Superoxide enhances photobleaching during cellular immune attack against fluorescent lipid monolayer membranes.

Lipid hapten-containing monolayer membranes with bound, anti-hapten antibody molecules serve as model immunological target membranes. Targets with bound-IgG trigger guinea pig macrophages to (a) adhere, (b) spread, (c) release lysosomal enzymes, and (d) increase cyanide-insensitive oxygen consumption. When the target membranes are derivatized with fluorescein, there is a 2-3-fold enhancement in the rate of fluorescein photobleaching in regions of cell-monolayer contact. This effect is due to release of O2- from macrophages, as shown by inhibition with superoxide dismutase and by the fact that enhanced photobleaching is not observed with cells of the RAW264 macrophage line, which undergo responses (a)-(d), but do not release O2- extracellularly. The O2- dependent photobleaching reaction appears to be relatively specific for fluorescein, as it did not occur with two other fluorophores, 4-nitrobenz-2-oxa-1,3-diazole and tetramethyl-rhodamine. Because stimulated neutrophils release large quantities of O2-, the photobleaching of fluorescein-labeled target membranes in response to neutrophils was examined. Monolayer membranes with specifically bound IgG caused neutrophils to adhere and become markedly motile during incubation at 37 degrees C. Like macrophages, neutrophils induced O2- -dependent photobleaching of fluorescein-labeled IgG in regions of cell-monolayer contact. In addition, neutrophils gave rise to a slower, nonphotochemical loss of fluorescence in the same contact regions. The latter effect is apparently due to cleavage of target-bound fluorescent IgG by proteolytic enzymes secreted by the neutrophils in response to the target surface.