DEMONSTRATION OF FUNCTIONALLY DISTINCT HUMAN POLYMORPHONUCLEAR LEUKOCYTE FRACTIONS BY SIMULTANEOUS MEASUREMENT OF PHAGOCYTOSIS AND OXYGEN RADICAL GENERATION

Phagocytosis and oxygen radical generation by human polymorphonuclear leukocytes (PMNLs) were studied by a two-color flow cytometric analysi s, where the red fluorescent product(s) of hydroethidine was used as a n indicator of intracellular generation of oxygen radicals and opsoniz ed zymosan (OZ) as an indicator of phagocytosis. Unstimulated cells fo rmed a single population of cells without any significant fluorescence . PMNLs stimulated by OZ exhibited a high red fluorescence. Most PMNLs phagocytosed OZ and generated oxygen radicals when stimulated by zymo san particles opsonized with human AB serum at concentrations of more than 10%, whereas three distinct subpopulations (designated as R1, R2 and R3) appeared when stimulated by zymosan particles opsonized with 3 % serum; R1 cells enhanced neither green nor red fluorescence, R2 cell s enhanced green fluorescence but not red fluorescence, and R3 cells e nhanced both green and red fluorescence. The R2 cells completely disap peared by the addition of Trypan blue. Most of the R3 cells disappeare d by the addition of cytochalasin B. These findings on the three fract ions were confirmed by the observation under fluorescence microscopy o f cells in each fraction obtained by sorting. In conclusion, PMNLs cou ld be separated into three functionally distinct subpopulations when s timulated by zymosan particles opsonized with a suboptimal concentrati on of serum. (C) 1997 Elsevier Science Ireland Ltd.