Absence of osteoclastogenic response to pooled polyclonal anti-citrullinated peptide/protein antibodies from patients with rheumatoid arthritis in vitro

Anti-citrullinated peptide/protein antibodies (ACPAs) are specific serological markers for rheumatoid arthritis (RA), but their direct role in promoting osteoclastogenesis remains controversial. This study aims to determine whether polyclonal ACPAs purified from RA patients can directly drive osteoclastogenesis in vitro. Polyclonal ACPAs were purified from pooled sera of 37 ACPA positive RA patients using Protein G and multi-citrullinated-epitope peptide (MCEP) conjugated affinity chromatography, with control IgG from 10 ACPA negative RA patients and a healthy donor. The purity and specific binding capability to citrullinated vimentin were verified by Coomassie Blue staining and western blot. CD14 + monocytes from healthy donors and RA patients were induced to differentiate into osteoclasts with M-CSF and RANKL, in the presence or absence of ACPAs. Osteoclast formation was evaluated by tartrate resistant acid phosphatase (TRAP) staining, gene expression of osteoclast-specific markers (TRAP, cathepsin K, MMP9) by quantitative PCR, and bone resorption activity by scanning electron microscopy. The purified ACPAs showed intact IgG structure of heavy and light chains, and specific binding to citrullinated vimentin, but not to native vimentin. However, ACPAs treatment did not significantly increase the number of TRAP-positive multinucleated cells, the expression of osteoclast-related genes, or bone resorption pit area compared to controls. Our findings demonstrate that pooled polyclonal ACPAs from RA patients, purified by MCEP affinity chromatography, do not directly promote osteoclastogenesis in vitro, suggesting that their osteoclastogenic effects may require a more complex microenvironment or depend on specific antibody properties not captured in this experimental setting.