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

Abstract

Fluorescence intensified/enhanced microscopy has been used to study the metabolic activation of living human neutrophils in time-lapse sequences. The autofluorescence associated with NAD(P)H's emission band was studied within individual quiescent and stimulated cells. Excitation of NAD(P)H-associated autofluorescence was provided by a high-intensity Hg-vapor lamp. The background-subtracted autofluorescence signals were computer enhanced. In some cases the ratio image of NAD(P)H-associated autofluorescence to tetramethyl-rhodamine methyl ester (TRME) fluorescence, which was found to be uniformly distributed within neutrophils, was calculated to normalize autofluorescence intensities for cell thickness. Activation of the NADPH oxidase by phorbol myristate acetate, F-, N-formyl-methionyl-leucyl-phenylalanine (FMLP), or tumor necrosis factor (TNF) dramatically reduced autofluorescence levels. Membrane solubilization with sodium dodecyl sulfate eliminated autofluorescence. Thus, control experiments indicated that most or all of the detectable NAD(P)H-associated autofluorescence was due to NAD(P)H, consistent with previous non-microscopic studies. To understand the metabolic events surrounding the internalization and oxidative destruction of targets, we have imaged the NAD(P)H-associated autofluorescence of neutrophils and the Soret band of antibody coated target erythrocytes during cell-mediated cytotoxicity. Absorption contrast microscopy of the erythrocyte's Soret band is an especially sensitive indicator of the entry of reactive oxygen metabolites into this target's cytosol. Thus, it is possible to spectroscopically dissect and image the substrate (NADPH) and product (O2-) reactions of the NADPH oxidase in living unlabeled neutrophils. During real-time experiments at 37 degrees C, the level of NAD(P)H-associated autofluorescence surrounding phagosomes greatly increases before the disappearance of the target's Soret band. NAD(P)H-associated autofluorescence in the vicinity of phagocytosed erythrocytes is greatly diminished after target oxidation. This suggests that NAD(P)H is translocated to the vicinity of phagosomes prior to the oxidation of targets. The apparent cytosolic redistribution of NAD(P)H was confirmed by ratio imaging microscopy to control for cell thickness. We suggest that NADPH including its sources and/or carriers accumulate near phagosomes prior to target oxidation and that local NADPH molecules are consumed during target oxidation.

RETRACTED: Dissipative metabolic patterns respond during neutrophil transmembrane signaling

“…This autofluorescence has been linked with neutrophil NAD(P)H, metabolism, function, and disease (7,8,19,(23)(24)(25)(26). Thus, NAD(P)H fluorescence was used as a convenient means of following intracellular metabolism in living cells and metabolic responses to receptor ligation.…”

The NADPH oxidase complex of phagocytic leukocytes: a biochemical and cytochemical view

“…Liang and Petty (1992) have developed a procedure for imaging the autofluorescence signal of NAD(P)H and monitoring the reduction of this autofluorescence following stimulation of the NADPH oxidase system. Microscopic analysis is a powerful procedure for monitoring dinucleotide levels in single cells and has also been applied in other situations, such as myocytes undergoing ischemic insult and skeletal muscle (Esumi et al 1991;Toth et al 1992).…”

Contribution of Neutrophils to Acute Lung Injury

“…Besides the well-known antimicrobicidal function of ROS during phagocytosis, low-level formation of ROS acts as intracellular signaling, so called "redox signaling" (196). ROS is released into the cytosol where they alter the redox state of the cell and modify other cell contents, such as proteins and lipids by oxidation (197). The membrane-bound multicomponent enzyme complex NADPH oxidase, which is dormant in resting cells and can be activated rapidly by chemoattractant peptides or chemokines, generates much ROS after activation.…”