Accumulating data indicate that bone marrow (BM) in rheumatoid arthritis (RA) patients participates in the pathogenesis of RA as a site of proinflammatory cytokine overproduction, lymphocyte homing and cell activation. IL-1b, IL-6, TNFα and IL-15 are among the proinflammatory cytokines elevated in RA in comparison with osteoarthritis (OA) patient BM. At present, it is unclear why the RA BM microenvironment produces more proinflammatory cytokines than OA BM. One possibility is that, in addition to physiological production, exogenous and/or endogenous factors further enhance innate immune responses in RA BM. Importantly, our recent data indicate that RA BM-derived B cells express functional Toll-like receptor 9 (TLR9), and therefore could respond to TLR9 agonist stimulation.

To test the effects of TLR9 agonists (oligodeoxynucleotides containing unmethylated CpG sequences (CpG-ODN)) on RA BM-derived B cells, and to estimate the presence of bacterial DNA in BM from RA and OA patients.

BM samples, obtained from RA patients during joint replacement surgery, served for bone marrow mononuclear cell isolation. Mature (CD20⁺) B cells were next purified from bone marrow mononuclear cells using a magnetic cell separation technique (MACS) and cultured in vitro in the presence of agonistic CpG-ODN or control GpC-ODN alone or with addition of IL-15. mRNA was isolated from purified B cells using Trizol and served as the template for RT-PCR analysis. The presence of bacterial DNA in BM plasma and bone marrow mononuclear cells was evaluated by DNA extraction and PCR amplification using primers specific for eubacterial 16S-ribosomal RNA. Flow cytometry was applied to assess the expression of intracellular proteins (TLR9 and proliferation marker Ki-67) and surface activation markers (CD86, CD54) on B lymphocytes. B-cell populations were distinguished according to CD19, CD20, CD27 and CD138 molecule expression. Specific ELISAs were used to measure TNFα and IL-6 in supernatants from cultured cells.

We found that BM-derived B lymphocytes isolated from RA patients express TLR9 at the mRNA and protein levels. Importantly, these TLR9 are functional. Stimulation of B cells by CpG-ODN, but not control GpC-ODN, in a dose-dependent manner enhanced expression of activation markers (CD86 and CD54), and stimulated IL-6 and TNFα secretion and cell proliferation in vitro. The specificity of CpG-ODN-triggered stimulation was confirmed in blocking experiments where cells cultured in the presence of chloroquine, a known inhibitor of TLR9 signaling, failed to respond to CpG-ODN. Moreover, in the presence of IL-15, stimulatory activities of TLR9 agonists were further enhanced. CpG-ODN, but not IL-15 alone, trigger differentiation of sorted B cells into CD19⁺CD20⁺CD27^high cells. Simultaneous addition of CpG-ODN and IL-15 promoted further differentiation of CD19⁺CD20⁺CD27^high cells toward plasma cells, as judged by upregulation of CD138 and downregulation of CD19 and CD20 expression. Finally, the presence of highly conserved regions of the eubacterial 16S-ribosomal RNA gene in samples of BM from RA patients was found at significantly higher frequency when compared with OA patients.

Our data indicate that BM of RA patients may represent an important secondary lymphoid organ that, especially during ongoing infection with the presence of bacterial DNA in blood and ultimately in BM, actively participates in the pathogenesis of RA.