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This article is part of the supplement: 25th European Workshop for Rheumatology Research

Poster presentation

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

PH Remans1, JM van Laar2, ME Sanders1, P-P Tak1 and KA Reedquist1

Author Affiliations

1 AMC, Amsterdam, The Netherlands

2 LUMC, Leiden, The Netherlands

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Arthritis Research & Therapy 2005, 7(Suppl 1):P130  doi:10.1186/ar1651

The electronic version of this article is the complete one and can be found online at:


Received:11 January 2005
Published:17 February 2005

© 2005 BioMed Central Ltd

Background

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.

Objectives

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

Methods

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.

Results

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.

Conclusion

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.