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Synovial monocytes mediate Rap1-dependent oxidative stress in rheumatoid arthritis T lymphocytes


Transient production of reactive oxygen species (ROS) plays an important role in optimizing transcriptional and proliferative responses to T-cell receptor signaling. Conversely, chronic oxidative stress leads to mitogenic hyporesponsiveness and enhanced transcription of inflammatory gene products. It has recently been demonstrated that constitutive activation of the small GTPase Ras and simultaneous inhibition of Rap1 in synovial fluid (SF) T cells results in high intracellular ROS production, which is thought to underlie many of the functional abnormalities observed in these cells in rheumatoid arthritis.


To identify the factor(s) responsible for modulation of intracellular ROS production in synovial T lymphocytes.


Purified rheumatoid arthritis peripheral blood (PB) T cells were incubated in the presence of different cytokines, in 50% autologous SF, or with autologous SF monocytes for 72 hours. Activation status of Ras and Rap1 GTPases were determined using activation-specific probes for these GTPases. Oxidation of the dye DCF by FACS analysis was used to measure intracellular ROS production.


Chronic stimulation of PB T cells for 72 hours with tumor necrosis factor alpha (TNF-α) or 50% autologous SF resulted in a slight increase in basal ROS production, but did not increase intracellular ROS production to levels found in SF T cells. Exposure of PB T cells to SF (but not PB) monocytes for 72 hours, however, led to a strong increase in ROS production in PB T cells, comparable with ROS levels in SF T cells. Moreover, similar Rap1 inhibition as found in SF T cells were observed in PB T cells after exposure to SF monocytes. To demonstrate that the inhibition of Rap1 is critical in the subsequent increase in ROS production, PB T cells were nucleofected with the constitutive active isoform of Rap1 (RapV12). In RapV12 nucleofected PB T cells the SF monocyte-induced ROS production was prevented. Cell–cell contact is critical, since in PB T cells separated from SF monocytes by a transwell membrane, the inhibition of Rap1 was relieved, concomitant with an absence in excess ROS production. Additionally, we found that addition of 10 μg/ml recombinant CTLA-4-Ig fusion protein also prevented oxidative stress in PB T cells exposed to SF monocytes, which suggested a central role for CD28. PB T cells were therefore stimulated with TNF-α, interferon gamma, IL-1β, or transforming growth factor beta, in the presence or absence of anti-CD28. Here we found that stimulation with anti CD28 by itself was sufficient to induce Rap1 inhibition and induce a moderate increase in ROS production. Co-incubation of PB T cells with TNF-α strongly enhanced the intracellular ROS production.


In vitro exposure of PB T cells from rheumatoid arthritis patients to synovial monocytes leads to a strong increase in intracellular ROS production. This is mediated by simultaneous Ras activation and inhibition of Rap1. Where Ras can be activated by a variety of stimuli, Rap1 inhibition is induced by SF monocytes through CD28 costimulatory signaling.

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Remans, P., van Laar, J., Sanders, M. et al. Synovial monocytes mediate Rap1-dependent oxidative stress in rheumatoid arthritis T lymphocytes. Arthritis Res Ther 7 (Suppl 1), P130 (2005).

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