Synovial detection and autoantibody reactivity of processed citrullinated isoforms of vimentin in inflammatory arthritides.
OBJECTIVE
To investigate the presence and characteristics of citrullinated vimentin in protein extracts of inflamed synovial tissue.


METHODS
Cytosolic protein extracts obtained from RA (n = 14) and SpA patients (n = 14) were analysed by gel electrophoresis and western blotting. Citrullinated vimentin isoforms were visualized by a combination of anti-modified citrulline (AMC) staining and anti-vimentin detections (V9, H-84). This was subsequently confirmed by immunoprecipitation. Autoantibody detection was verified using sera obtained form RA (n = 6) and SpA (n = 6) patients.


RESULTS
A specific cluster of spots displayed on the 2D gel images of cytosolic synovial tissue extracts, was identified by mass spectrometry as vimentin. Interestingly, our results suggested that these isoforms could be the result of caspase cleavage. In addition, these cleaved forms of vimentin were found to be citrullinated in synovial cytosolic protein extracts of inflammatory arthritides, mainly in RA patients. Caspase-3 is able to cleave vimentin at amino acid 85. Western blot analysis with a specific antibody against amino acids 1-84 of vimentin (H-84) confirmed that the citrullinated isoforms of vimentin were lacking this part of the protein. These results were also confirmed by immunoprecipitation of vimentin derived from cytosolic protein extracts of RA and SpA patients. Furthermore, the presence of autoantibodies against these citrullinated processed forms of vimentin was found to be predominantly associated with RA patients.


CONCLUSIONS
These findings show the presence of processed citrullinated vimentin in inflammatory arthritides, mainly in RA and suggest a possible origin of the ACPA immune response in RA.
Tilleman
K
K
Laboratory for Pharmaceutical Biotechnology, Ghent University, Harelbekestraat 72, B- 9000 Ghent, Belgium.
Van Steendam
K
K
Cantaert
T
T
De Keyser
F
F
Elewaut
D
D
Deforce
D
D
D000368:Aged; D000937:Antigen-Antibody Reactions; D001168:Arthritis / Q000276:immunology*; D001172:Arthritis, Rheumatoid / Q000276:immunology; D001323:Autoantibodies / Q000097:blood / Q000276:immunology*; D001324:Autoantigens / Q000276:immunology; D015153:Blotting, Western / Q000379:methods; D053148:Caspase 3 / Q000378:metabolism; D002956:Citrulline / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D047468:Immunoprecipitation; D008297:Male; D008875:Middle Aged; D020033:Protein Isoforms / Q000032:analysis / Q000276:immunology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013167:Spondylitis, Ankylosing / Q000276:immunology; D013583:Synovial Membrane / Q000737:chemistry* / Q000276:immunology; D014746:Vimentin / Q000032:analysis* / Q000276:immunology / Q000378:metabolism
D001323:Autoantibodies; D001324:Autoantigens; D020033:Protein Isoforms; D014746:Vimentin; D002956:Citrulline; D053148:Caspase 3