In vitro modulation of the cytokine network by MTX, increasing Th2 cytokines and decreasing Th1 cytokines, could explain its antiinflammatory and immunoregulatory actions in vivo during the treatment of RA.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by inflammation of the synovial membrane of multiple joints. This inflammatory microenvironment allows fibroblast-like synoviocytes (FLS) to express or enhance several adhesion or costimulatory molecules. This phenotypic shift, under proinflammatory cytokines, seems to be related to functional consequences for antigen presentation to T cells. The sensory neuropeptide substance P (SP), present at high levels, is able to act on FLS proliferation and enzyme secretion. These data led us to investigate whether SP could also provoke a phenotypic change of FLS. Using flow cytometry and a three-step cellular ELISA method, we determined whether SP has an influence on the expression of MHC class II, intercellular adhesion molecule-1 (ICAM-1), VCAM-1, LFA-3, CD40, B7.1 or B7.2 molecules on RA FLS incubated with interferon-gamma (IFN-gamma) or IL-1beta or tumour necrosis factor-alpha (TNF-alpha) with or without SP. Our results indicate that SP potentiates the effect of proinflammatory cytokines on the expression of VCAM-1 on RA FLS. We verified the presence of specific SP (NK1) receptor mRNA. Using reverse transcription-polymerase chain reaction, we showed that RA FLS of patients express NK1 receptor mRNA. These results suggest that SP increase of cytokine-induced VCAM-1 expression acts via this specific SP receptor. Thus, during chronic inflammation RA FLS are at the interface between the immune and the nervous systems.

In a previous study, we showed that the T cell repertoire is biased in the synovial membrane (SM) compared with peripheral blood during rheumatoid arthritis (RA). The same bias was observed in different joints from the same patient and seems to be the same over time. To discover whether this bias was due to expansion of a clonal subset resulting from activation by conventional Ag(s) or to polygonal stimulation by superantigen(s), we sequenced more than 650 TCRBV-D-J junctional regions from freshly isolated SM and peripheral blood of two DR4-RA patients. From each patient, two SM were obtained on the same day, and a third was obtained later. Several dominant clones were found in SM but not in peripheral blood. Some of them were found only at the first time point in anatomically different SM, the majority persisted over time, and others were detected only at the second time point. Analysis of the complementarity-determining region 3 (CDR3) showed a bias in TCRBD and amino acid usage. Valine, encoded by randomly inserted N nucleotides, was used by 45% of dominant clones compared with 18% in the control population (p less than 0.001). In addition, GXXG and TSG motifs were frequently observed in the CDR3 of these dominant clones. These data indicate a dynamic TCR selection process during the perpetuation phase of RA. The dynamic changes of dominant clones also suggest a determinant spreading mechanism during RA.