Background 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 signalling 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 intracellular level of the anti-oxidant (GSH) by treatment of cells with N-acetyl-cysteine (NAC) seems to partly restore the observed signalling defects.
Objectives 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 patients with RA.
Methods T cells from healthy donors and PB and SF from RA patients were isolated and 5 min after stimulation with either CD3 antibodies or PMA+ionomycine, whole cell lysates were prepared in Ral lysis buffer. GTP-bound Rap1 was precipitated 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.
Results Our data show that in T cells isolated from healthy controls, Rap1 activation was sensitive to redox balance alterations. Upregulation of intracellular GSH by incubation with 10 mM NAC for 48 h resulted in diminished Rap1 activation, while depletion of GSH by pre-incubation with 200mM BSO resulted in increased 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, while the PB T cells of RA patients could be normally activated, Rap1 remained inactive in SF T cells upon stimulation. 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 seems to reverse the hypo responsiveness of the SF T cells, this had no effect on the defective rap1 activation.
Rap1 activation is defective in SF T cells from RA patients, and cannot be restored by the replenishment of GSH with NAC.
This defective activation is not due to increased levels of the Rap1 GTPase activating proteins RapGAP or Spa1.