Rheumatoid arthritis (RA) is characterized by chronic inflammation of the synovial joints leading to irreversible joint destruction. To prevent and/or slow down progressive joint destruction, the diagnosis of RA at early stage in the disease is important. The autoantibodies against citrullinated proteins (aCCP) provide such ability, representing a marker for early RA. However, a remarkable variety in the reactivity against different citrulline-containing peptides has been described, which indicates that not only citrulline but also its flanking amino acid sequences play a role in the antigenicity and that autoantibodies recognizing such targets are poly-clonal. Furthermore, B cells isolated from synovial fluid of aCCP-positive RA patients were shown to produce aCCP antibodies spontaneously, whereas peripheral blood B cells require a stimulation to produce these antibodies. The predominance of IgG production in synovial fluid and synovial tissue was observed in comparison with serum. These findings altogether provide the evidence for the local antigen-driven maturation of B cells into aCCP-producing plasma cells within inflamed RA synovium due to the presence of citrullinated synovial proteins.
The aim of the study was to analyze the occurence of the aCCP-specific B cells in peripheral blood (PB) and synovial tissue (ST) of RA patients. Three citrulline-containing peptides derived from human natural proteins were used in order to test the hypothesis of polyclonal production of aCCP autoantibodies.
PB and ST were collected from nine patients with RA. Biotinylated citrulline-containing peptides derived from human filaggrin (P1), aggrecan (P3) and fibrin (P0428), and flow cytometry were employed to analyze B-cell populations specific for citrullinated peptides. The serum levels of aCCP autoantibodies were evaluated by ELISA kit (Mark2). Statistical analysis was performed by Pearson and paired t tests using GraphPad software (GraphPad, San Diego, CA, USA).
The serum titres of aCCP autoantibodies ranged from 0.0 to 1300.0 U/ml (median 276.6; 95% confidence interval 34.99–880.4). The CD19+ B cells recognizing individual synthetic citrullinated peptides were detected in PB and ST of RA patients, in most cases showing the reaction with at least two of three tested peptides. The frequencies of these B cells differed for both individual citrullinated peptides and patients, especially in ST, and the observed maximum was 2.4% for peptide P1 and P3 in PB and 6.4% for peptide P3 in ST. In some cases, the frequencies of CD19+ B cells specific for citrullinated peptides were increased in ST as compared with PB of the same patient; for example, 2.07% in ST versus 0.75% in PB for peptide P0428 or 6.35% in ST versus 0.1% in PB for peptide P3. However, the frequencies of analyzed citrulline-specific B cells did not correlate to serum aCCP autoantibodies titers. The data might indicate the presence of several B-cell subsets specific for different citrullinated substrates within the inflamed joint and the ongoing autoantigen-driven B-cell activation in the synovium. However, the clonal relation between B cells specific for the used individual citrullinated peptides remains unclear and will be subsequently analyzed.
The authors thank Prof. WJ van Venrooij (University of Nijmegen, Nijmegen, The Netherlands) and Dr JW Drijfhout (Leiden University Medical Center, Leiden, The Netherlands) for the biotinylated citrullinated peptides, provided as a kind gift. This work was supported by grant NK/7273-3 of the Ministry of Health of Czech Republic.