Flow cytometric studies of oxidative product formation by neutrophils: a graded response to membrane stimulation.
Flow cytometric studies of oxidative product formation by neutrophils: a graded response to membrane stimulation.
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DOI：
10.4049/jimmunol.130.4.1910
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发表时间：
1983-04
影响因子：
4.4
通讯作者：
D. Bass;J. W. Parce;L. Dechatelet;P. Szejda;M. Seeds;M. Thomas
中科院分区：
文献类型：
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作者：
D. Bass;J. W. Parce;L. Dechatelet;P. Szejda;M. Seeds;M. Thomas
我们开发了一种定量检测方法，利用流式细胞术的单细胞分析来监测多形核白细胞（PMNL）的氧化爆发（H2O2产生），并研究了PMNL是否对膜刺激有氧化爆发或无氧化爆发的反应。在与正常中性粒细胞孵育期间，双乙酸二氯荧光素扩散到细胞中，被水解成2',7'-二氯荧光素（DCFH），从而被捕获在细胞内。细胞内的DCFH是一种非荧光荧光素类似物，在PMNL的刺激下被氧化成高荧光的2',7'-二氯荧光素（DCF）。pma刺激PMNL的激发/发射光谱和质谱分析表明氧化产物为DCF。正常静息和pma刺激的PMNL在15分钟内每个细胞分别氧化6.9 +/- 0.7原子和160 +/- 13原子的DCF。100 ng/ml PMA刺激PMNL形成DCF时，缺乏钙、镁离子和/或添加2 mM EDTA均未抑制DCF的形成。由于EDTA阻止PMNL聚集（即使在100 ng/ml PMA刺激下），这将妨碍准确的流式细胞分析，因此在培养基中使用EDTA进行进一步的实验。使用PMNL有不同严重程度氧化代谢缺陷的患者的细胞，证明了DCFH平均氧化与单磷酸己糖分流刺激之间的密切相关。胞内DCFH也可被H2O2试剂或葡萄糖氧化酶+葡萄糖或黄嘌呤氧化酶+乙醛生成的氧衍生物氧化；过氧化氢酶对DCFH的氧化有抑制作用，但超氧化物歧化酶对DCFH的氧化没有影响。数据表明，DCFH氧化实验与PMNL氧化代谢爆发有定量关联，强烈提示该反应是由PMNL产生的H2O2介导的。PMNL与不同浓度的PMA孵育引起所有PMNL的分级反应；也就是说，1 ng/ml PMA在单个PMNL群体中引起34%的最大平均反应（而不是像全有或无假设预测的那样，66%的静息和34%的完全刺激）。因此，在这些实验条件下，PMNL的氧化产物形成是对PMA刺激膜的分级反应。
We have developed a quantitative assay to monitor the oxidative burst (H2O2 production) of polymorphonuclear leukocytes (PMNL) using single cell analysis by flow cytometry, and have examined whether PMNL respond to membrane stimulation with an all-or-none oxidative burst. During incubation with normal neutrophils, dichlorofluorescin diacetate diffused into the cells, was hydrolyzed to 2',7'-dichlorofluorescin (DCFH) and was thereby trapped within the cells. The intracellular DCFH, a nonfluorescent fluorescein analogue, was oxidized to highly fluorescent 2',7'-dichlorofluorescein (DCF) by PMNL stimulated by phorbol myristate acetate (PMA). That the oxidative product was DCF was shown by excitation/emission spectra and by mass spectrometry of the product from PMA-stimulated PMNL. Normal resting and PMA-stimulated PMNL oxidized 6.9 +/- 0.7 and 160 +/- 13 attomoles DCF per cell, respectively, in 15 min. Absence of calcium and magnesium ions and/or addition of 2 mM EDTA did not inhibit DCF formation by PMNL stimulated by 100 ng/ml PMA. Since EDTA prevented aggregation of PMNL (even when stimulated by 100 ng/ml PMA), which would prevent accurate flow cytometric analysis, further experiments were performed with EDTA in the medium. A close correlation between average DCFH oxidation and hexose monophosphate shunt stimulation was demonstrated using cells from patients whose PMNL had oxidative metabolic defects of varying severity. Intracellular DCFH was also oxidized by reagent H2O2 or oxygen derivatives generated by glucose oxidase + glucose or by xanthine oxidase + acetaldehyde; DCFH oxidation by these systems was inhibited by catalase but unchanged by superoxide dismutase. The data indicate that the DCFH oxidation assay is quantitatively related to the oxidative metabolic burst of PMNL, and they strongly suggest that the reaction is mediated by H2O2 generated by the PMNL. Incubation of PMNL with varying concentrations of PMA caused graded responses by all PMNL present; i.e., 1 ng/ml PMA caused a mean response of 34% maximal with a single population of responding PMNL (rather than 66% resting and 34% fully stimulated as predicted by the all-or-none hypothesis). Thus, with these assay conditions, oxidative product formation by PMNL occurs as a graded response to membrane stimulation by PMA.