To characterize the molecules responsible for synovial fibroblast-T lymphocyte (TL) cross-talk in rheumatoid arthritis (RA), synovial fibroblasts from patients with established RA (RASFibs) were cocultured with TLs from peripheral blood of early RA patients (RAPBTL). TLs from peripheral blood of healthy controls and from synovial fluid of RA served as controls. Adhesion molecules and cytokines were determined by flow cytometry, ELISA, and real-time PCR. RAPBTL (n = 20) induced an up-regulation of ICAM-1, intracellular IL-8, IL-6, IL-15, and surface IL-15 in cocultured RASFibs. In turn, RAPBTL showed an up-regulation of TNF-alpha, IFN-gamma, IL-17, CD25, and CD69 expression. Responses seen with TLs from peripheral blood of healthy controls (n = 20) were significantly lower, whereas responses with TLs from synovial fluid of RA (n = 20) were maximal. Blocking Abs to IL-15 and CD54, but not an isotype-control Ab, down-regulated the increased TL cytokine and activation marker expression. Abs to CD69, CD11a, IL-17, TNF-alpha, and IFN-gamma significantly decreased the up-regulation of RASFib cytokine and CD54 expression. Cocultures using 0.4- micro m inserts did not result in up-regulation of surface molecules or cytokines. Methotrexate significantly inhibited RASFib/TL cross-talk signals and decreased adhesion of TL to RASFibs. In summary, RASFib production of IL-15 induces the proinflammatory cytokines TNF-alpha, IFN-gamma, and IL-17 in cocultured TLs through a cell contact-dependent mechanism. In turn, these cytokines stimulate the expression of IL-15, IL-8, and IL-6 in RASFibs, thereby creating a feedback loop that favors persistent synovial inflammation. Methotrexate seems to disrupt this loop by decreasing cell adhesion.