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 incompletely understood, although much evidence suggests that both the mitogenic and the death-promoting effects of these agents involve the biochemical cascade initiated by the CD3/T-cell antigen receptor (TCR) complex. Reactive oxygen species (ROS) and in particular H(2)O(2) have been shown to have a role in cell response to cytokines and growth factors. Here we report that the proliferation of mouse thymocytes in response to the mitogenic lectin concanavalin A (ConA) is strongly and selectively inhibited by the intracellular ROS scavenger N-acetylcysteine (NAC) and by diphenyleneiodonium (DPI), a potent inhibitor of NADPH-dependent membrane oxidases activated by surface receptors. A rapid 'burst' of intracellular oxygen radicals was observed in mouse thymocytes stimulated by ConA, with kinetics that paralleled the appearance of tyrosine-phosphorylated proteins. This burst was abrogated by the pretreatment of cells with NAC or DPI. Only a modest increase in intracellular oxygen species was found in thymocytes stimulated by strong cross-linking of TCR together with CD4 or CD28. Pharmacological interference with ROS production in ConA-stimulated thymocytes resulted in a decreased tyrosine phosphorylation of multiple protein species, including a 38 kDa band able to recruit the adapter protein Grb2 and corresponding to the recently identified transducer LAT (linker for activation of T-cells), a molecule involved in linking activated TCR to the production of interleukin 2 and the proliferation of T-cells. Furthermore, ROS inhibition markedly attenuated the activation of stress-activated protein kinase/JNK-1 (c-Jun N-terminal kinase 1) in response to lectins. Taken together, these results identify ROS as important modulators of the signalling cascade initiated by mitogenic lectins in thymocytes and, by extension, as a novel class of mediators downstream of antigen receptors.
Pani
G
G
Institute of General Pathology, Catholic University Medical School, Largo F. Vito 1, 00168 Rome, Italy. ibipg@rm.unicatt.it
Colavitti
R
R
Borrello
S
S
Galeotti
T
T
D000111:Acetylcysteine / Q000494:pharmacology*; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D015704:CD4 Antigens / Q000502:physiology; D016827:CD8 Antigens / Q000502:physiology; D002478:Cells, Cultured; D003208:Concanavalin A / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D008213:Lymphocyte Activation; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D048055:Mitogen-Activated Protein Kinase 8; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009861:Onium Compounds / Q000494:pharmacology; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000502:physiology*; D017260:Receptor-CD3 Complex, Antigen, T-Cell / Q000502:physiology; D013601:T-Lymphocytes / Q000187:drug effects / Q000201:enzymology* / Q000276:immunology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000975:Antioxidants; D015704:CD4 Antigens; D016827:CD8 Antigens; D004791:Enzyme Inhibitors; D009861:Onium Compounds; D017382:Reactive Oxygen Species; D017260:Receptor-CD3 Complex, Antigen, T-Cell; D003208:Concanavalin A; D019000:Phosphotyrosine; C007517:diphenyleneiodonium; D048055:Mitogen-Activated Protein Kinase 8; D020928:Mitogen-Activated Protein Kinases; D013755:Tetradecanoylphorbol Acetate; D000111:Acetylcysteine