Measurement of Su peroxide Release from Single Pulmonary Alveolar Macrophages

"K. A. DIGREGORIO, E. v. CILENTO, AND R. c. LANTZ. Measurement of superoxide release from single pulmonary alueolar macrophages. Am. J . Physiol. 252 (Cell Physiol. 21 ): C677-C683, 1987.-An electrooptical method was developed to quantify superoxide (02) release from single rat pulmonary alveolar macrophages (PAM) during adherence to the bottom of a culture dish. This was done by measuring the reduction of nitro blue tetrazolium (NBT) to a diformazan precipitate at 550 nm from videorecorded images of individual cells. Temporal changes in cell optical density, which are proportional to the mass of diformazan produced, were calculated from videophotometric measurements of the change in light intensity over individual cells. Total diformazan produced increased 78 and 1263 with an increase in NBT from 0.5 to 1.0 and 2.0 mg/ml, respectively. Total diformazan produced and maximum rate of production among individual PAM varied two- to threefold providing strong evidence for heterogeneity in 02 production. Specific inhibition of 02 production by superoxide dismutase, iodoacetate, and chlorpromazine significantly reduced the total diformazan produced and maximum rate of diformazan production. Hydrogen peroxide was not involved in NBT reduction, since catalase alone did not significantly change diformazan production. This novel method to quantify 02 release from single PAM should be valuable in analyzing heterogeneity and single cell kinetics of O; production, in assessing the effects of exposure of cells to particulates on O; release, and in relating release to electrophysiological measurements.rat; nitro blue tetrazolium; macrophage heterogeneity; single cell kinetic PULMONARY ALVEOLAR MACROPHAGES (PAM) protect the lungs by phagocytizing foreign debris and bacteria (24). This process involves ingestion as well as destruction of foreign matter. Phagocytosis is usually accompanied by a respiratory burst that increases cellular oxygen consumption and glucose metabolism in the hexose monophosphate shunt (7), which leads to the release of highly reactive oxygen metabolites at the surface of the · plasma membrane (6, 27). These agents have been implicated in the killing of bacteria by phagocytes (22), but have also been shown to be toxic and have been linked to cancer, emphysema, arthritis, diabetes, and other diseases (17, 22, 30). Toxic effects may be a result of either an abnormally low production of metabolites, resulting in damage to lung tissue by respirable bacteria and dusts, or an abnormally high production, resulting in direct damage to lung tissue by metabolites themselves."