Crosstalk between synovial fibroblasts and T lymphocytes in rheumatoid arthritis: effect of methotrexate

Objective

Our objective was to characterize the molecules responsible for crosstalk between synovial fibroblasts (SFibs) and T lymphocytes (TLs) in RA and test the effect of methotrexate (MTX).

Methods

SFibs were obtained from RA patients. TLs were isolated from peripheral blood of healthy controls and RA patients, and from RA synovial fluid by Ficoll–Hypaque gradient and magnetic depletion of non-T cells; purity was 99%. Cocultures were established in six-well plates; adhesion molecules and surface and intracellular cytokines were determined by flow cytometry.

Results

Synovial fluid TLs (SFTLs) from RA patients (n = 10) and peripheral blood TLs (PBTLs) from untreated recent-onset RA (n = 10) induced an up-regulation of intercellular adhesion molecule (ICAM)-1, intracellular IL-8, IL-15 and surface IL-15 in SFibs. SFTLs and PBTLs showed an up-regulation of IFN-γ, IL-17, CD25 and CD69 when cocultured with SFibs. Freshly isolated PBTLs from healthy controls (n = 10) did not induce SFib cytokine production. Pretreatment of SFs with MTX (0.1–1 μM) prevented the TL-stimulated increase of IL-8 and IL-15. Pretreatment of TLs with MTX (0.1–1 μM) prevented the SFib-induced up-regulation of IFN-γ, IL-17, CD25 and CD69. Blocking MoAbs to IL-15, but not an isotype-control antibody, prevented the increased TL secretion of IFN-γ and IL-17 induced by SFibs. Blocking MoAbs to IL-17 significantly decreased the TL-induced up-regulation of IL-8 and IL-15 in SFibs. Cocultures of TLs and SFibs using 0.4μM plate inserts did not induce a significant up-regulation of adhesion molecules or cytokines. Paraformaldehide-fixed TLs did up-regulate IL-8 and IL-15 in SFibs. This suggests that direct cell contact is an important contributor to the observed effects.

Conclusion

SFib production of IL-8 and IL-15 may contribute to the influx and proliferation of TL in the RA joint. In turn, TL cytokines IFN-γ and IL-17 stimulate the production of IL-8 and IL-15 by Sfibs, thereby creating a feedback loop that leads to persistent synovial inflammation. MTX seems to disrupt this loop, acting at both the SFib and TL level.