Endogenous oxygen radicals modulate protein tyrosine phosphorylation and JNK-1 activation in lectin-stimulated thymocytes

Molecular events mediating the T-lymphocyte response to lectins are still i ncompletely understood, although much evidence suggests that both the mitog enic and the death-promoting effects of these agents involve the biochemica l cascade initiated by the CD3/T-cell antigen receptor (TCR) complex. React ive oxygen species (ROS) and in particular H2O2 have been shown to have a r ole in cell response to cytokines and growth factors. Here we report that t he proliferation of mouse thymocytes in response to the mitogenic lectin co ncanavalin A (ConA) is strongly and selectively inhibited by the intracellu lar ROS scavenger N-acetylcysteine (NAC) and by diphenyleneiodonium (DPI), a potent inhibitor of NADPH-dependent membrane oxidases activated by surfac e receptors. A rapid 'burst' of intracellular oxygen radicals was observed in mouse thymocytes stimulated by ConA, with kinetics that paralleled the a ppearance of tyrosine-phosphorylated proteins. This burst was abrogated by the pretreatment of cells with NAC or DPI. Only a modest increase in intrac ellular oxygen species was found in thymocytes stimulated by strong cross-l inking of TCR together with CD4 or CD28. Pharmacological interference with ROS production in ConA-stimulated thymocytes resulted in a decreased tyrosi ne phosphorylation of multiple protein species, including a 38 kDa band abl e to recruit the adapter protein Grb2 and corresponding to the recently ide ntified transducer LAT (linker for activation of T-cells), a molecule invol ved in linking activated TCR to the production of interleukin 2 and the pro liferation of T-cells. Furthermore, ROS inhibition markedly attenuated the activation of stress-activated protein kinase/JNK-1 (c-Jun N-terminal kinas e 1)in response to lectins. Taken together, these results identify ROS as i mportant modulators of the signalling cascade initiated by mitogenic lectin s in thymocytes and, by extension, as a novel class of mediators downstream of antigen receptors.