Proteomic analysis of circulating immune complexes in juvenile idiopathic arthritis reveals disease-associated proteins.
OBJECTIVE Immune complexes (IC) have been detected in the majority of sera of patients with juvenile rheumatoid arthritis (JRA). Our aim was to determine the possible constituents of these IC. METHODS One hundred and four sera from patients with JRA were separated on a Sepharose 4B column to which were bound F(ab')2 fragments of goat anti-human IgM antibody to separate IgM-containing IC. The column was sequentially eluted with 1 M ammonia and 0.1 M glycine-HCl buffer, pH 3. The eluted material was treated with sodium dodecyl sulfate (SDS) and simultaneously reduced with 2-mercaptoethanol. Individual components were then separated by SDS-gradient polyaclylamide gel electrophoresis and were transferred to nitrocellulose by Western blotting. The nitrocellulose strips were overlaid with the patient's own serum and developed with specific antiserum to human IgM and IgG. RESULTS Four bands were noted in the majority of JRA patients, including a band in the 70-80 kD area to the IgM heavy chain and in the 50 kD area to the IgG heavy chain. Additional bands specific for JRA patients' sera were noted in the 40 kD and 60 kD areas. Overall, 61 of 104 sera demonstrated all four bands; 78 showed the 40 kD band and 73 the 60 kD band. CONCLUSION These IC were previously shown to contain IgM rheumatoid factor (RF), IgG-RF, and IgG in addition to the 40 and/or 60 kD constituents present in the isolates. Preliminary studies on these constituents suggest the possibility of the 60 kD band being the human 60 kD mitochondrial heat shock protein. Further investigations of these immune complex components may aid in the understanding of the pathophysiology of JRA.