Tetsushi Aita

Production of interleukin-7 and interleukin-15 by fibroblast-like synoviocytes from patients with rheumatoid arthritis

OBJECTIVE To examine the ability of fibroblast-like synoviocytes in rheumatoid arthritis (RA) to produce interleukin-7 (IL-7) and IL-15, and the ability of these cytokines to induce the proliferation of synovium-infiltrating T cells. METHODS Messenger RNA (mRNA) and protein levels of IL-7 and IL-15 in synovial tissue cells and fibroblast cell lines were determined by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. T cell-enriched populations from RA synovial tissues were isolated by deleting adherent cells after a 14-hour incubation in plastic dishes or by expanding T cells during a 14-day incubation of tissue cells with IL-2 alone, and their proliferative responses to IL-7, IL-15, and IL-2 were measured by 3H-thymidine incorporation. RESULTS Freshly isolated cells from RA synovial tissues more strongly expressed mRNA for both IL-7 and IL-15 compared with the cells from osteoarthritis tissues, and could spontaneously release greater amounts of these cytokine proteins in culture. Fibroblast cell lines prepared from RA patients were able to produce large amounts of IL-15 and small amounts of IL-7 at both the transcriptional and protein levels, and their cytokine production was significantly elevated when stimulated with IL-1 and tumor necrosis factor alpha. Purified synovial tissue macrophages spontaneously released IL-15 but not IL-7, and synovial T cells did not produce either cytokine. IL-7 and IL-15, similar to IL-2, stimulated the proliferation of synovial tissue T cells from RA patients; IL-7 was less potent than IL-15 or IL-2. CONCLUSION These results indicated that fibroblast-like synoviocytes are an important source of the cytokines with IL-2-like activity, IL-15 and IL-7, in RA joints, and that IL-15 may be mainly responsible for local T cell activation and expansion in the presence of deficient IL-2 production by T cells.

Differential in vitro effects of IL-4, IL-10, and IL-13 on proinflammatory cytokine production and fibroblast proliferation in rheumatoid synovium.

Abstract The purpose of this study was to compare the potential of interleukin-4 (IL-4), IL-10, and IL-13 to interrupt two major inflammatory pathways in rheumatoid arthritis (RA), i.e., overexpression of proinflammatory cytokines and cytokine-mediated fibroblast growth. IL-4, IL-10, and IL-13 were all able to significantly inhibit the production of IL-1β, tumor necrosis factor-α (TNF-α), IL-6, and IL-8 by freshly isolated RA synovial tissue cells; IL-10 was most effective in terms of IL-1β and TNF-α reduction. The IL-1 receptor antagonist was enhanced by IL-4 and IL-13, but only slightly enhanced by IL-10. Spontaneous interferon-γ secretion was diminished by IL-4 and IL-10 but not by IL-13. Addition of anti-IL-10 neutralizing antibody to RA synovial tissue cells resulted in a substantial increase in IL-1β and TNF-α levels, whereas neither anti-IL-4 nor anti-IL-13 antibody had a significant effect. IL-1β-stimulated proliferation of RA synovial fibroblast cell lines was inhibited by IL-4 and IL-13, but not by IL-10; IL-4 was over tenfold more effective than IL-13. These results suggest that IL-4, IL-10, and IL-13 all have the therapeutic potential to regulate the disease activity mediated by proinflammatory cytokines in RA, but each cytokine may have different potencies.

The production of CXCR3-agonistic chemokines by synovial fibroblasts from patients with rheumatoid arthritis

The inflamed synovial tissue of rheumatoid arthritis (RA) is characterized by an infiltration with Th1 cells that predominantly express the chemokine receptors CXCR3 and CCR5. In this study, we investigated the production of the CXCR3-agonistic chemokines CXCL9, CXCL10, and CXCL11 by synovial tissue cells and synovial fibroblast-cell lines (fourth or fifth passage) from RA patients. Concentrations of all CXCR3 ligands in synovial fluids were markedly higher in RA patients than in osteoarthritis (OA) patients. Synovial tissue cells from RA patients more strongly expressed mRNAs for CXCR3 ligands and spontaneously secreted larger amounts of these chemokine proteins than the cells from OA patients. The mRNA expression of all CXCR3 ligands was induced in synovial fibroblasts from RA patients after stimulation with interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), or interleukin-1 beta (IL-1β). However, synovial fibroblasts significantly secreted CXCL9 and CXCL10 proteins, but not CXCL11 protein, after IFN-γ stimulation and secreted only CXCL10 protein after TNF-α or IL-1β stimulation. When stimulated with a combination of IFN-γ and TNF-α, these cells were able to secrete large amounts of all three chemokines. These results indicate that synovial fibroblasts may be involved in perpetuating the Th1 immune response by producing the Th1-associated CXCR3 ligands, and the synergistic effect of IFN-γ and TNF-α may be important for their chemokine production in RA joints.

Resistance to IL-10 inhibition of interferon gamma production and expression of suppressor of cytokine signaling 1 in CD4+ T cells from patients with rheumatoid arthritis

IL-10 has been shown to block the antigen-specific T-cell cytokine response by inhibiting the CD28 signaling pathway. We found that peripheral blood CD4+ T cells from patients with active rheumatoid arthritis (RA) were able to produce greater amounts of interferon gamma after CD3 and CD28 costimulation in the presence of 1 ng/ml IL-10 than were normal control CD4+ T cells, although their surface expression of the type 1 IL-10 receptor was increased. The phosphorylation of signal transducer and activator of transcription 3 was sustained in both blood and synovial tissue CD4+ T cells of RA, but it was not augmented by the presence of 1 ng/ml IL-10. Sera from RA patients induced signal transducer and activator of transcription 3 phosphorylation in normal CD4+ T cells, which was mostly abolished by neutralizing anti-IL-6 antibody. Preincubation of normal CD4+ T cells with IL-6 reduced IL-10-mediated inhibition of interferon gamma production. Blood CD4+ T cells from RA patients contained higher levels of suppressor of cytokine signaling 1 but lower levels of suppressor of cytokine signaling 3 mRNA compared with control CD4+ T cells, as determined by real-time PCR. These results indicate that RA CD4+ T cells become resistant to the immunosuppressive effect of IL-10 before migration into synovial tissue, and this impaired IL-10 signaling may be associated with sustained signal transducer and activator of transcription 3 activation and suppressor of cytokine signaling 1 induction.