Flow cytometric quantitation of oxidative product formation by polymorphonuclear leukocytes during phagocytosis

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.