Functional Toll-like receptor 9 modulates the activity of bone marrow B cells isolated from rheumatoid arthritis patients

Background

Toll-like receptors (TLRs), a family of pathogen recognition receptors, represent an important component of the innate immune system that also contributes to the development of acquired immunity. TLR9 expressed in the cytoplasm of several cell types including B cells, and recognize and are activated by unmethylated CpG-rich, pathogen-derived DNA sequences. This CpG stimulation triggers B-cell proliferation and promotes Th1 response. Moreover, DNA containing CpG-rich motifs, acting as polyclonal stimuli, participates in the maintenance of serological memory by human memory B cells. Recent data indicate that bone marrow in rheumatoid arthritis (RA) patients may actively participate in the pathogenesis of RA as a secondary lymphoid organ via overproduction of proinflammatory cytokines and a site of effective antigen presentation.

Objective

To test the hypothesis that RA bone-marrow-derived B cells express functional TLR9.

Methods

Bone marrow mononuclear cells (BMMC) were isolated from RA bone marrow samples obtained during joint replacement surgery. The expression of TLR9 protein on B lymphocytes, gated as CD19-positive cells in BMMC preparation, was assessed by intracellular staining and flow cytometric analysis. BMMC were stimulated in vitro with agonistic (CpG-ODN) or control (GpC-ODN) oligodeoxynucleotides (15–30 μg/ml). In blocking experiments chloroquine (2–3 μg/ml) was added to the culture 50 min before stimuli. The expression of activation markers CD86 and CD54 on B lymphocytes (CD19⁺) were analyzed after 48 and 72 hours in culture using flow cytometry. Ki-67 and CFSE staining (flow cytometry) was applied to evaluate B-cell proliferation after 72 and 120 hours of culture with oligodeoxynucleotides. The concentrations of oligodeoxynucleotides and chloroquine used in experiments were not toxic to BMMC as judged by colorimetric lactate dehydrogenase assay. The presence of bacterial DNA in bone marrow plasma and BMMC was evaluated by DNA extraction and PCR amplification. Primers used for PCR recognize highly conserved regions of the eubacterial 16S-ribosomal RNA gene.

Results

RA bone-marrow-derived B lymphocytes express TLR9 protein that could be detected via intracellular staining and flow cytometric analysis. Importantly, these TLR9 are functional. CpG-ODN, but not control GpC-ODN, in a dose-dependent manner enhanced the expression of activation markers (CD86 and CD54) on B cells in BMMC cultured in vitro. The specificity of CpG-ODN triggered expression of CD86 was confirmed in experiments where cells cultured in the presence of chloroquine, a known inhibitor of TLR9-triggered signal tranduction, failed to respond to CpG-ODN. Moreover, CpG-ODN triggers bone marrow B-cell proliferation in vitro, as judged by enhanced expression of proliferation marker Ki-67 and a diminished level of CFSE dye. Interestingly, our preliminary data indicate the presence of bacterial DNA in several samples of bone marrow tissues isolated from RA patients. We could amplify DNA encoding bacterial 16S-ribosomal RNA in one bone marrow plasma and three BMMC out of total five analyzed samples.

Conclusions

Our results indicate that CpG oligodeoxynucleotides, the potent agonists of TLR9, modulate the activity of B cells from bone marrow of RA patients via induction of costimulatory and adhesion molecules (CD86, CD54) expression, and via cell proliferation. Thus, our data suggest that bone marrow of RA patients may represent an important secondary lymphoid organ that actively participates in the pathogenesis of RA.