Early degranulation of human neutrophils: immunocytochemical studies of surface and intracellular phagocytic events.

Degranulation of azurophil and specific granules after phagocytic challenge with E. coli for 5 sec to 10 min was investigated in the human polymorphonuclear neutrophil (PMN). PMN were stained simultaneously with fluorescein and rhodamine-labeled monospecific antisera to myeloperoxidase (MPO) and lactoferrin (LF) to identify azurophil and specific granules, respectively, within single cells. Fixation was designed to preserve or disrupt differential permeability of cell membrane to fluorescent conjugates in order to study granule translocation. Within 5 sec after phagocytic challenge, MPO and LF appeared on the cell surface coating the bacteria as granule contents leaked from the incompletely formed phagolysosomes. The phagocytic cup, shown by scanning electron microscopy as large and circular, appeared by immunofluorescent markers to be outlined by curvilinear staining for both granule markers, and was always coincident with bacterial localization. MPO and LF appeared singly or simultaneously on the cell surface, suggesting that degranulation to the surface was random. Sequential phagocytic events were demonstrated by comparing staining intensities for each granule marker on the surface and intracellularly within single cells. LF sometimes appeared on the cell surface independent of the nascent phagosome, suggesting that perturbation of the cell membrane by bacteria may cause some specific granule extrusion not limited to the phagosome. These results imply that bacteria make contact with granule-associated anti-microbial substances within 5 sec after phagocytosis is initiated and that free communication of granule constituents occurs between the newly forming phagolysosome and the extracellular space.