Thiol-mediated redox regulation of neutrophil apoptosis

Background. Intracellular glutathione, an endogenous antioxidant, protects cellular function against oxidative stress. Because oxidative stress has been implicated in neutrophil apoptosis, we hypothesized that reduced thiol levels may induce apoptosis through an alteration in cellular redox state. Methods. Human polymorphonuclear leukocytes (PMNs), were incubated with medium or with increasing concentrations of the reduced glutathione (GSH)-depleting agents diethylmaleate and diamide and buthionine sulfoximine, an inhibitor of GSH synthesis. Apoptosis was assessed by means of flow cytometry with propidium iodide DNA staining and confirmed morphologically. GSH was measured colorimetrically, and tyrosine phosphorylation was assessed by means of immunoblotting. Results. Diethylmaleate and diamide induced a dose-dependent reduction in GSH and a corresponding increase in PMN apoptosis. This effect could be reversed with N-acetylcysteine, suggesting that diethylmaleate induces apoptosis through the depletion of GSH. The antioxidant pyrolidine dithiocarbamate had no effect. Because oxidants can mediate intracellular signaling via tyrosine phosphorylation, we therefore evaluated the effects of the tyrosine kinase inhibition on diethylmaleate-induced PMN apoptosis. Both genistein and herbimycin A reduced diethylmaleate-induced apoptosis and tyrosine phosphorylation. Conclusions. Sulfhydryl oxidation by diethylmaleate alone induces apoptosis, providing evidence of a redox-sensitive, thiol-mediated pathway of apoptosis. Furthermore, tyrosine phosphorylation appears to play an important role in this process. Because apoptosis is a critical mechanism regulating PMN survival in vivo, manipulation of PMN intracellular thiols may represent a novel therapeutic target for the regulation of cellular function.