Redox Balance Alterations and Hyporesponsiveness of Synovial T Cells in Rheumatoid Arthritis

In rheumatoid arthritis (RA), the functional status of T lymphocytes is incompletely understood. Synovial fluid (SF) T lymphocytes display phenotypic evidence of former activation, but there is hardly any production of T cell derived cytokines in the synovium. Moreover, the in vitro proliferative responsiveness of SF T lymphocytes is decreased compared with that of peripheral blood (PB) T lymphocytes.

Previous studies have revealed reduced intracellular Ca²⁺ responses and a decreased overall tyrosine phosphorylation pattern in SF T lymphocytes upon TCR/CD3 simulation [1]. Specifically, the tyrosine phosphorylation of the TCR zeta chain, one of the most proximal events in TCR signaling, was clearly diminished in RA SF T lymphocytes [2]. Moreover, the phosphorylation of a 36kDa protein was virtually absent in RA SF T lymphocytes. Here we report that the 36kDa protein is identified as LAT (linker for activation of T cells). In healthy T lymphocytes, LAT is heavily phosphorylated on tyrosine residues by ZAP-70 (zeta-associated protein of 70kDa) upon TCR engagement, which is required for the activation of PLCg1 and the subsequent influx of Ca²⁺ [3]. Using FACS analysis, we show that the expression of LAT is reduced in both SF and PB T lymphocytes from RA patients compared with T lymphocytes from healthy controls.

LAT is normally a membrane-bound protein due to the presence of a short α -helical structure. Using immuno-fluorescence staining and microscopy, we found that LAT is displaced from the membrane in SF but not PB T lymphocytes from RA patients. The synovium provides an environment of oxidative stress for the SF T lymphocytes, which are severely depleted in their intracellular levels of the antioxidant glutathione (GSH). The replenishment of GSH in SF T lymphocytes by treatment with NAC (N-acetyl-L-cysteine) restores the membrane localization of LAT, and also the phosphorylation of LAT and the expression of IL-2 after TCR stimulation.

Conclusively, it is demonstrated that the hyporesponsiveness of synovial T lymphocytes in RA is associated with the membrane-displacement of LAT. The membrane localization of LAT is sensitive to intracellular GSH alterations [4]. The displacement of LAT fails to bring ZAP-70 in its proximity after TCR stimulation, whereby LAT remains unphosphorylated and the TCR-mediated signaling pathway is abrogated. Hence, these results suggest a role for the membrane-displacement of LAT in the hyporesponsiveness of SF T lymphocytes as a consequence of local oxidative stress.

Affiliations

1. Leiden, The Netherlands

   • Ferdinand C Breedveld
   • , SI Gringhuis
   • , MM Maurice
   •  & CL Verweij

Reprints and Permissions