G. Pani et al., Endogenous oxygen radicals modulate protein tyrosine phosphorylation and JNK-1 activation in lectin-stimulated thymocytes, BIOCHEM J, 347, 2000, pp. 173-181
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.