Immune Recognition of Citrullinated Proteoglycan Aggrecan Epitopes in Mice with Proteoglycan-Induced Arthritis and in Patients with Rheumatoid Arthritis

PLoS One. 2016 Jul 28;11(7):e0160284. doi: 10.1371/journal.pone.0160284. eCollection 2016.


Background: Rheumatoid arthritis (RA) is an autoimmune inflammatory disease affecting the joints. Anti-citrullinated protein antibodies (ACPA) are frequently found in RA. Previous studies identified a citrullinated epitope in cartilage proteoglycan (PG) aggrecan that elicited pro-inflammatory cytokine production by RA T cells. We recently reported the presence of ACPA-reactive (citrullinated) PG in RA cartilage. Herein, we sought to identify additional citrullinated epitopes in human PG that are recognized by T cells or antibodies from RA patients.

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

Results: Splenocytes 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 found in PGIA serum. PBMC from ACPA+ and ACPA- RA patients, but not from healthy controls, responded to Cit49 with robust cytokine production. High levels of anti-Cit49 antibodies were found in the plasma of a subset of ACPA+ RA patients. Another PG peptide (Cit13) similar to the previously described T-cell epitope induced greater cytokine responses than R13 by control (but not RA) PBMC, however, anti-Cit13 antibodies were rarely detected in human plasma.

Conclusions: We identified a novel citrullinated PG epitope (Cit49) that is highly immunogenic in mice with PGIA and in RA patients. We also describe T-cell and antibody reactivity with Cit49 in ACPA+ RA. As citrullinated PG might be present in RA articular cartilage, Cit PG epitope-induced T-cell activation or antibody deposition may occur in the joints of RA patients.

Publication types

  • Validation Study

MeSH terms

  • Aggrecans / immunology
  • Aggrecans / metabolism*
  • Amino Acid Sequence
  • Animals
  • Arthritis, Experimental / chemically induced
  • Arthritis, Experimental / immunology*
  • Arthritis, Rheumatoid / immunology*
  • B-Lymphocytes / cytology
  • B-Lymphocytes / immunology
  • Case-Control Studies
  • Cell Proliferation
  • Cells, Cultured
  • Citrulline / metabolism*
  • Cytokines / biosynthesis
  • Cytokines / metabolism
  • Enzyme-Linked Immunosorbent Assay
  • Epitopes / chemistry
  • Epitopes / immunology*
  • Flow Cytometry
  • Humans
  • Mice
  • Proteoglycans / chemistry
  • Proteoglycans / toxicity*
  • Spleen / cytology
  • Spleen / metabolism
  • T-Lymphocytes / cytology
  • T-Lymphocytes / immunology


  • Aggrecans
  • Cytokines
  • Epitopes
  • Proteoglycans
  • Citrulline