Objective In the majority of patients with rheumatoid arthritis (RA), antibodies specifically recognize citrullinated autoantigens that are generated by peptidylarginine deiminases (PADs). Neutrophils express high levels of PAD and accumulate in the synovial fluid (SF) of RA patients during disease flares. This study was undertaken to test the hypothesis that neutrophil cell death, induced by either NETosis (extrusion of genomic DNA-protein complexes known as neutrophil extracellular traps [NETs]) or necrosis, can contribute to production of autoantigens in the inflamed joint. Methods Extracellular DNA was quantified in the SF of patients with RA, patients with osteoarthritis (OA), and patients with 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 those with OA. Results Extracellular DNA was detected at significantly higher levels in RA SF than in OA SF (P < 0.001) or PsA SF (P < 0.05), and its expression levels correlated with neutrophil concentrations and PAD activity in RA SF. Necrotic neutrophils released less soluble extracellular DNA compared to NETotic cells in vitro (P < 0.05). Higher PAD activity was detected in RA SF than in OA SF (P < 0.05). The citrullinated proteins PAD2 and PAD4 were found attached to NETs and also freely diffused in the supernatant. PAD enzymatic activity was detected in supernatants of neutrophils undergoing either NETosis or necrosis. Conclusion Release of active PAD isoforms into the SF by neutrophil cell death is a plausible explanation for the generation of extracellular autoantigens in RA.
Bibliographical note© 2015 The Authors. Arthritis & Rheumatolgy published by Wiley Periodicals, Inc. on behalf of the American College of Rheumatology. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Funding Information: European Union Seventh Framework Programme. Grant Numbers: HEALTH‐2010‐261460, HEALTH‐F2‐2012‐305549
NIHR Wellcome Trust Clinical Research Facility at University Hospitals Birmingham NHS Foundation Trust
Arthritis Research UK Clinician Scientist Award. Grant Number: 18547
Medical Research Council Confidence in Concepts. Grant Number: MC_PC_12011
Medical Research Council Developmental Pathway Funding Scheme. Grant Number: MR/M007669/1
University Hospital Birmingham Charities. Grant Number: 17‐3‐690
The Arthritis Research UK Rheumatoid Arthritis Pathogenesis Centre of Excellence (RACE)
Arthritis Research UK. Grant Number: 20298