title: Release of Active Peptidyl Arginine Deiminases by Neutrophils Can Explain Production of Extracellular Citrullinated Autoantigens in Rheumatoid Arthritis Synovial Fluid.
abstract: 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.
  
  
  CONCLUSIONS
  Release of active PAD isoforms into the SF by neutrophil cell death is a plausible explanation for the generation of extracellular autoantigens in RA.
authors.affiliation: Arthritis Research UK Centre of Excellence for Rheumatoid Arthritis Pathogenesis and University of Birmingham, Birmingham, UK.
authors.forename: Julia
authors.initials: J
authors.lastname: Spengler
authors.affiliation: Arthritis Research UK Centre of Excellence for Rheumatoid Arthritis Pathogenesis and University of Birmingham, Birmingham, UK.
authors.forename: Božo
authors.initials: B
authors.lastname: Lugonja
authors.affiliation: Karolinska University Hospital and Karolinska Institutet, Stockholm, Sweden.
authors.forename: A Jimmy
authors.initials: AJ
authors.lastname: Ytterberg
authors.affiliation: Karolinska Institutet, Stockholm, Sweden.
authors.forename: Roman A
authors.initials: RA
authors.lastname: Zubarev
authors.affiliation: University of Birmingham, Birmingham, UK.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Creese
authors.affiliation: Arthritis Research UK Centre of Excellence for Rheumatoid Arthritis Pathogenesis and University of Birmingham, Birmingham, UK.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Pearson
authors.affiliation: University of Birmingham, Birmingham, UK.
authors.forename: Melissa M
authors.initials: MM
authors.lastname: Grant
authors.affiliation: University of Birmingham, Birmingham, UK.
authors.forename: Michael
authors.initials: M
authors.lastname: Milward
authors.affiliation: Karolinska University Hospital and Karolinska Institutet, Stockholm, Sweden.
authors.forename: Karin
authors.initials: K
authors.lastname: Lundberg
authors.affiliation: Arthritis Research UK Centre of Excellence for Rheumatoid Arthritis Pathogenesis, University of Birmingham, and Sandwell and West Birmingham Hospitals NHS Trust, Birmingham, UK.
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Buckley
authors.affiliation: Arthritis Research UK Centre of Excellence for Rheumatoid Arthritis Pathogenesis, University of Birmingham, and University Hospitals NHS Foundation Trust, Birmingham, UK.
authors.forename: Andrew
authors.initials: A
authors.lastname: Filer
authors.affiliation: Arthritis Research UK Centre of Excellence for Rheumatoid Arthritis Pathogenesis, University of Birmingham, and Sandwell and West Birmingham Hospitals NHS Trust, Birmingham, UK.
authors.forename: Karim
authors.initials: K
authors.lastname: Raza
authors.affiliation: University of Birmingham, Birmingham, UK.
authors.forename: Paul R
authors.initials: PR
authors.lastname: Cooper
authors.affiliation: University of Birmingham, Birmingham, UK.
authors.forename: Iain L
authors.initials: IL
authors.lastname: Chapple
authors.affiliation: Arthritis Research UK Centre of Excellence for Rheumatoid Arthritis Pathogenesis and University of Birmingham, Birmingham, UK.
authors.forename: Dagmar
authors.initials: D
authors.lastname: Scheel-Toellner
mesh_terms: D000328:Adult; D000368:Aged; D015535:Arthritis, Psoriatic / Q000276:immunology; D001172:Arthritis, Rheumatoid / Q000276:immunology*; D001324:Autoantigens / Q000276:immunology* / Q000378:metabolism; D015153:Blotting, Western; D016022:Case-Control Studies; D016923:Cell Death / Q000276:immunology; D002956:Citrulline / Q000378:metabolism*; D004247:DNA / Q000032:analysis*; D065206:Extracellular Traps; D005260:Female; D005455:Fluorescent Antibody Technique; D006801:Humans; D006867:Hydrolases / Q000276:immunology* / Q000378:metabolism; D008297:Male; D013058:Mass Spectrometry; D008875:Middle Aged; D009504:Neutrophils / Q000166:cytology / Q000276:immunology*; D020370:Osteoarthritis, Knee / Q000276:immunology; D000080064:Protein-Arginine Deiminase Type 2; D000076342:Protein-Arginine Deiminases; D013582:Synovial Fluid / Q000166:cytology / Q000276:immunology
chemical_list: D001324:Autoantigens; D002956:Citrulline; D004247:DNA; D006867:Hydrolases; C000620352:PADI2 protein, human; D000080064:Protein-Arginine Deiminase Type 2; D000076342:Protein-Arginine Deiminases