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 represents a novel therapeutic target for the regulation of cellular function.
Watson
R W
RW
Department of Surgery, Toronto Hospital, University of Toronto, Canada.
Rotstein
O D
OD
Nathens
A B
AB
Dackiw
A P
AP
Marshall
J C
JC
D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D019328:Buthionine Sulfoximine; D003958:Diamide / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000378:metabolism; D005434:Flow Cytometry; D005978:Glutathione / Q000494:pharmacology; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008298:Maleates / Q000494:pharmacology; D008717:Methionine Sulfoximine / Q000031:analogs & derivatives / Q000378:metabolism; D009504:Neutrophils / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D010084:Oxidation-Reduction; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D013438:Sulfhydryl Compounds / Q000502:physiology*; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D000975:Antioxidants; D004791:Enzyme Inhibitors; D008298:Maleates; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D003958:Diamide; D008717:Methionine Sulfoximine; D019328:Buthionine Sulfoximine; D005982:Glutathione Transferase; D011505:Protein-Tyrosine Kinases; C014476:diethyl maleate; D005978:Glutathione