CTLA-4IG suppresses reactive oxygen species by preventing synovial adherent cell-induced inactivation of Rap1, a Ras family GTPASE mediator of oxidative stress in rheumatoid arthritis T cells.

OBJECTIVE: Oxidative stress contributes to the inflammatory properties of rheumatoid arthritis (RA) synovial T lymphocytes. This study was undertaken to investigate the mechanisms leading to production of reactive oxygen species (ROS) and oxidative stress in RA synovial T lymphocytes. METHODS: ROS production in T lymphocytes from the peripheral blood (PB) of healthy donors and from the PB and synovial fluid (SF) of RA patients was measured by ROS-dependent fluorescence of 6-carboxy-2',7'-dichlorofluorescein. Rap1 GTPase activation was assessed by activation-specific probe precipitation. Proliferation of RA PB and SF T lymphocytes was assayed by 3H-thymidine incorporation. In some experiments, RA PB T cells were preincubated with autologous SF or with PB or SF adherent cells. Experiments were performed in the absence or presence of transwell membranes or CTLA-4Ig fusion proteins. Short- and long-term stimulations of healthy donor PB T lymphocytes were performed with inflammatory cytokines, in the absence or presence of activating anti-CD28 antibodies. RESULTS: T lymphocyte ROS production and Rap1 inactivation were mediated by cell-cell contact with RA synovial adherent cells, and this correlated with T cell mitogenic hyporesponsiveness. CTLA4-Ig blockade of synovial adherent cell signaling to CD28 T cells reversed the inhibition of Rap1 activity and prevented induction of ROS. Introduction of active RapV12 into T cells also prevented induction of ROS production. Coincubation of T cells with stimulating anti-CD28 antibodies and inflammatory cytokines synergistically increased T cell ROS production. CONCLUSION: Cell-cell contact between T cells and RA synovial adherent cells mediates Rap1 inactivation and subsequent ROS production in T lymphocytes following exposure to inflammatory cytokines. This process can be blocked by CTLA4-Ig fusion protein.

Intracellular free radical production in synovial T lymphocytes from patients with rheumatoid arthritis.

OBJECTIVE: To investigate the cellular and molecular sources of oxidative stress in patients with rheumatoid arthritis (RA) through analysis of the production of reactive oxygen species (ROS) in synovium. METHODS: Cytochemical procedures based on the 3,3'-diaminobenzidine (DAB)-Mn2+ deposition technique were used on unfixed cryostat sections of synovium from RA patients and rheumatic disease controls. For immunophenotyping, sections were incubated, fixed, and stained with fluorescein isothiocyanate-labeled antibodies. Fluorescence-activated cell sorter analysis of the ROS-reactive dye 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate-di(acetoxymethyl ester) was used to measure intracellular ROS in T lymphocytes from peripheral blood and synovial fluid. To determine which enzymes produced ROS, different inhibitors were tested. RESULTS: Large quantities of DAB precipitated in the majority of RA synovial T lymphocytes, indicative of intracellular ROS production. These ROS-producing T lymphocytes were observed throughout the synovium. Polymerization of DAB was observed to a lesser extent in other forms of chronic arthritis, but was absent in osteoarthritis. DAB staining of cytospin preparations of purified RA synovial fluid T cells confirmed the presence of ROS-producing cells. One of the ROS involved appeared to be H2O2, since catalase suppressed intracellular ROS production. Superoxide dismutase, which uses superoxide as a substrate to form H2O2, diphenyleneiodonium (an inhibitor of NADPH oxidase), N(G)-monomethyl-L-arginine (an inhibitor of nitric oxide synthesis), nordihydroguaiaretic acid (an inhibitor of lipoxygenase), and rotenone (an inhibitor of mitochondrial ROS production) failed to suppress ROS production. CONCLUSION: Our findings show that chronic oxidative stress observed in synovial T lymphocytes is not secondary to exposure to environmental free radicals, but originates from intracellularly produced ROS. Additionally, our data suggest that one of the intracellularly generated ROS is H2O2, although the oxidase(s) involved in its generation remains to be determined.

Nutrient supplementation with polyunsaturated fatty acids and micronutrients in rheumatoid arthritis: clinical and biochemical effects.

OBJECTIVE: To investigate in a double-blind placebo-controlled, parallel group study, the effects of a nutrient supplement, containing, among other ingredients, the omega-3 fatty acids eicosapentaenoic acid (1.4 g EPA), docosahexaenoic acid (0.211 g DHA), omega-6 fatty acid gamma-linolenic acid (0.5 g GLA) and micronutrients in patients with active rheumatoid arthritis (RA). DESIGN, SUBJECTS AND INTERVENTION: RA patients were randomized to receive either daily liquid nutrient supplementation or placebo for 4 months. The primary end point was the change in tender joint count at 2 and 4 months. Other clinical variables included swollen joint count, visual analogue scales for pain and disease activity, grip strength, functionality score and morning stiffness. Biochemical parameters included plasma concentrations of PUFA and vitamins C and E. SETTING: Outpatient university clinic. RESULTS: In all, 66 patients enrolled, 55 completed the study. No significant change from baseline in tender joint count or any of the other clinical parameters was detected in either group. Patients receiving nutrient supplementation, but not those receiving placebo, had significant increases in plasma concentrations of vitamin E (P=0.015), and EPA, DHA and docosapentaenoic acid concomitant with decreases of arachidonic acid (P=0.01). Intergroup differences for PUFA and vitamin E were significantly different (P=0.01 and 0.03, respectively). CONCLUSIONS: This double-blind, placebo-controlled study in RA patients did not show superior clinical benefit of daily nutrient supplementation with EPA, GLA and micronutrients at the doses tested as compared to placebo. The study adds information regarding doses of omega-3 fatty acids, below which anti-inflammatory effects in RA are not seen.

Displacement of linker for activation of T cells from the plasma membrane due to redox balance alterations results in hyporesponsiveness of synovial fluid T lymphocytes in rheumatoid arthritis.

The T lymphocytes that reside in the synovium of the inflamed joints in patients with rheumatoid arthritis display severe hyporesponsiveness upon antigenic stimulation, which is probably due to their constant subjection to high levels of oxidative stress. Here we report that the synovial fluid T lymphocytes exert severely impaired phosphorylation of the adaptor protein linker for activation of T cells (LAT), a crucial component of the TCR-mediated signaling pathways. In healthy T lymphocytes, LAT is a membrane-bound protein and becomes phosphorylated by zeta-associated protein of 70 kDa (ZAP-70) upon TCR engagement. The molecular basis underlying the deficient phosphorylation of LAT and consequently the hyporesponsiveness of the synovial fluid T lymphocytes lies in the membrane displacement of LAT. We demonstrate that the subcellular localization of LAT is sensitive to changes in the intracellular levels of the antioxidant glutathione. The membrane anchorage of LAT, and consequently the phosphorylation of LAT and the cellular activation of the synovial fluid T lymphocytes upon TCR engagement, is restored in synovial fluid T lymphocytes after supplementation of the intracellular glutathione levels with N-acetyl-l -cysteine. These data suggest a role for the membrane displacement of LAT in the hyporesponsiveness of the synovial fluid T lymphocytes as a consequence of oxidative stress.