Szejda P, Parce J W, Seeds M S, Bass D A
J Immunol. 1984 Dec;133(6):3303-7.
Stimulation of the oxidative metabolic burst of human polymorphonuclear leukocytes (PMNL) may occur by an all-or-none trigger mechanism or by a graded response to increasing stimulation of an individual cell. If the proposed all-or-none mechanism occurred during phagocytosis, a PMNL would expend all of its metabolic potential at once, yet PMNL can proceed to ingest multiple organisms. This study employed dual laser flow cytometry to correlate the number of cell-associated organisms with oxidative product formation in individual PMNL. Intracellular oxidation of nonfluorescent 2',7'-dichlorofluorescein (DCFH) to highly fluorescent 2',7'-dichlorofluorescein (DCF) provided a quantitative assay of H2O2-dependent oxidative product formation generated by the cell's oxidative metabolic burst. Staphylococcus aureus were fixed and stained with Texas red to allow simultaneous monitoring of bacteria (red fluorescence, greater than 580 nm) and DCF (green fluorescence, 510 to 550 nm) content of each cell. Computer correlation of bacterial and DCF fluorescence allowed determination of the DCF formation by PMNL containing specific numbers (0 to 15) of bacteria. Oxidative product formation was directly related to the number of bacteria ingested in a time-dependent manner (mean per cell of 6.4, 12.8, 19.1, and 24.4 attomoles (amol) DCF formed per cell per bacterium after 15, 30, 45, and 60 min, respectively. Opsonization of bacteria with fresh normal serum (primarily C3b opsonization) or with specific IgG demonstrated qualitatively similar responses, except that the response per IgG-opsonized organism was, on the average, more than twice the response to bacteria opsonized with serum. Thus, sequential phagocytosis of multiple bacteria elicits an incremental oxidative response of human PMNL.
人类多形核白细胞（PMNL）氧化代谢爆发的刺激可能通过全或无触发机制发生，也可能通过对单个细胞刺激增加的分级反应发生。如果所提出的全或无机制在吞噬作用期间发生，PMNL会立即耗尽其所有代谢潜能，但PMNL可以继续吞噬多个生物体。本研究采用双激光流式细胞术将单个PMNL中与细胞相关的生物体数量与氧化产物形成相关联。非荧光的2',7'-二氯荧光素（DCFH）在细胞内氧化为高荧光的2',7'-二氯荧光素（DCF），为细胞氧化代谢爆发产生的依赖H2O2的氧化产物形成提供了定量测定方法。金黄色葡萄球菌经固定并用德克萨斯红染色，以便同时监测每个细胞中的细菌（红色荧光，大于580nm）和DCF（绿色荧光，510至550nm）含量。细菌和DCF荧光的计算机相关性允许确定含有特定数量（0至15个）细菌的PMNL的DCF形成。氧化产物的形成与摄入细菌的数量呈时间依赖性直接相关（分别在15、30、45和60分钟后，每个细胞每吞噬一个细菌形成的DCF平均为6.4、12.8、19.1和24.4阿托摩尔（amol））。用新鲜正常血清（主要是C3b调理）或特异性IgG对细菌进行调理显示出定性相似的反应，只是平均而言，每个IgG调理的生物体的反应是血清调理细菌反应的两倍多。因此，人类PMNL对多个细菌的顺序吞噬引发递增的氧化反应。
