To examine synovial fluid as a site for generating citrullinated antigens, including the candidate autoantigen citrullinated α‐enolase, in rheumatoid arthritis (RA).

Synovial fluid was obtained from 20 patients with RA, 20 patients with spondylarthritides (SpA), and 20 patients with osteoarthritis (OA). Samples were resolved using sodium dodecyl sulfate–polyacrylamide gel electrophoresis, followed by staining with Coomassie blue and immunoblotting for citrullinated proteins, α‐enolase, and the deiminating enzymes peptidylarginine deiminase type 2 (PAD‐2) and PAD‐4. Proteins from an RA synovial fluid sample were separated by 2‐dimensional electrophoresis, and each protein was identified by immunoblotting and mass spectrometry. Antibodies to citrullinated α‐enolase peptide 1 (CEP‐1) and cyclic citrullinated peptide 2 were measured by enzyme‐linked immunosorbent assay.

Citrullinated polypeptides were detected in the synovial fluid from patients with RA and patients with SpA, but not in OA samples. Alpha‐enolase was detected in all of the samples, with mean levels of 6.4 ng/μl in RA samples, 4.3 ng/μl in SpA samples, and <0.9 ng/μl in OA samples. Two‐dimensional electrophoresis provided evidence that the α‐enolase was citrullinated in RA synovial fluid. The citrullinating enzyme PAD‐4 was detected in samples from all 3 disease groups. PAD‐2 was detected in 18 of the RA samples, in 16 of the SpA samples, and in none of the OA samples. Antibodies to CEP‐1 were found in 12 of the RA samples (60%), in none of the SpA samples, and in 1 OA sample.

These results highlight the importance of synovial fluid for the expression of citrullinated autoantigens in inflammatory arthritis. Whereas the expression of citrullinated proteins is a product of inflammation, the antibody response remains specific for RA.