title: NADPH oxidase-dependent oxidation and externalization of phosphatidylserine during apoptosis in Me2SO-differentiated HL-60 cells. Role in phagocytic clearance.
abstract: Resolution of inflammation requires clearance of activated neutrophils by phagocytes in a manner that protects adjacent tissues from injury. Mechanisms governing apoptosis and clearance of activated neutrophils from inflamed areas are still poorly understood. We used dimethylsulfoxide-differentiated HL-60 cells showing inducible oxidase activity to study NADPH oxidase-induced apoptosis pathways typical of neutrophils. Activation of the NADPH oxidase by phorbol myristate acetate caused oxidative stress as shown by production of superoxide and hydrogen peroxide, depletion of intracellular glutathione, and peroxidation of all three major classes of membrane phospholipids, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. In addition, phorbol myristate acetate stimulation of the NADPH oxidase caused apoptosis, as evidenced by apoptosis-specific phosphatidylserine externalization, increased caspase-3 activity, chromatin condensation, and nuclear fragmentation. Furthermore, phorbol myristate acetate stimulation of the NADPH oxidase caused recognition and ingestion of dimethylsulfoxide-differentiated HL-60 cells by J774A.1 macrophages. To reveal the apoptosis-related component of oxidative stress in the phorbol myristate acetate-induced response, we pretreated cells with a pancaspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk), and found that it caused partial inhibition of hydrogen peroxide formation as well as selective protection of only phosphatidylserine, whereas more abundant phospholipids, phosphatidylcholine and phosphatidylethanolamine, were oxidized to the same extent in the absence or presence of z-VAD-fmk. In contrast, inhibitors of NADPH oxidase activity, diphenylene iodonium and staurosporine, as well as antioxidant enzymes, superoxide dismutase/catalase, completely protected all phospholipids against peroxidation, inhibited expression of apoptotic biomarkers and externalization of phosphatidylserine, and reduced phagocytosis of differentiated HL-60 cells by J774A.1 macrophages. Similarly, zymosan-induced activation of the NADPH oxidase resulted in the production of superoxide and oxidation of different classes of phospholipids of which only phosphatidylserine was protected by z-VAD-fmk. Accordingly, zymosan caused apoptosis in differentiated HL-60 cells, as evidenced by caspase-3 activation and phosphatidylserine externalization. Finally, zymosan triggered caspase-3 activation and extensive SOD/catalase-inhibitable phosphatidylserine exposure in human neutrophils. Overall, our results indicate that NADPH oxidase-induced oxidative stress in neutrophil-like cells triggers apoptosis and subsequent recognition and removal of these cells through pathways dependent on oxidation and externalization of phosphatidylserine.
authors.affiliation: Department of Environmental and Occupational Health, Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, PA 15260, USA.
authors.forename: Antonio
authors.initials: A
authors.lastname: Arroyo
authors.forename: Martin
authors.initials: M
authors.lastname: Modrianský
authors.forename: F Behice
authors.initials: FB
authors.lastname: Serinkan
authors.forename: Rosario I
authors.initials: RI
authors.lastname: Bello
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Matsura
authors.forename: Jianfei
authors.initials: J
authors.lastname: Jiang
authors.forename: Vladimir A
authors.initials: VA
authors.lastname: Tyurin
authors.forename: Yulia Y
authors.initials: YY
authors.lastname: Tyurina
authors.forename: Bengt
authors.initials: B
authors.lastname: Fadeel
authors.forename: Valerian E
authors.initials: VE
authors.lastname: Kagan
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002454:Cell Differentiation; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D002843:Chromatin / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D053938:DNA Fragmentation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D005978:Glutathione / Q000378:metabolism; D018922:HL-60 Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D015227:Lipid Peroxidation; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D019255:NADPH Oxidases / Q000378:metabolism*; D009504:Neutrophils / Q000378:metabolism; D018384:Oxidative Stress; D010100:Oxygen / Q000378:metabolism*; D010586:Phagocytes / Q000378:metabolism*; D010587:Phagocytosis; D010718:Phosphatidylserines / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism; D013463:Sulfuric Acid Esters / Q000494:pharmacology*; D013997:Time Factors; D015054:Zymosan / Q000494:pharmacology
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D002843:Chromatin; D004791:Enzyme Inhibitors; D010718:Phosphatidylserines; D010743:Phospholipids; D013463:Sulfuric Acid Esters; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D015054:Zymosan; D006861:Hydrogen Peroxide; D019255:NADPH Oxidases; C505407:CASP3 protein, human; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases; D005978:Glutathione; C033522:methyl sulfate; D010100:Oxygen