Characterization of Autoreactive T Cells to the Autoantigens Heterogeneous Nuclear Ribonucleoprotein A2 (RA33) and Filaggrin in Patients with Rheumatoid Arthritis

Abstract

The role of autoimmune reactions in the pathogenesis of rheumatoid arthritis (RA) is poorly understood. To address this issue we have investigated the spontaneous T cell response to two well-characterized humoral autoantigens in RA patients and controls: 1) the heterogeneous nuclear ribonucleoprotein A2, i.e., the RA33 Ag (A2/RA33), and 2) filaggrin in unmodified and citrullinated forms. In stimulation assays A2/RA33 induced proliferative responses in PBMC of almost 60% of the RA patients but in only 20% of the controls (patients with osteoarthritis or psoriatic arthritis and healthy individuals), with substantially stronger responses in RA patients (p < 0.00002). Furthermore, synovial T cells of seven RA patients investigated were also clearly responsive. In contrast, responses to filaggrin were rarely observed and did not differ between RA patients and controls. Analysis of A2/RA33-induced cytokine secretion revealed high IFN-γ and low IL-4 production in both RA and control PBMC, whereas IL-2 production was mainly observed in RA PBMC (p < 0.03). Moreover, A2/RA33-specific T cell clones from RA patients showed a strong Th1 phenotype and secreted higher amounts of IFN-γ than Th1 clones from controls (p < 0.04). Inhibition experiments performed with mAbs against MHC class II molecules showed A2/RA33-induced T cell responses to be largely HLA-DR restricted. Finally, immunohistochemical analyses revealed pronounced overexpression of A2/RA33 in synovial tissue of RA patients. Taken together, the presence of autoreactive Th1-like cells in RA patients in conjunction with synovial overexpression of A2/RA33 may indicate potential involvement of this autoantigen in the pathogenesis of RA.

Immunodominant T‐cell epitopes of hnRNP‐A2 associated with disease activity in patients with rheumatoid arthritis

“…The four peptides 10-26, 50-70, 120-133, and 152-170 were found to fulfill this criterium, although 10-26 bound weakly to DR Ã 0101, 120-133 weakly to DR Ã 0404, and 152-170 weakly to DR Ã 0401. Therefore, these epitopes, followed by peptides [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][177][178][179][180][181][182][183][184][185][186][187][188][189][190][191][192][193], and 289-306, were considered best candidates to detect hnRNP-A2 specific T cells in patients with RA.…”

“…HPLC purified peptides of varying length were also synthesized. Recombinant hnRNP-A2 protein was prepared as previously described [8]. Purified tuberculin protein derivate (PPD) was purchased from Statens Serum institute (Copenhagen, Denmark), tetanus toxoid (TT) was obtained from Pasteur Merieux Connaught (Willowdale, ON, Canada), and PHA was from Gibco-Invitrogen.…”

“…Results are given as stimulation index (SI) defined by the ratio of (mean cpm obtained in cultures with antigen with or without Ab): (mean cpm obtained in cultures with medium only). An SI Z2 was regarded as positive response [8].…”

“…HnRNP-A2 appears to be ubiquitously expressed, although the level of expression may greatly vary between different tissues. Interestingly, hnRNP-A2 is overexpressed in RA synovial tissue, where it is detectable not only in the nucleus but also in the cytoplasm of macrophages and fibroblast-like synoviocytes [8]. Autoantibodies to hnRNP-A2 (which are also known as anti-RA33 Ab) are present in approximately 30% of RA patients [9], but also in patients with systemic lupus erythematosus (SLE) and mixed connective tissue disease [9].…”

“…Remarkably, however, epitope recognition was found to differ between the three disorders [10]. Furthermore, also T cells from peripheral blood and synovial fluid of RA patients were found to react to hnRNP-A2, in about 60% of the patients [8]. Interestingly, autoimmunity to hnRNP-A2 has been observed in TNF-transgenic (Tg) mice [11], which develop arthritis spontaneously, and is a dominant immunological event in pristane-induced arthritis in rats [12].…”