NADPH oxidase-dependent oxidation and externalization of phosphatidylserine during apoptosis in Me2SO-differentiated HL-60 cells -: Role in phagocytic clearance
NADPH oxidase-dependent oxidation and externalization of phosphatidylserine during apoptosis in Me2SO-differentiated HL-60 cells -: Role in phagocytic clearance
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DOI：
10.1074/jbc.m204513200
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发表时间：
2002-12-20
影响因子：
4.8
通讯作者：
Kagan, VE
中科院分区：
文献类型：
--
作者：
Arroyo, A;Modriansky, M;Kagan, VE
炎症的消退需要吞噬细胞以保护邻近组织免受损伤的方式清除活化的中性粒细胞。调控细胞凋亡和清除炎症区域活化中性粒细胞的机制仍然知之甚少。我们使用二甲基亚砜分化的HL-60细胞显示诱导型氧化酶活性，研究NADPH氧化酶诱导的中性粒细胞典型的凋亡途径。佛波醇肉豆蔻酸酯乙酸酯激活NADPH氧化酶引起氧化应激，如超氧化物和过氧化氢的产生，细胞内谷胱甘肽的耗尽，以及所有三种主要类型的膜磷脂，磷脂酰胆碱，磷脂酰乙醇胺和磷脂酰丝氨酸的过氧化反应所示。此外，佛波酯肉豆蔻酸酯刺激NADPH氧化酶引起细胞凋亡，证明了凋亡特异性磷脂酰丝氨酸外化，增加半胱天冬酶-3活性，染色质凝聚，和核碎裂。此外，佛波醇肉豆蔻酸酯乙酸酯-NADPH氧化酶的刺激引起J774 AA巨噬细胞对二甲基亚砜分化的HL-60细胞的识别和摄取。为了揭示肉豆蔻酸佛波酯诱导的反应中氧化应激的凋亡相关成分，我们用pancaspase抑制剂苄氧羰基-Val-Ala-Asp-氟甲基酮预处理细胞，（z-VAD-fetamine），并发现其引起过氧化氢形成的部分抑制以及仅磷脂酰丝氨酸的选择性保护，而更丰富的磷脂、磷脂酰胆碱和磷脂酰乙醇胺，在不存在或存在z-VAD-fetoxy的情况下氧化至相同程度。相比之下，NADPH氧化酶活性的抑制剂，二苯碘鎓和星形孢菌素，以及抗氧化酶，超氧化物歧化酶/过氧化氢酶，完全保护所有的磷脂对过氧化，抑制细胞凋亡的生物标志物的表达和磷脂酰丝氨酸的外化，并减少分化的HL-60细胞的吞噬J774A.1巨噬细胞。类似地，酵母聚糖诱导的NADPH氧化酶的活化导致超氧化物的产生和不同类别的磷脂的氧化，其中只有磷脂酰丝氨酸被z-VAD-fatomy保护。因此，酵母聚糖引起分化的HL-60细胞凋亡，证明了半胱天冬酶-3激活和磷脂酰丝氨酸外化。最后，酵母多糖触发caspase-3激活和广泛的SOD/过氧化氢酶可降解的磷脂酰丝氨酸暴露在人类中性粒细胞。总体而言，我们的研究结果表明，NADPH氧化酶诱导的氧化应激在嗜酸性粒细胞样细胞触发凋亡和随后的识别和清除这些细胞通过依赖于氧化和磷脂酰丝氨酸的外化途径。
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 J774AA 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-fluoromethyI 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.