Background/Purpose:

Several citrullinated proteins have been detected in the serum and synovial fluid (SF) of rheumatoid arthritis (RA) patients. However, few studies have evaluated citrullination of proteins in juvenile idiopathic arthritis (JIA). It has been proposed that anti-cyclic citrullinated peptide (anti-CCP) antibodies play a pathogenic role in the development of anti-CCP antibody positive arthritis, and it is expected that these antibodies would be present at higher concentrations at the site of inflammation. We evaluated SF from JIA patients to investigate the presence of anti-CCP antibody isotypes and identified specific citrullinated autoantibodies in JIA SF.

Methods:

Anti-CCP antibody isotypes (IgA, IgG, IgM, IgA/IgG) were measured by ELISA in the SF of 47 JIA patients. As non-inflammatory controls, SF from 10 osteoarthritis (OA) patients was used. Twenty-four SF samples from patients with various diagnoses were treated and transferred to PVDF membranes and probed with antibodies to native-α enolase, native fibrinogen, and anti-modified citrulline (AMC). SF samples were immunoprecipitated with antibodies to fibrinogen and α-enolase, and citrullinated antibodies were then detected with AMC assay.

Results:

Eleven of 47 (23%) JIA SF samples were positive for at least one anti-CCP antibody isotype. IgM anti-CCP antibodies were positive in 9 JIA patients, IgG anti-CCP antibodies were positive in 8 patients, and 2 were positive for IgA anti-CCP antibodies. Three JIA patients were positive for the combined IgA/IgG anti-CCP antibodies. Polyarthritis patients demonstrated significantly higher levels of all anti-CCP antibody isotypes compared to other JIA subtypes (p<0.05), and even higher levels when only evaluating IgM rheumatoid factor (RF)-positive polyarthritis patients. Serum-matched anti-CCP antibody data was available on 27 JIA patients and 5 controls. All IgM RF-positive polyarthritis patients were positive for IgG anti-CCP antibodies in both serum and SF. Two JIA patients positive for IgG anti-CCP antibodies in SF were negative in serum. More JIA patients were positive for IgA anti-CCP antibodies in serum than in SF, while SF showed increased positivity for IgM anti-CCP antibodies compared to serum.

Western blot analysis revealed multiple citrullinated proteins in JIA that were not detected in OA. Immunoprecipitation with α-enolase and fibrinogen, followed by AMC detection showed presence of these specific citrullinated proteins in JIA and no detection in OA

Conclusion:

All anti-CCP antibodies were detected in the SF of JIA patients. The most commonly found anti-CCP antibody isotype in JIA SF was IgM, which may be partly indicative of defective B cell class switching in these patients. Measurement of only IgG anti-CCP antibodies would have overlooked 4 JIA patients with elevated levels of IgA and/or IgM anti-CCP antibodies, indicating the importance of measuring all 3 isotypes. The abundance of citrullinated proteins in JIA SF may be characteristic of inflammation, as seen in RA and spodyloarthritis. We have shown the detection of citrullinated proteins at the site of inflammation, specifically identifying citrullinated fibrinogen and α-enolase in SF.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/anti-cyclic-citrullinated-peptide-antibody-isotyping-and-identification-of-citrullinated-proteins-in-the-synovial-fluid-of-juvenile-idiopathic-arthritis-patients/