In rheumatoid arthritis (RA), constitutive activation of integrins promotes retention of oxidatively stressed, activated T lymphocytes in synovial tissue where they perpetuate inflammation through cell–cell contact with synovial stromal cells, macrophages and plasma cells. We have previously demonstrated that activation of Ras GTPases, coupled with inactivation of the related GTPase Rap1, is responsible for oxidative stress in RA synovial fluid (SF) T lymphocytes . Inactivation of Rap1, however, is incongruous with the observation that Rap1 signaling is absolutely required for integrin function in T lymphocytes. Because T lymphocytes express multiple Ras homologs (H-Ras, K-Ras, and N-Ras), we explored the possibility that these proteins may make distinct contributions to reactive oxygen species (ROS) regulation and integrin function in T lymphocytes.
To determine which Ras homologs are activated in RA SF T lymphocytes, to determine whether Ras homologs selectively regulate ROS production and integrin-dependent adhesion in T lymphocytes, and to use pharmacological inhibitors to characterize downstream signaling pathways by which Ras homologs may mediate these effects.
Activation of H-Ras, K-Ras, and N-Ras homologs in RA SF T lymphocytes were determined by activation-specific GTPase precipitation and immunoblot analysis. T-lymphocyte intracellular ROS production was measured by FACS-based analysis of DCFA oxidation. Control, active Ras, and RapGAP constructs were introduced into human Jurkat T cells and healthy donor peripheral blood T lymphocytes by electroporation and nucleofection techniques, respectively. For ROS analysis, cells were cotransfected with RFP to detect transfected cells. For static adhesion assays, a luciferase reporter plasmid was used to detect transfected cells: cells were allowed to adhere to immobilized fibronectin or recombinant human intracellular adhesion molecule, non-adherant cells washed off, and adherant cells quantitated by luciferase assays. In some experiments, cells were preincubated with the pharmacological inhibitors BAPTA-AM, LY294002, catalase, and DPI prior to ROS or adhesion analysis.
All three homologs of Ras were found to be constitutively activated in RA SF T lymphocytes. Active H-Ras, but not K-Ras or N-Ras, stimulated ROS production in both Jurkat and peripheral blood T lymphocytes. H-Ras-induced ROS production was sensitive to BAPTA-AM and catalase, but not LY294002 or DPI, indicating a calcium-sensitive hydrogen peroxide source of ROS that does not require a functional NADPH oxidase. These results are consistent with our previous observations in purified RA SF T lymphocytes. In contrast, N-Ras, but not H-Ras or K-Ras, induced integrin-dependent adhesion in Jurkat T cells and peripheral blood T lymphocytes. N-Ras-induced adhesion was sensitive to LY294002, indicative of PI3-kinase signaling. Additionally, inactivation of Rap1 with RapGAP had no effect on N-Ras-induced adhesion. This represents the first (patho)physiological signaling pathway by which integrins can be activated independently of Rap1. Our results suggest that selective targeting of H-Ras and N-Ras may allow specific modulation of T-cell ROS production and integrin activation in RA T lymphocytes.
Remans PHJ, Gringhuis SI, van Laar JM, Sanders ME, Papendrect-van der Voort EAM, Zwartkruis FJT, Nivine Levarht EW, Rosas M, Coffer PJ, Breedveld FC, et al.: Rap1 signaling is required for suppression of Ras-generated reactive oxygen species and protection against oxidative stress in T lymphocytes.