Anti–citrullinated protein antibodies bind surface‐expressed citrullinated Grp78 on monocyte/macrophages and stimulate tumor necrosis factor α production

Abstract Objective Anti–citrullinated protein antibodies (ACPAs), which are the most specific autoantibody marker in patients with rheumatoid arthritis (RA), correlate with disease activity; however, the role of ACPAs in RA pathogenesis has not been elucidated. We hypothesized that ACPAs may directly stimulate mononuclear cells to produce inflammatory cytokines. Thus, we identified cognate antigens of ACPAs on monocyte/macrophages and examined their immunopathologic roles in the pathogenesis of RA. Methods ACPAs were purified from pooled ACPA‐positive RA sera by cyclic citrullinated peptide–conjugated affinity column. After coculture of U937 cells with ACPAs, the tumor necrosis factor α (TNFα) production and NF‐κB DNA binding activity of the cells were measured by enzyme‐linked immunosorbent assay. The cognate antigens of ACPAs on the U937 cell surface were probed by ACPAs, and the reactive bands were examined via proteomic analysis. Results ACPAs specifically enhanced TNFα production and increased the DNA‐binding activity of NF‐κB in U937 cells. Proteomic analysis revealed that Grp78 protein (72 kd) was one of the cognate antigens of ACPAs. The truncated form of cell surface–expressed Grp78 (55 kd) on U937 cells contained citrulline capable of binding with ACPAs. After citrullination, glutathione S‐transferase–tagged recombinant Grp78 (97.52 kd) became a 72‐kd fragment and bound with ACPAs. ACPAs also bound to human monocytes and lymphocytes to promote TNFα production. Conclusion We clearly demonstrated that ACPAs enhance NF‐κB activity and TNFα production in monocyte/macrophages via binding to surface‐expressed citrullinated Grp78.