Background/Purpose:  Rheumatoid arthritis (RA) is an autoimmune disease leading to the inflammatory destruction of synovial joints. Anti-citrullinated protein antibodies (ACPA) are frequently detected in the serum of RA patients and may contribute to the pathogenesis of RA. Citrullination, which may involve several endogenous (self) proteins, is a post-translational modification of arginine residues in the protein backbone. Previous studies identified a citrullinated epitope in cartilage proteoglycan (PG) aggrecan, which elicited pro-inflammatory cytokine production by RA T cells in vitro. Moreover, we have recently reported the presence of ACPA-reactive (citrullinated) PG in RA cartilage. The purpose of the present study was to identify novel citrullinated epitopes in human PG that are recognized by T cells and/or antibodies from RA patients.

Methods:  We used spleen cells from mice with PG-induced arthritis (PGIA) as screening tools to select citrulline (Cit)-containing PG peptides that were more immunogenic than their arginine (R)-containing counterparts. The Cit-R pairs of selected peptides were then tested for induction of pro-inflammatory T-cell cytokine production in RA and healthy control peripheral blood mononuclear cell (PBMC) cultures using cytokine ELISA and flow cytometry methods. Anti-Cit and anti-R peptide antibodies in mouse serum or human plasma were detected by ELISA.

Results:  Spleen cells from mice with PGIA exhibited greater T-cell cytokine secretion in response to the Cit than the R version of PG peptide 49 (P49) and anti-P49 antibodies were detected in PGIA serum. PBMC from RA patients, but not PBMC from healthy controls responded to the citrullinated form of P49 (designated Cit49) with robust cytokine production in vitro. Importantly, high levels of anti-Cit49 antibodies were found in the plasma of ACPA-positive RA patients (n = 32). However, there was no correlation between the levels of Cit49-induced T-cell cytokines and anti-Cit49 antibodies in ACPA-positive RA. Another PG peptide (Cit13) similar to the previously described T-cell epitope induced greater cytokine responses than R13 by healthy control (but not RA) PBMC, however, anti-Cit13 antibodies were rarely detected in human plasma.

Conclusion:  We have identified a novel citrullinated human PG epitope (Cit49), which is highly immunogenic both in mice with PGIA and patients with RA. The lack of correlation between T-cell and antibody reactivity with Cit49 in the ACPA-positive RA group suggests that this peptide is recognized as either a strong T-cell epitope or a strong B-cell epitope in different subsets of ACPA-positive patients. As citrullinated PG may be present in RA articular cartilage, Cit PG epitope-induced T-cell activation or deposition of Cit PG epitope-specific antibodies can occur in the joints of ACPA-positive RA patients, potentially contributing to cartilage destruction.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/immune-recognition-of-a-novel-citrullinated-epitope-of-cartilage-proteoglycan-aggrecan-in-mice-with-proteoglycan-induced-arthritis-and-in-patients-with-rheumatoid-arthritis/