title: Synovial fluid is a site of citrullination of autoantigens in inflammatory arthritis.
abstract: OBJECTIVE
  To examine synovial fluid as a site for generating citrullinated antigens, including the candidate autoantigen citrullinated alpha-enolase, in rheumatoid arthritis (RA).
  
  
  METHODS
  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, alpha-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 alpha-enolase peptide 1 (CEP-1) and cyclic citrullinated peptide 2 were measured by enzyme-linked immunosorbent assay.
  
  
  RESULTS
  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/microl in RA samples, 4.3 ng/microl in SpA samples, and <0.9 ng/microl in OA samples. Two-dimensional electrophoresis provided evidence that the alpha-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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Imperial College London, London, UK.
authors.forename: Andrew
authors.initials: A
authors.lastname: Kinloch
authors.forename: Karin
authors.initials: K
authors.lastname: Lundberg
authors.forename: Robin
authors.initials: R
authors.lastname: Wait
authors.forename: Natalia
authors.initials: N
authors.lastname: Wegner
authors.forename: Ngee Han
authors.initials: NH
authors.lastname: Lim
authors.forename: Albert J W
authors.initials: AJ
authors.lastname: Zendman
authors.forename: Tore
authors.initials: T
authors.lastname: Saxne
authors.forename: Vivianne
authors.initials: V
authors.lastname: Malmström
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Venables
mesh_terms: D001172:Arthritis, Rheumatoid / Q000276:immunology / Q000378:metabolism*; D001324:Autoantigens / Q000378:metabolism*; D014408:Biomarkers, Tumor / Q000378:metabolism; D016022:Case-Control Studies; D002956:Citrulline / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D006801:Humans; D006867:Hydrolases / Q000378:metabolism; D010003:Osteoarthritis / Q000276:immunology / Q000378:metabolism*; D010456:Peptides, Cyclic / Q000378:metabolism; D010751:Phosphopyruvate Hydratase / Q000378:metabolism; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases; D025241:Spondylarthritis / Q000276:immunology / Q000378:metabolism*; D013582:Synovial Fluid / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000378:metabolism
chemical_list: D001324:Autoantigens; D014408:Biomarkers, Tumor; D004268:DNA-Binding Proteins; D010456:Peptides, Cyclic; D025521:Tumor Suppressor Proteins; C487763:cyclic citrullinated peptide; D002956:Citrulline; D006867:Hydrolases; C000629235:PADI4 protein, human; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases; C095157:ENO1 protein, human; D010751:Phosphopyruvate Hydratase