Redox-sensitive changes in conformation and cellular localization of LAT and downstream TCR signaling lead to hyporesponsiveness of synovial fluid T cells in rheumatoid arthritis

Abstract

In rheumatoid arthritis (RA), the synovial fluid (SF) T lymphocytes present in the inflamed joints, display hyporesponsiveness upon engagement of the TCR/CD3 complex despite phenotypic evidence of former activation. We have previously shown that the central and crucial adaptor protein LAT (linker for activation of T cells), which plays a central and crucial role in the T cell receptor (TCR)-mediated signaling pathways, exhibits deficient phosphorylation due to displacement of the integral membrane protein from the plasma membrane in SF T lymphocytes. SF T lymphocytes exhibit several features of chronic oxidative stress, e.g. severely decreased intracellular levels of glutathione (GSH), and our previous studies have indicated that the subcellular localization of LAT is sensitive to changes in the intracellular GSH levels. The cysteine-to-serine substitutions of several cysteine residues (C26/29 or C117) within LAT creates LAT mutants that are resistant to reduced intracellular GSH levels and remain membrane-anchored in GSH-depleted cells. In this study, we have used the redox-insensitive LAT mutants to study the effect of redox balance alterations, like in SF T lymphocytes, on TCR signaling pathways downstream from LAT and on CD28 signaling pathways. In co-transfection experiments, we show that the presence of the redox-insensitive LAT mutants allows for the partial restoration of the TCR-mediated signaling pathways, but not the signaling pathways induced through the CD28 receptor. The data are indicative that the Raf1-ERK and the calcium-calcineurin pathways leading to transcriptional activation of AP-1 and NFAT, respectively, are very sensitive to reduced intracellular GSH levels, while the activation of the p38/Mpk2 pathway leading to AP-1-mediated transcription is mostly unaffected by chronic oxidative stress. A very proximal event in the CD28-mediated signaling pathways seems to be extremely sensitive to GSH depletion since costimulation did not affect the transcriptional activity of either AP-1 or NF-kappaB. We conclude that the signaling pathways in SF T lymphocytes from RA patients are affected at several levels by chronic oxidative stress, all contributing to the observed hyporesponsiveness of these cells.