Background/Purpose: Rheumatoid arthritis (RA) is a systemic autoimmune disease, which causes joint inflammation and bone loss. Inflammation-mediated joint damage is linked to the imbalance of bone-resorbing osteoclast (OC) and bone-depositing osteoblast (OB) activity. Pro-inflammatory cytokines present in the synovium have been shown to directly or indirectly stimulate OC differentiation. Although the effect of T cell, macrophage and synoviocyte-derived cytokines on bone resorption is well characterized, less is known about the impact of B cell-derived cytokines. Tumor necrosis factor α (TNFα) is a pro-inflammatory cytokine that promotes osteoclastogenesis and inhibits OB differentiation. In this study, we examined TNFα expression by B cells in RA patients and healthy controls.

Methods: PBMCs were isolated from peripheral blood of healthy controls (HC) or RA patients (n=6 each) by Ficoll-Hypaque density gradient centrifugation. RA patients fulfilled 1987 American College of Rheumatology diagnostic criteria and were CCP+. B cells were isolated by CD19+ magnetic bead selection from peripheral blood and stimulated with α-Igs (α-IgA, α-IgG, and α-IgM) and CpG2006 for 4 or 24 hours. TNFα production was measured by qPCR, flow cytometry, and ELISA. Statistical significance was determined by Mann-Whitney test. B cell populations were categorized based on the differential expression of CD27 and IgD, as naïve (CD19+CD27-IgD+), double negative (CD19+CD27-IgD-), unswitched (CD19+CD27+IgD+) and switched memory B cells (CD19+CD27+IgD-). RA synovial biopsies were immuno-stained with antibodies against CD20, CD27 and TNF.

Results: Short-term stimulation of healthy control (HC) B cells with α-Igs and CpG2006 (4 hours) induced significant production of TNFα compared to unstimulated B cells at both the mRNA (64.4±8.2 fold, P<0.001) and secreted protein level (ng/uL TNFα: 2.86±0.60 vs. 0.0124±0.0047, P<0.02) at 4 hours. At 24 hours, the secreted protein level did not further increase (ng/uL TNFα: 3.032±.40 for stimulated vs. 0.0078±0.0023 for unstimulated, P<0.005), and the mRNA levels were actually lower (9.1 fold over unstimulated control), suggesting the mRNA concentration peaks closer to the 4 hour time point. There was no statistically significant difference in TNFα production by HC vs. RA B cells at the mRNA (64.4±8.2 fold in HC vs 52.4±14.5 fold in RA, n=6) or protein level at 4 hours (ng/uL TNFα: 2.86±0.60 in HC vs 2.23±0.84 in RA, n=5) or 24 hours (ng/uL TNFα: 3.032±0.40 in HC vs 2.66±0.69 in RA, n=6). The synovium was enriched for memory B cells (CD27+IgD- memory 55.15±10.41 vs naïve 8.47±2.64, n=7, P<0.001) expressing TNFα on immuno-staining. In agreement with memory B cells being the prime producers of TNFα, preliminary data indicates that peripheral blood memory B cells have an increased propensity to produce TNFα under stimulation compared to the double negative or naïve B cell subsets (%TNFα+: 17.1 vs 8.4 and 1.2, respectively). 

Conclusion: Our results suggest that TNF- producing memory B cells are enriched in the RA synovial microenvironment, potentially stimulating OC production/activity and thus enhancing bone erosion.

Funding: NIAID R01 AI077674, P01 AI078907, and ACE U19 AI563262

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/b-cells-are-prime-producers-of-tumor-necrosis-factor-alpha-in-rheumatoid-arthritis/