Rheumatoid Arthritis and citrullinated proteins: immunological memory or ongoing immune response to persisting antigen?
Rheumatoid arthritis (RA) is one of the most common autoimmune diseases.
Antibodies to citrullinated proteins are a very specific marker of RA, and target several deiminated proteins, such as fibrin, collagen, enolase: they are collectively referred to as ACPA (anti citrullinated protein/peptides). Recently our group described a new target of ACPA, derived from the Epstein Barr Virus Nuclear Protein 1; the method based on this peptide (VCP: viral citrullinated peptide) is able to detect RA patients with a sensitivity of 50% and a specificity of 95%.
The production of ACPA shows some peculiarities: ACPA of the IgM isotype are found years after the disease onset, the levels of ACPA are not clearly associated with disease activity, and they do not disappear from the circulation after B cell depleting therapies.
During an immune response to exogenous antigens, antibodies of the IgM isotype are produced in the very early phase; then class switch and affinity maturation take place, with the production of IgG antibodies, showing an increased affinity for the antigen; these processes occur in the germinal center of lymphoid organs. In autoimmune diseases, germinal center-like structures are found in targeted organs, furthermore class switch and affinity maturation may take place outside the germinal centers. Humoral memory is sustained by the production of specific antibodies from long lived plasmacells or by activated memory B cells or both.
The aim of this work is to characterize the immune response to citrullinated antigens in RA patients, trying to dissect if the continuous ACPA production is the result of “immunological memory” (sustained by long lived plasmacells or by B cell memory reactivation) or is rather due to an ongoing immune response to persisting antigens.
We first studied the isotype distribution of anti-VCP in RA patients and in patients suffering from other chronic diseases; IgG, IgA, IgM anti-VCP are found almost exclusively in RA patients, IgM anti-VCP can be the only isotype in serum, and they are present also many years after the disease onset, suggesting a continuous priming of naive B cells; IgG anti-VCP display greater affinity to VCP in a liquid phase inhibition assay, suggesting the development of immunological memory. We then compared three different ACPA and antibodies to exogenous antigens in a follow-up study of RA patients: the levels and variations of the three ACPA were correlated in a significant way, and showed no correlation with the other antibodies examined.
No memory T cells, or precursor memory B cells specific for VCP were found in the peripheral blood of RA patients; instead, anti-VCP producing plasmacells were detected in the synovial fluid, but not in the peripheral blood of RA patients. The culture of PBMC on fibroblast like synoviocytes (FLS) monolayer led to the production of anti-VCP, even in the absence of polyclonal stimulation; notably, FLS from RA patients and FLS from healthy donors displayed the same effect.
Our data suggest that ACPA production is the result of a continuous recruitment of B cells from peripheral blood to inflamed joints; they can find in the synovium a microenvironment able to sustain their activation, maturation, and differentiation to plasmacells; FLS could also provide plasmacells with survival factors even in the inactive phase of the disease, as suggested by the observation that FLS derived from RA or normal subjects have the same effects.
