Free radicals regulate glucocorticoid-induced apoptosis in T lymphocytes

Background

Glucocorticoid-induced apoptosis is a phenomenon of considerable biological and clinical significance. In particular, lymphoid cells are susceptible to glucocorticoid-induced apoptosis. In patients with rheumatoid arthritis, one of the critical hallmarks of synovial T cells is that they suffer from severe oxidative stress. During the past decade, reduction–oxidation (redox) reactions that generate reactive oxygen species (ROS) have been identified as important chemical mediators in the regulation of signal transduction processes. In particular, free radicals appear to have a central role in the balance between cell growth, survival and apoptosis.

Objective

To investigate the role of free radicals in corticoid-induced apoptosis.

Materials and methods

T lymphocytes were isolated from peripheral blood (PB) and synovial fluid through a negative isolation procedure (Dynal). The cells were treated with different concentrations of methylprednisolone, in combination with different inhibitors and substrates of oxidases. Apoptosis was assessed by annexine V/PI staining, and oxidation of the dye DCF was used to measure ROS production. Control, active Ras (RasV12) and inhibitory Rap1 constructs (RapGAP) were introduced in PB T lymphocytes by nucleofection techniques.

Results

A time-dependent and concentration-dependent apotosis is induced in T lymphocytes after treatment with methylprednisolone. But where concentrations over 200 μM methylprednisolone were required to induce apoptosis in T cells isolated from PB from healthy donors and RA patients, apoptosis in T cells isolated from the synovial fluid could be readily detected at concentrations of 5–10 μM. To test whether the disturbed redox balance plays a central role in sensitising T cells for glucocorticoid-induced apoptosis, PB T cells were first incubated for 12 hours with subapoptotic concentrations of H₂O₂ (5–50 μM), and for 48 hours with BSO (which results in intracellular gluthathione depletion) and NAC (which increases intracellular gluthathione). Here, both pretreatment of the T cells with H₂O₂ and BSO sensitised the T cells for apoptosis, whereas NAC protected T cells from glucocorticoid-induced apoptosis. Moreover, other T cells that are known to suffer from oxidative stress also showed increased apoptosis following treatment with methylprednisolone: both HIV-infected CD4 lymphocytes that were isolated from patients diagnosed with AIDS (n = 3) and T cells that were nucleofected with RasV12 and Rap1GAP also showed apoptosis at 5–10 μM methylprednisolone.

Concomittant with the apoptosis, a 1.5-fold to 2.5-fold increase in intracellular ROS production was measured in the pre-apoptotic cells. This increase of intra-cellular ROS seems to be a critical process in the apoptotic process as incubation in oxygen-deprived conditions inhibited the apoptosis. Also, addition of rotenone and catalase inhibited both ROS increase and apoptosis after methylprednisolone treatment, suggestive of a mitochondrial source of the free radicals.

Conclusions

Cells suffering from oxidative stress are sensitised to glucocorticoid-induced apoptosis.