Optical microscopy of antibody-dependent phagocytosis and lysis of erythrocytes by living normal and chronic granulomatous disease neutrophils: A role of superoxide anions in extra- and intra-cellular lysis

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Abstract
Novel optical microscopic techniques have been developed to observe neutrophil-mediated effector functions at the level of individual cells. Conventional absorption spectrophotometry has shown that exposure of hemoglobin to superoxide anions decreases the intensity of the Soret band and shifts it to lower wavelengths. This oxidative event can be visualized within intact erythrocytes using bright-field microscopy in conjunction with violet illumination at 430 nm. The sequential oxidation of lgG-opsonized sheep erythrocytes bound to normal human neutrophils can be observed. Chronic granulomatous disease (CGD) neutrophils which do not generate superoxide anions were not capable of influencing target absorption at 430 nm. Cytolytic events were visualized by fluorescence microscopy. Cytosolic or membrane compartments of sheep erythrocytes were labeled with eosin Y or fluorescein isothiocyanate, respectively. Time-dependent studies of erythrolysis show that targets are lysed extra- and intra-cellularly. The fluorescent diffusion gradient generated at the site of membrane rupture suggests that a pore of approximately 30 nm in diameter is formed in the target membrane. The site of pore formation is not found at the target-effector cell interface. CGD neutrophils did not display these cytolytic phenomena. Furthermore, the cytosolic label eosin Y could be followed into an associated granule compartment; we suggest that the phenomenon of piranhalysis may participate in antibody-dependent effector mechanisms. Phagocytosis can also be observed using fluorescently-labeled erythrocytes. Determinations of phagocytic index are more reliable with this approach. These microscopical methods are both simple and efficient. To our knowledge, these are the first direct microscopic studies of effector cell-mediated target cell oxidation and cytolysis. These experiments provide a fresh approach to the study of phagocyte effector functions at the cellular level and illuminate the importance of superoxide anions in antibody-dependent erythrolysis.

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