TRANSCRIPTION FACTORS AS TARGETS FOR OXIDATIVE SIGNALING DURING LYMPHOCYTE-ACTIVATION

We previously have demonstrated a requirement for oxidative events during cell cycle entry in T lymphocytes and have hypothesised that reactive oxygen species may act as intracellular signalling agents during lymphocyte activation. In the current study, cysteamine, an aminothiol compound with antioxidant activity, has been used to further investigate the role of oxidative signalling during lymphocyte activation. Treatment of normal human peripheral blood lymphocytes with cysteamine in vitro was found to inhibit proliferation in a dose-dependent manner, with essentially complete inhibition occurring at a dose of 400 mu M. This inhibitory effect was limited to the first 2 h after mitogenic activation, localizing the time-frame of action of cysteamine to within the commitment period. It therefore was of interest to establish which, if any, commitment events were affected by oxidative signalling during cell cycle entry. Taking the IL-2 gene as a candidate, we examined the effect of cysteamine treatment on early gene expression during lymphocyte activation, and on the activity of transcription factors AP-1, NF-kappa B, NF-AT and Oct1, whose functions are required for expression of the IL-2 mRNA. Cysteamine treatment inhibited both expression of the IL-2 mRNA and secretion of IL-2 into the culture medium. The inhibitory effect of cysteamine may be mediated at least in part by an effect on transcription factor function, as the DNA binding activities of AP-1 and NF-kappa B extracted from mitogen-stimulated cells were significantly inhibited by cysteamine treatment. Interestingly, Oct1 and NF-AT DNA binding activity were not affected by cysteamine treatment, suggesting that oxidative signalling processes operate in a selective manner. The identification of regulatory proteins, such as transcription factors, as molecular targets for oxidative signalling provides further evidence to implicate oxidative signalling as being intimately involved in the G(0) to G(1) phase transition in T lymphocytes.