Background/Purpose: Rheumatoid arthritis (RA) is a systemic inflammatory disorder that often leads to joint damage. Several lines of evidence suggests the role of B cells in joint destruction including the efficacy of B cell depletion therapy as a treatment and the presence of B cell aggregates in RA synovium and subchondral bone. The aim of this study was to investigate the mechanisms by which B cells contribute to joint destruction in RA.

Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood or synovial fluid by Ficoll-Hypaque density gradient centrifugation from healthy controls (HC) or RA patients. Purified B cells were obtained by CD19 magnetic isolation. Cells were stimulated with anti-CD40 (2.5 µg/ml) and PMA (20 ng/ml) for 48 hours. RANKL expression was detected by cell surface staining and multi-color flow cytometry. Several markers to identify B cells and for T cell exclusion were employed including CD19, CD27, IgD, CD95, CD21 and CD3. For osteoclast formation assay, purified B cells were cultured for 7 days with anti-CD40 and PMA in the first 48 hours. Normal bone marrow derived osteoclast precursors (OCPs) were co-cultured with stimulated B cells in the presence of M-CSF and 10ng RANKL for 4-7 days. Cells were stained with TRAP and multi-nucleated TRAP+ cells were enumerated.

Results: Upon stimulation of PBMCs with anti-CD40 and PMA, the percentage of RANKL+ B cells was significantly higher than cells cultured with medium alone (6.854±1.097 vs. 0.684±0.14, n=9, p< 0.001). The same result was observed with purified B cells (data not shown). CD27+ memory B cells (unswitched CD27+IgD+ and switched CD27+ IgD-) had a greater propensity to produce RANKL in comparison to CD27- B cells (9.41±0.62 vs. 4.93±0.69, p=0.0084). Flow sorting B cells into naïve, transitional, switched and unswitched memory cells further verified the propensity of memory B cells to produce RANKL. Notably, the majority of RANKL-expressing B cells appeared to express the activation marker CD95 (79.04±6.47% CD95+ vs. 20±6.47% CD95-, p= 0.001). We also observed that PB from RA patients contained elevated B cells of an activated memory phenotype (CD95+ on switched memory in RA [n=13] 50.6+19.5 vs. HC [n=14] 29.6+9.5, p=0.005). In accord with this activated memory expansion, the frequency of RANKL+ PB B cells after stimulation was higher in RA when compared to HC (18.4+5.1 vs. 6.9+0.8, p=0.09). Remarkably, RA synovial fluid B cells produced even higher RANKL (23.2+7.6%, n=4) and also spontaneously produced RANKL. Finally, B cells supported osteoclast differentiation, and RA B cells are more efficient than HC (n=4, p<0.05).

Conclusion: Our data support the hypothesis that B cells play a key role in RA disease pathology and joint destruction in part by RANKL production. Activated memory B cells are expanded in RA and have a propensity to produce RANKL, an important factor in bone resorption.

Supported in part by the University of Rochester Autoimmunity Center of Excellence U19 AI563262

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/a-link-between-b-cells-and-bone-erosion-in-ra-rankl-production-by-memory-b-cells/