Nitric Oxide-Mediated Augmentation of Neutrophil Reactive Oxygen and Nitrogen Species Formation: Critical Use of Probes
Nitric Oxide-Mediated Augmentation of Neutrophil Reactive Oxygen and Nitrogen Species Formation: Critical Use of Probes
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DOI：
10.1002/cyto.a.20975
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发表时间：
2010-11-01
期刊：
影响因子：
3.7
通讯作者：
Dikshit, Madhu
中科院分区：
文献类型：
--
作者：
Kumar, Sachin;Patel, Satyananda;Dikshit, Madhu
本实验室和其他实验室以前的报告表明NO介导的NADPH氧化酶的双相调节和中性粒细胞活性氧产生的衰减，而最近我们报道了在DCF荧光增强NO治疗后。这些差异似乎是由于利用不同的探针/方法来评估NO对活性氧和氮物质（ROS/RNS，活性物质）产生的影响。本研究旨在通过使用多种探针、人中性粒细胞/HL 60细胞和各种干预措施来研究这一点并评估NO介导的酶反应性物质的形成。向PMNs悬浮液中添加NO供体、SNP或SNAP（100 nM-1 mM）表现出DCF荧光的浓度和时间依赖性增强，但降低DHE荧光。通过增强DMPO-硝酮加合物、二酪氨酸和罗丹明-123以及东莨菪内酯的淬灭证实了活性物种的集体产生。NO也增强了细菌的杀伤作用，而不改变吞噬作用。NO对缺乏功能性NADPH氧化酶的HL-60细胞的作用增强了活性物质的形成，表明其他酶也很重要。NADPH氧化酶抑制剂（DPI）、MPO抑制剂（ABAH）或NOS抑制剂（7-NI）可显著降低NO依赖性ROS/RNS的产生。然而，7-NI降低MPO活性，这意味着NOS在反应性物种形成中的重新评价这些报告。所获得的结果表明NO介导的反应性物种在人类中性粒细胞扩增。此外，超氧化物清除NO似乎是减少DHE荧光的关键过程，并建议DCF作为最合适的探针来测量NO介导的活性氧调节，特别是在各种病理情况下的有用性。(C0)2010年国际细胞计数促进学会
Previous reports from this laboratory and others demonstrated NO-mediated biphasic modulation of NADPH oxidase and attenuation of neutrophil reactive oxygen species generation, whereas recently we reported augmentation in DCF fluorescence following NO treatment. These discrepancies seem to be due to utilization of different probes/methods to assess effect of NO on reactive oxygen and nitrogen species (ROS/RNS, reactive species) generation. This study aims to look into this and evaluate NO-mediated enzymatic reactive species formation by using multiple probes, human neutrophils/HL60 cells and various interventions. Addition of NO donor, SNP or SNAP (100 nM-1 mM) to PMNs suspension, exhibited a concentration- and time-dependent augmentation in DCF fluorescence, but reduced DHE fluorescence. Collective generation of reactive species was confirmed by enhanced DMPO-nitrone adduct, dityrosine and rhodamine-123 and quenching of scopoletin. NO also enhanced bacterial killing, without altering phagocytosis. Addition of NO to HL-60 cells lacking functional NADPH oxidase enhanced reactive species formation, indicating importance of other enzyme(s) too. NO-dependent ROS/RNS generation was substantially reduced by NADPH oxidase inhibitor (DPI), MPO inhibitor (ABAH), or NOS inhibitor (7-NI). However, 7-NI reduced MPO activity, warranting reappraisal of those reports, which implied NOS in reactive species formation. The results obtained demonstrated NO-mediated reactive species augmentation in human PMNs. Furthermore, superoxide scavenging by NO seems to be the key process in the decrease of DHE fluorescence and suggest usefulness of DCF as the most appropriate probe to measure the NO-mediated modulation of reactive oxygen species in particular in various pathological situations. (C0) 2010 International Society for Advancement of Cytometry