Deregulated Ras and Rap1 signaling in rheumatoid arthritis T cells leads to persistent production of free radicals

Background

We have previously found that key Ras family signaling molecules, H-Ras and Rap1, are deregulated in SF T lymphocytes: Ras is constitutively active, whereas Rap1 can not be activated. Here we report that similar signaling events in Jurkat cells lead to the persistent production of intracellular reactive oxygen species (ROS), mimicking the hyporesponsiveness of synovial T lymphocytes.

Methods

Jurkat cells were cotransfected with pCMV-CD20 expression plasmid to differentiate between transfected and untransfected cells and 20 μg expression plasmid encoding for RasV12, RasN17; RasV12/G37, RasV12/E38, RasV12/C40, Rlf-CAAX, RalV23, RalN; RapV12, RapGAP, RapN17 RasV12/G37, RasV12/E38, RasV12/C40. For ROS determination, cells were first stained with CyCr CD 20 for detection of positive transfected cells. CD20-pos cells were analysed on a FACScan for the mean fluorescence intensity of oxidated DCF (di-chlorofluorescin), as a measurement for the presence of intracellular ROS.

Results

1) Treatment of either PB T lymphocytes or Jurkat cells with either anti-CD3 antibodies or PMA/I leads to a rapid and transient generation of intracellular ROS which was maximal 2–5 minutes poststimulation. 2)In Jurkat, expression of Ras is sufficient to induce the generation of ROS, in a Ral dependent manner and Ras is required for TCR and PMA/ionomycine induced ROS3. Consitutively active Rap1 suppressed TCR, TPA and Ras-induced ROS production in a PI3-kinase dependent manner, while inactivation of the Rap signalling pathway sensitized T cells to agonist-induced ROS generation.

Conclusions

1) Rap1 GTPase plays a critical role in the termination of transient ROS production in T lymphocytes following agonist stimulation. This is unlikely due to competitive sequestration of Ras effectors by Rap1 as Rap1-dependent inhibition of ROS is PI 3-kinase-dependent, while Ras-induced ROS generation is Ral-dependent. 2)As the chronic oxidative stress observed in SF T cells can be mimicked by transient transfection of Jurkat cells with interfering Rap1 proteins, we hypothesize that deregulation of Rap and Ras are the critical events leading to the disturbed intra-cellular redox balance that underlies the hyporesponsiveness of Synovial T cells in rheumatoid arthritis.