Ultracytochemical study on the localization of superoxide producing sites in stimulated rat neutrophils

Superoxide anion production in neutrophils plays an important role in the m icrobicidal defense system in the body. In this study, isolated rat neutrop hils were stimulated experimentally and examined by electron microscopy to determine the site of superoxide production and its subsequent translocatio n during different cell stimulation time periods. Blood and peritoneal neut rophils were incubated for periods of 5, 10, and 15 min with phorbol 12-myr istate 13-acetate (PMA), N-formyl-Met-Leu-Phe (fMLP), and combinations of P MA and cytochalasin B (CB) and fMLP and CB. Ultracytochemical detection of O-2(-) was performed with the 3,3'-diaminobenzidine-manganese (DAB/Mn) cyto chemical method and cationized ferritin (CF) particles were added to stimul ation media to monitor endocytotic events that occurred during neutrophil s timulation. Unstimulated neutrophils were devoid of O-2(-) activity in cyto plasmic granules and at the plasma membrane surface. After 5 min stimulatio ns with PMA, PMA + CB, or fMLP + CB, electron-dense DAB/Mn reaction product was detected in small, centrally located tubular compartments within the n eutrophils. CF particles which were added to the stimulation media became i nternalized in endocytotic vesicles after 5 min stimulation; these vesicles were devoid of O-2(-) activity,At 10 min stimulation with PMA, O-2(-)-posi tive granules subsequently fused with:each other and translocated to sub-pl asma membrane regions where they either contacted the plasma membrane or fu sed with CF-containing endocytotic vesicles. Little reaction product was ob served on the surface of the neutrophils. Spectrophotometric comparison of the stimulatory effects of PMA, fMLP, and fMLP + CB revealed different rate s and yields of O-2(-) production. Results from this study suggest that the O-2(-)-producing sites of rat neutrophils originate intracellularly and tr anslocate to the plasma membrane surface following stimulation with PMA, PM A + CB, and fMLP + CB, but not with fMLP or CB alone. Furthermore?, these c ompartments appear to possess the ability to fuse with endocytotic vesicles , a process that may be linked to intracellular microbicidal activity in ci rculating and tissue neutrophils. Anat Rec 258:156-165, 2000. (C) 2000 Wile y-Liss, Inc.