title: Synovial intracellular citrullinated proteins colocalizing with peptidyl arginine deiminase as pathophysiologically relevant antigenic determinants of rheumatoid arthritis-specific humoral autoimmunity.
abstract: OBJECTIVE
  To address the ongoing debate concerning the specificity of synovial citrullinated proteins for rheumatoid arthritis (RA) and to analyze their pathophysiologic relevance to the induction or perpetuation of the RA-specific anti-citrullinated protein antibodies (ACPAs).
  
  
  METHODS
  Synovium of 19 RA patients and 19 non-RA controls was immunostained for the presence of citrullinated proteins with a mouse monoclonal antibody (F95), for the citrullinating enzyme peptidyl arginine deiminase type 2 (PAD-2), and for the free citrulline-producing enzyme inducible nitric oxide synthase (iNOS). Extending the RA cohort to 61 patients, the findings of anticitrulline staining in synovium were related to serum and synovial fluid ACPA levels, as measured by enzyme-linked immunosorbent assay.
  
  
  RESULTS
  F95 staining indicated the presence of synovial intracellular citrullinated proteins in 53% of RA samples versus 5% of control samples, whereas extracellular staining was not RA specific. Immunoblotting and inhibition experiments confirmed that the antibody recognized citrullinated proteins but not free citrulline. Accordingly, iNOS was equally found in RA and control synovium and in intracellular citrullinated protein-positive and intracellular citrullinated protein-negative samples. In contrast, intracellular citrullinated proteins colocalized with PAD-2, which was found in 59% of RA samples versus 17% of control samples. Independent of local disease activity, the presence of the RA-specific synovial intracellular citrullinated proteins was associated with significantly higher systemic and local ACPA levels and with local ACPA production in the joint.
  
  
  CONCLUSIONS
  These data confirm the presence of RA-specific intracellular citrullinated proteins in synovium. The link with PAD-2 and local and systemic ACPA levels emphasizes their pathophysiologic relevance for RA-specific humoral autoimmunity.
authors.affiliation: Ghent University Hospital, Belgium.
authors.forename: Leen
authors.initials: L
authors.lastname: De Rycke
authors.forename: Anthony P
authors.initials: AP
authors.lastname: Nicholas
authors.forename: Tineke
authors.initials: T
authors.lastname: Cantaert
authors.forename: Elli
authors.initials: E
authors.lastname: Kruithof
authors.forename: Joshua D
authors.initials: JD
authors.lastname: Echols
authors.forename: Bart
authors.initials: B
authors.lastname: Vandekerckhove
authors.forename: Eric M
authors.initials: EM
authors.lastname: Veys
authors.forename: Filip
authors.initials: F
authors.lastname: De Keyser
authors.forename: Dominique
authors.initials: D
authors.lastname: Baeten
mesh_terms: D000917:Antibody Formation; D001172:Arthritis, Rheumatoid / Q000276:immunology*; D015551:Autoimmunity; D016022:Case-Control Studies; D002956:Citrulline / Q000276:immunology* / Q000378:metabolism; D000939:Epitopes; D006801:Humans; D006867:Hydrolases / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism*; D000076342:Protein-Arginine Deiminases; D011506:Proteins / Q000276:immunology* / Q000378:metabolism; D013583:Synovial Membrane / Q000378:metabolism*; D014018:Tissue Distribution
chemical_list: D000939:Epitopes; D011506:Proteins; D002956:Citrulline; D006867:Hydrolases; D000076342:Protein-Arginine Deiminases