA. One of the objectives of the French multicenter prospective cohort, ESPOIR [11], is to determine the specific biological features of early RA by comparing serum samples from patients with either early RA or other early arthritis who are naïve to disease-modifying antirheumatic drugs (DMARDs) and steroids. In this study, we assessed baseline levels of several markers of nonspecific B-cell activation, such as beta2-microglobulin, immunoglobulin FLCs, IgG, IgA, and IgM as well as serum BAFF. Since the BAFF 871T>C polymorphism is reported to be correlated with serum BAFF level in various diseases [12–14], patients were also genotyped for this polymorphism. Our findings show that baseline serum markers of B-cell activation are higher in patients with early RA than in patients with undifferentiated arthritis (UA). In addition, their increase is correlated with disease activity but is independent of serum BAFF levels and the BAFF gene polymorphism.

These findings from ESPOIR, the prospective French multicenter cohort of early arthritis patients, demonstrate that serum markers of B-cell activation are elevated in patients with early RA compared with undifferentiated early arthritis. They also show that markers of B-cell activation are correlated with disease activity. Lastly, they indicate that serum BAFF might not drive the specific B-cell activation we observed in early RA. ESPOIR offered us the opportunity to investigate markers of B-cell activation in patients with early arthritis who had not yet been exposed to corticosteroids or DMARDs at enrollment. The disease-related increase of markers of B-cell activation might otherwise have been masked by these treatments, which can modulate these marker levels [16, 17]. Another obvious advantage of performing such a study with this cohort was the centralization of biological, immunological, and radiological examinations and the prospective follow-up, which allows us to determine whether markers of B-cell activation can serve as predictive factors of long-term clinical activity.

The main limitations of this study were the short duration of follow-up (1 year) so far, since ACR criteria for RA might be fulfilled later, and the absence of validated criteria for early RA. Although the 1987 ACR criteria lack specificity for early RA diagnosis [18], they remain widely used in patients with early arthritis in the absence of any other international criteria for early RA. The percentage of patients who fulfilled these 1987 ACR criteria of RA is higher in the ESPOIR cohort (72% at inclusion and 79% at 1 year) than in other early arthritis cohorts [19, 20]. This may be related to the inclusion criteria of ESPOIR, which were more stringent (at least two swollen joints and 6 weeks of duration) than those of some other early arthritis cohorts. It has been suggested that the specificity of ACR criteria in early RA may improve when the physician's level of confidence in the RA diagnosis is high [18]. At the ESPOIR 1-year follow-up visit, the rheumatologists completed a visual analog scale (VAS) assessing their confidence in the RA diagnosis for each patient. We were therefore able to conduct a separate analysis of the 367 of the 578 patients fulfilling 1987 ACR criteria for RA for whom this VAS was equal to or greater than 75 mm on a scale of 100 mm. Even when these more stringent criteria for RA were used, levels of IgG, IgA, and kappa FLCs remained higher in early RA than in undifferentiated early arthritis (data not shown). Lastly, since few serum markers of other immune cells are available in routine, only markers of B-cell activation were assessed in the present study, which did not address the pathogenic role of other immune cells.

The ESPOIR cohort confirms the pathogenic activation of autoreactive B cells in early RA, reflected by the association between anti-CCP and IgA-RF with early radiological erosions, also observed in previous early RA cohorts [21, 22]. The originality of the present study is to also assess markers of activation of alloreactive B cells in early arthritis, such as total levels of immunoglobulins, FLCs of immunoglobulins, and beta2-microglobulin.

The first important finding is that serum level markers of alloreactive B-lymphocyte activation are higher in early RA than in UA. Some features of B-cell activation were nonetheless observed in patients with UA, including BAFF levels that were higher than in healthy donors, elevated IgG in 55% of the patients, and elevated levels of immunoglobulin FLCs in 22% (compared with 0% in healthy donors, according to previous studies [9, 23]). During follow-up, it will be interesting to study whether patients with UA and high levels of markers of B-cell activation may more frequently develop RA or another autoimmune disease, such as systemic lupus erythematosus (SLE) or primary Sjögren syndrome, both of which feature elevated levels of markers of B-cell activation [9]. The markers we assessed reflect different stages of B-lymphocyte activation. All of them except IgM and BAFF were elevated in early RA. Beta2-microglobulin, present at the surface of all nucleated cells in association with major histocompatibility complex (MHC) class I molecules, is secreted mainly by B cells and plasma cells, while plasmablasts and plasma cells secrete immunoglobulin FLCs. Increased levels of the latter thus suggest that plasmablasts might also be activated in early RA. The normality of the kappa-to-lambda ratio in nearly all study patients confirmed that B-cell activation is polyclonal in RA. As opposed to IgM, IgG and IgA are secreted by mature plasma cells, which are a terminal stage of B-cell differentiation, after isotype class switching. A case-control study observed that, more than 10 years before RA development, future patients had elevated levels of IgG and IgA, but not of IgM, as in our patients with early RA [8].

Our second important finding was the absence of an association between the BAFF gene polymorphism and either RA or any of the baseline clinical, immunological, or radiological characteristics of RA. The lack of an association with RA is consistent with a previous Japanese genetic study [24]. We found no association between the BAFF polymorphism and serum BAFF levels, contrary to findings of our group and others in primary Sjögren syndrome and hematological disorders [12–14], in which the BAFF 871T>C allele was associated with higher serum levels of the cytokine. This discrepancy may be due to much higher serum BAFF levels in the diseases in which this association has been observed. The polymorphism may thus predispose patients to higher BAFF levels in diseases in which environmental stimulations trigger BAFF secretion.

Serum BAFF level was higher than in healthy blood donors, as previously reported for patients with established disease [25, 26]. However, BAFF levels did not differ significantly between patients with early RA and those with undifferentiated early arthritis. Serum BAFF level was not associated with total Ig, RF, or anti-CCP levels, disease activity, or initial radiological erosions. These results are consistent with previous reports showing no difference in serum BAFF levels for patients with RA and other arthritides, including SLE, spondylarthropathy, crystal-induced arthritis, and osteoarthritis [26, 27]. One study found an association between serum BAFF and early RA but in a much smaller population (n = 48) and with a different technique for BAFF assessment [28].

Thus, the specific increase of markers of B-cell activation in early RA might not be related to the increase of BAFF secretion. Membrane-bound BAFF or post-translational changes of BAFF (glycosylation, trimerization with a proliferation-inducing ligand [APRIL] or delta BAFF) might affect serum B-cell activation independently of the quantity of BAFF secreted and detected in the serum. The role of local BAFF production in arthritic joints cannot be ruled out either since BAFF levels are reported to be higher in synovial fluid than in serum [27]. As in serum, however, these levels in synovial fluid do not differ for patients with RA and with other inflammatory arthritides [27].

Our results thus suggest that serum BAFF does not drive B-cell activation in early RA, at least not directly or solely. The triggering forces of B-cell activation in early RA may depend on T lymphocytes. A specific pattern of T-lymphocyte activation was recently reported in RA, with Th2 (interleukin [IL]-4, IL-13) and IL-17 expression increasing temporarily in the synovial fluid of patients with very early RA [29]. Th2 profiles favor B-cell activation and antibody secretion. Among the factors unrelated to T cells, the role of innate immune cytokines, such as IL-6, should also be considered.

Interestingly, our results showed elevated serum IgA levels in early RA. This might be related to synovial, T cell-independent activation of B lymphocytes, which might be locally triggered by resident cells of autoimmunity, like synoviocytes, as described in mucosa [30]. In regard to the cytokine triggers of IgA increase, transforming growth factor-beta, which enhances IgA class switching [31], might be involved. T cell-independent class IgA class switching might also be favored by APRIL, a cytokine of the TNF family that shares two of the three receptors of BAFF: B-cell maturation antigen (BCMA) and TACI (transmembrane activator and CAML [calcium modulator and cyclophilin ligand] interactor) [10]. APRIL levels are elevated in synovial fluid from patients with established RA [27], and synovial expression of APRIL (but not of BAFF) is reported to be highest in patients with germinal center-like structures in the synovium, a form of lymphoid organization known to enhance B-cell activation [32]. Investigation of the role of APRIL in the B-cell activation of patients with early RA therefore appears useful.

Lastly, one of the important results of the study was the association observed between the increase in some markers of B-cell activation and disease activity (assessed by DAS28) and between the increase in some markers of B-cell activation (total IgA and kappa FLCs) and disease severity (early radiological erosion at inclusion). The relevance of markers of B-cell activation as prognostic markers will be further investigated throughout the cohort study, planned to last for 10 years to ensure assessment of patients' long-term clinical and radiological course. From a therapeutic perspective, these results suggest the potential value of B-cell depletion in early RA, which is currently under evaluation in controlled trials.

The elevation of markers of B-cell activation in very early RA demonstrates that B-cell activation is an early pathogenic event in RA. B-cell activation is not directly driven by a systemic increase of BAFF in early RA. The increase of some markers of B-cell activation is associated with disease activity, autoantibody secretion, and early radiological erosion. This study thus sheds new light on the early pathogenic role of B lymphocytes in RA and suggests that targeting these cells might be a useful therapeutic strategy in early RA.