Release of active peptidyl arginine deiminases by neutrophils can explain production of extracellular citrullinated autoantigens in RA synovial fluid

Objective: In the majority of patients with rheumatoid arthritis (RA), antibodies specifically recognize citrullinated autoantigens that are generated by peptidylarginine deiminases (PAD). Neutrophils express high levels of PAD and accumulate in the synovial fluid (SF) of RA patients during disease flares. Here, we tested the hypothesis that neutrophil cell death, by release of neutrophil extracellular traps (NETosis) and necrosis, can contribute to production of autoantigens in the inflamed joint. Methods: Extracellular DNA was quantified in SF of patients with RA, osteoarthritis (OA) and psoriatic arthritis (PsA). Release of PAD from neutrophils was investigated by western blotting, mass spectrometry, immunofluorescence staining and PAD activity assays. PAD2 and PAD4 protein expression, as well as PAD enzymatic activity, were assessed in the SF of patients with RA and OA.Results: Extracellular DNA was detected in SF from RA patients at significantly higher levels than in OA SF (p<0.001) or PsA SF (p<0.05) and correlated with neutrophil concentrations and PAD activity in the SF. Necrotic neutrophils released less soluble extracellular DNA than NETotic cells in vitro (p<0.05). Higher PAD activity was detected in SF from RA patients compared to OA patients (p<0.05). Citrullinated proteins, PAD2 and PAD4 were found both attached to NETs as well as free in the supernatant. PAD enzymatic activity was detected both in supernatants of neutrophils undergoing NETosis and necrosis.Conclusion: Release of active PAD isoforms into SF by neutrophil cell death is a plausible explanation for the generation of extracellular autoantigens in RA.