Secretion of anti-citrulline-containing peptide antibody by B lymphocytes in rheumatoid arthritis

Objective To understand the regulation of anti–citrulline-containing peptide antibody (anti-CCP) production in rheumatoid arthritis (RA), production of anti-CCP by B cells derived from peripheral blood (PB), bone marrow (BM), and synovial fluid (SF) was examined. Methods Purified PB and SF B cells were isolated by negative selection and then cultured in the absence or presence of L–CD40 ligand cells and interleukin-10 or anti-CD3–activated T cells. Total IgM and IgM–anti-CCP were detected after 14 days of culture by enzyme-linked immunosorbent assay. Enzyme-linked immunospot assays were performed to analyze the frequency of cells that spontaneously produced IgM–anti-CCP in BM and SF B cells. Results IgM–anti-CCP autoantibodies were induced in PB B cells from healthy controls and RA patients following coculture with activated T cells or application of the CD40 activation system, whereas no production could be detected when PB B cells were cultured in the absence of a stimulus. SF and BM B cells from anti-CCP–seropositive RA patients, but not anti-CCP–seronegative patients, actively produced IgM–anti-CCP without stimulation. The frequency of spontaneous production of IgM–anti-CCP among the IgM-secreting cells ranged from 2.2% to 25%. Conclusion These results indicate the presence of B cell precursors for anti-CCP autoantibodies that are able to produce antibodies upon stimulation in the PB B cell repertoire of healthy controls and patients with RA. In contrast, B cells that actively secreted anti-CCP were specifically present in the BM and SF compartment of anti-CCP–seropositive RA patients. The local presence of anti-CCP–secreting cells in the inflamed joints provides evidence for an antigen-driven maturation of CCP-specific B cells at the site of inflammation in RA.