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Arthritis Res. 2001; 3(Suppl A): P041.
Published online Jan 26, 2001. doi:  10.1186/ar210
PMCID: PMC3273224
Defective Rap1 activation in synovial fluid T cells from patients with rheumatoid arthritis
PHJ Remans,1 SI Gringhuis,1 K Reedquist,2 FC Breedveld,1 JM van Laar,1 and CL Verweij1
1Rheumatology, LUMC, 2300 RC Leiden, The Netherlands
2Dept. Physiological Chemistry, and Center of Biomedical Genetics, UMC, 3584 CG Utrecht, The Netherlands
Supplement
21st European Workshop for Rheumatology Research
Conference
21st European Workshop for Rheumatology Research
1-4 March 2001
Vienna, Austria
Received January 15, 2001
Rap1 is a small G-protein, member of the Ras GTPase family. Rap1 has been linked to T cell anergy and it is suggested that Rap1 has the potential to inhibit Ras-mediated oncogenic or growth promoting activity. Synovial fluid (SF) T cells from patients with rheumatoid arthritis (RA) display a defective phosphorylation pattern of several pivotal signaling proteins and severe hyporesponsiveness upon antigenic stimulation. Our previous data showed that the hyporesponsive state of the SF T cells in RA correlates with markers of oxidative stress and replenishment of the intra-cellular level of the anti-oxidant glutathione (GSH) by treatment with N-acetyl-L-cysteine (NAC) restores the observed signaling defects.
Objective
To determine Rap1 activation and its correlation with oxidative stress in T cells from healthy controls compared to peripheral blood (PB) and SF T cells isolated from RA patients.
Methods
T cells from healthy donors and PB and SF T cells from 5 RA patients were isolated and after 5 minutes of stimulation with either anti-CD3 antibodies or PMA+ionomycin, whole cell lysates were prepared in Ral lysis buffer. GTP-bound Rap1 was isolated using the bacterial expressed fusion protein GST-RalGDS and detected by ECL Western blotting. In whole cell lysates, total Rap1, rapGAP and Spa1 were detected by ECL western blotting as well.
Our data show that in T cells isolated from healthy controls, Rap1 was activated in a redox-dependent way. Upregulation of intracellular GSH levels by incubation with 10 mM NAC for 48 h resulted in diminished Rap1 activation, while depletion of GSH by pre-incubation for 48 h with 200 μM BSO resulted in enhanced Rap1 activation. We also observed that treatment of T cells with H2O2 led to the rapid activation of Rap1.
Despite an environment of oxidative stress in the inflamed joints of RA patients, we found that in SF T cells Rap1 was present in its inactive GDP-bound state. Furthermore, upon stimulation Rap1 remained inactive in SF T cells while in contrast Rap1 could be activated in PB T cells from RA patients. The defective Rap1 activation was not due to increased levels of the Rap1 GTPase activating proteins RapGAP or Spa1. While restoration of the intracellular redox balance does reverse the hyporesponsiveness of the SF T cells, the replenishment of GSH with NAC had no effect on the defective Rap1 activation.
Conclusions
1. In healthy T cells the small GTPase Rap1 is activated by H2O2; 2. Rap1 activation after T cell stimulation is redox-dependent; 3. Rap1 activation is defective in SF T cells from RA patients, and cannot be restored by the replenishment of GSH with NAC.
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