Heterogeneity in superoxide production as measured by nitro blue tetrazolium reduction from individual PAM

Heterogeneity in superoxide (O₂^-·) production (as determined by the reduction of nitro blue tetrazolium to a diformazan precipitate), cell volume, and cell spreading were measured from single rat pulmonary alveolar macrophages (PAM). Of the 330 cells that produced O₂^-· due to stimulation by adherence, the maximum diformazan produced (MAX) varied between 1.8 and 47.2 fmol and the maximum (initial) rate of diformazan production (R) varied between 2.2 and 81.7 x 10^-3 fmol/s. Only six PAM of 336 analyzed failed to produce O₂^-· suggesting that O₂^-· production is not a specific function of a small subpopulation of PAM but rather that all PAM are capable of producing O₂^-·. Importantly, O₂^-· production by single cells showed extensive heterogeneity that did not correlate (r² < 0.12) with individual cell volumes, suggesting that the observed heterogeneity may not be due to differences in maturation and/or differentiation. However, when the single cell data were grouped into cell volume subfractions small but increasing linear trends (r² > 0.81) in MAX and R were found. This discrepancy suggests that PAM heterogeneity in O₂^-· fraction cannot be estimated adequately by measurements on subpopulations of cells.