Heterogeneity in Superoxide Production as Measured by Nitro Blue Tetrazolium Reduction from Individual PAM

"PULMONARY ALVEOLAR macrophages (PAM) protect the lungs by providing a first line of defense against inhaled foreign debris and bacteria. In this role PAM phagocytize foreign matter, secrete enzymes that augment microbial killing and digestion, and produce reactive oxygen me-tabolites (2, 3, 18, 19, 22, 26). However, in addition to this antibacterial function, PAM participate in a diversity of functions, such as tissue repair, lipoprotein metabolism, and immunoregulatory activities (11, 22).Studies have suggested the existence of a heterogeneous population of PAM with respect to immunologic function, enzyme release, phagocytosis, morphological characteristics, expression of membrane receptors, ad-herence, and superoxide (O2•) release (4-7, 10, 16, 21, 23, 28). This heterogeneity has been observed by analyz¬ing subfractions of PAM separated by cell density frac-tionation. For example, using guinea pig PAM, Dauber et al. (7) showed heterogeneity in cell density and size, morphological characteristics, and cytochemical properties, whereas Holian and co-workers (16) reported variations in migration, chemotaxis, O2• release, and pinocytosis among subfractions collected. Similarly, O'Neill et al. (21) observed functional heterogeneity in rat PAM with regard to phagocytosis, O2 • release, and lysosomal enzyme activity.Several investigations have reported a correlation between PAM activity and cell volume. Most studies have shown that O2 release from PAM increased with decreasing cell size (15, 21, 25), although Bielefeldt Ohmann and Babiuk (4) reported a decreasing trend. However, Gallin (13) has suggested that significant variability may even exist within density-defined or cell-volume subfractions that could lead to misinterpretation of results concerning PAM heterogeneity.A better understanding of the heterogeneity in O2 production may be obtained by using single cell meas-urements to evaluate whether functionally distinct sub-populations actually do exist. As such, the present study was designed to examine heterogeneity in O2 • release from single PAM using a recently developed electrooptical technique (8). The initial oxygen metabolite, O2•, produced by NADPH oxidase at the surface of the plasma membrane, was measured by quantifying the reduction of nitro blue tetrazolium (NBT I to a diformazan precipitate. Measurements were made from video recordings of individual cells stimulated by their adherence to the bottom of a culture dish. The cell volume and degree of spreading also were determined directly from the same video recordings, and the data were correlated with the initial rate and maximum amount of diformazan produced by the same cell."