BAFF is a cytokine that plays a pivotal role in B-cell survival, differentiation, and activation. We and others recently demonstrated that resident cells of the synovial membrane synthesize and release BAFF in response to stimulation of innate immune receptors such as integrin α5β1
and IFN-γ receptors [20
]. Beside this established key BAFF, other molecules might influence directly or indirectly B cells such as TSLP and SLPI. Direct evidence of the capacity of these factors to promote B-cell activation was demonstrated following TLR activation of oral epithelial cells [22
As an increasing body of evidence suggested that MPs have potent proinflammatory activities and are potentially important mediators of inflammatory and autoimmune diseases [29
], we have investigated in this study the role of MPs on BAFF, TSLP, and SLPI secretion by FLSs isolated from RA patients. MPs are produced during cell death but they may also arise during cell activation. They can be produced by virtually all cell types, but in contrast to MPs isolated from blood, MPs isolated from synovial fluids from RA patients are derived mainly from inflammatory and immune cells [23
We first evaluated MP levels in synovial fluids obtained from patients with RA (7), OA (5), MC (3), and AR (5). All synovial fluids contained MPs although their levels seemed to be higher in RA and MC synovial fluids. These results are in accordance with observations indicating that the number of MPs is increased during inflammatory states in vivo
As Distler and colleagues [16
] demonstrated that MPs serve as important triggering elements to promote cytokine, chemokine, and MMP release from RA synovial fibroblasts, we then explored the role of RA and OA synovial fluid-derived MPs in inducing BAFF synthesis by activated FLSs. In the present study, we demonstrated a new mechanism by which FLSs could contribute to the adaptative autoimmune response. We showed that MPs, which are produced in synovial fluids during RA and OA, are potent stimuli of BAFF synthesis in a similar degree to IFN-γ, which is used as positive control. To our knowledge, this is the first time that BAFF induction by MPs has been demonstrated. This effect was observed with both RA and OA MPs, suggesting that MPs isolated from joints of patients with degenerative joint diseases such as OA have the same effect as MPs present in the joints of patients with RA and therefore that this action is not disease-dependent. The main difference regarding the assessed effect concerns the levels of MPs, which are much lower in synovial fluids of OA than of RA. However, it can be speculated that other types of cytokines and other functional effects of MPs which were not assessed in the present study might be different. It must be noted that, in contrast to other studies, this work was performed with purified MPs free of exosomes, which are preformed vesicles of endosomal origin which are stored intracellularly in multivesicular bodies and released by exocytosis. Exosomes, which are investigated mainly in the regulation of immune responses, do not expose phosphatidyserine, they share a common set of membrane molecules like tetraspanins, and they harbor unique subsets of proteins linked to cell type-associated functions [34
]. Results suggest that MPs could contribute to the interplay between FLSs and B cells through BAFF synthesis. This induction of BAFF might also contribute to the increased proliferation of FLSs in RA, which might also be related to the autocrine effect of BAFF on FLSs. Indeed, FLSs not only secrete BAFF, they also bear BAFF receptors [36
]. Berckmans and colleagues [15
] found that, in patients with RA, most of the MPs present in the synovial fluid are produced by monocytes/macrophages, T cells, and granulocytes. MPs deriving from B cells, platelets, and erythrocytes are present only in low numbers.
To gain further information about the parental cells possibly involved in MP-mediated BAFF synthesis, we explored the effect of MPs isolated from CEM lymphocyte and THP-1 cells activated under various conditions. As previously shown by studies performed with LPS-treated U937 cells [38
], THP-1 cell-derived MPs exhibit strong proinflammatory activities. Moreover, they were able to induce BAFF release by activated FLSs. In view of the abundance of these cells in the synovial cavity in RA, our results suggest that macrophages could serve as important triggering elements in promoting the inflammatory response and cooperation with B cells through the release of MPs. In contrast, we observed that MPs derived from activated CEM lymphocytes, which are inducers of IL-6 and IL-8 release, did not promote BAFF synthesis. BAFF release was not observed even with a 10-fold increase in MP concentration. T cells also occur abundantly in synovium and synovial fluids; nevertheless, our data demonstrate that MPs eventually produced by activated lymphocytes T in vivo
cannot be considered key contributors in the induction of BAFF. We cannot rule out that MPs released by other cells present in the synovial cavity such as FLSs could interfere in this process. In fact, we have performed some preliminary experiments with MPs isolated from LPS-activated FLSs and have observed that these MPs act in an autocrine pathway and induce IL-6 release by activated FLSs (data not shown).
We reported also that MPs isolated from synovial fluids, CEM lymphocytes, or THP-1 cells were strong inducers of TSLP. TSLP is an IL-7-like cytokine that stimulates dendritic cells to produce more BAFF and constitutes a Th2-independent pathway for antibody production. It was demonstrated that epithelial cells lining tonsillar crypts released AID (activation-induced cytidine deaminase)-inducing factors, including BAFF, IL-10, and TSLP, after sensing viral RNA through TLR3 [22
]. The resultant class switching caused the production of broadly IgG and IgA antibodies, including antibodies to self antigens. RA FLSs also release TSLP in response to LPS and poly I:C, and this effect is downregulated by IFN-γ and dexamethasone [39
]. Our findings indicate that, like LPS and poly I:C, MPs could indirectly participate in B-cell activation by activating dendritic cells through TSLP release by RA FLSs and may be involved in the physiopathology of inflammatory arthritis.
However, we also showed that RA FLSs activated with MPs isolated from synovial fluids, CEM lymphocytes, or THP-1 cells release SLPI. SLPI was originally identified as a protein synthesized by macrophages which antagonized LPS activation of NF-κB. In B lymphocytes, SLPI inhibits class switching by interfering with NF-κB-dependent pathways and with the upregulation of AID induced by BAFF and viral RNA. As SLPI is released at a later time point, it may restrain the intrasynovial production of potentially pathogenic IgG antibodies. This needs to be demonstrated.