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

Abstract

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 3 H-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 longterm stimulations of healthy donor PB T lymphocytes were performed with inflammatory cytokines, in the absence or presence of activating anti-CD28 antibodies. 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. Results. T lymphocyte ROS production and

The Presumed Hyporesponsive Behavior of Rheumatoid Arthritis T Lymphocytes Can Be Attributed to Spontaneous Ex Vivo Apoptosis rather than Defects in T Cell Receptor Signaling

“…Similar effects are observed in PB T cells chronically exposed to TNF-␣ (20,30). Additionally, stimulation of PB T cells with a number of inflammatory cytokines, in combination with CD28 ligation, can reproduce oxidative stress observed in synovial T cells (37).…”

“…Lysates were cleared by centrifugation at 13,000 rpm for 15 min and protein expression was analyzed by standard Western blotting procedures as previously reported in detail (37). Proteins were resolved by SDS-PAGE, transferred to polyvinylidene difluoride membrane (Bio-Rad), and blots were probed with Abs against Noxa (Imgenex), Bim (Chemicon International), Bcl-2 (Alexis), Bcl-x L (BD Transduction Laboratories), Mcl-1 (BD Biosciences Pharmingen), and ␤-actin and ERK1/2 (Santa Cruz Biotechnology).…”

Aberrant Reactive Oxygen and Nitrogen Species Generation in Rheumatoid Arthritis (RA): Causes and Consequences for Immune Function, Cell Survival, and Therapeutic Intervention

“…Extracellular ROS production may influence the oxidation state of surface proteins, but endogenous sources of oxidants (e.g., mitochondria) or endosomal ROS production may play an important role in the regulation of intracellular signaling. Remans et al (244)(245)(246) have undertaken studies to determine how intracellular pathways that are associated with ROS production may be regulated. They identified that a failure of RapI to regulate the RaI-mediated increase in ROS production, which lies downstream from Ras activation, may contribute to a hyporesponsive T cell phenotype; using a Jurkat T cell line transformed to overexpress ras constructs, this group determined that following anti-CD3 driven activation of T cells, intracellular ROS levels increased, as demonstrated by dichlorofluorescein diacetate (DCF) fluorescence.…”

Sustained Rap1 activation in autoantigen-specific T lymphocytes attenuates experimental autoimmune encephalomyelitis

“…A potential role for Rap1 in human T cell-mediated autoimmune disease was first suggested by the fact that Rap1 is inactivated in RA synovial fluid T cells and that this is associated with the persistent production of reactive oxygen species (ROS) and hyper-responsive cytokine production following TCR triggering (Remans et al, 2004(Remans et al, , 2006Abreu et al, 2009). Similarly to Ras, Rap1 is activated by TCR and adhesion molecule ligation, as well as by chemokine stimulation (Minato et al, 2007).…”