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

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 IgG-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.

Apparent role of traveling metabolic waves in oxidant release by living neutrophils

“…Previous findings suggested that ROMs are not released uniformly about the perimeter of neutrophils (22). Moreover, neutrophils with multiple attached targets have been shown to expose each target cell to ROMs sequentially, not simultaneously (32). Thus, ROMs may be released vectorially from neutrophils, but a mechanism to account for this has remained elusive.…”

Superoxide‐mediated lysis of erythrocytes: The role of colloid‐osmotic forces

“…As time passes the erythrocytes disappear at this wavelength indicating cytosolic oxidation (data not shown; see Petty et al, 1992 for further kinetic Soret imaging studies). Since leakage of hemoglobin was not observed, the disappearance a t 430 nm is due to hemoglobin oxidation (Francis et al, 1988a). This result suggests that oxidant-induced erythrocyte shape change precedes cytosolic oxidation and cytolysis.…”

Imaging neutrophil activation: Analysis of the translocation and utilization of NAD(P)H‐associated autofluorescence during antibody‐dependent target oxidation

“…Previous studies in this laboratory (Francis et al, 1988;Petty et al, 1989) have shown that the entry of oxidative molecules into target erythrocytes can be conveniently monitored by imaging the Soret band at its 430 nm edge using transmitted light microscopy. We have combined these two methods to spatially and temporally resolve the metabolic changes in neutrophils that accompany the oxidative destruction of targets.…”