Plasma membrane and phagosome localisation of the activated NADPH oxidase in elicited peritoneal macrophages of the guinea‐pig

Abstract

The subcellular distribution of the NADPH oxidase of guinea-pig peritoneal-elicited macrophages was investigated. Post-nuclear supernatants obtained from PMA-stimulated macrophages were fractionated in discontinuous sucrose gradients. The NADPH oxidase was found to be enriched at the interface between 20 and 34 per cent sucrose. This interface was also enriched in 5'-nucleotidase, a plasma membrane marker and in glucose-6-phosphatase and NADPH-cytochrome c reductase, two endoplasmic reticulum markers. The distribution in the gradient of beta-glucuronidase, a marker of lysosomes and of succinate dehydrogenase, a marker of mitochondria was clearly different from that of NADPH oxidase and of the markers of plasma membrane and of endoplasmic reticulum. These results indicated that in stimulated-elicited macrophages the NADPH oxidase is associated with a membrane fraction. With the fractionation technique employed it was not possible to clarify whether the oxidase is located in the plasma membrane or in the endoplasmic reticulum. In order to clarify this matter the isolation of phagosomes was performed. NADPH oxidase was found to be enriched in the phagosomal fraction. Phagosomes were also found to be enriched in the plasma membrane marker 5'-nucleotidase. Glucose-6-phosphatase,, a marker of endoplasmic reticulum, and beta-glucuronidase, a marker of lysosomes were not enriched in the phagosomal fraction. The results obtained clearly suggest that the activated NADPH oxidase of peritoneal elicited macrophages of guinea pig is located in the plasma membrane.

Immunotheranostic Polymersomes Modularly Assembled from Tetrablock and Diblock Copolymers with Oxidation-Responsive Fluorescence

“…This minimalist in vitro experiment verifies sensitivity of the polymersome fluorescence to ROS, but it should be noted that in vivo these shifts in I 550/640 ratio may vary extensively by cell type, activation state, and mechanism of endocytosis due to the dynamic and complex mixtures of ROS present in endosomal compartments. 6, 7, 12-14 …”

“…To carry out these activities, macrophages trigger a respiratory burst during phagocytosis, rapidly generating reactive oxygen species (ROS) mainly at the plasma membrane. 7, 8 Rapid degradation of engulfed materials is further enhanced by massive recruitment of V-ATPase, which acidifies their phagosomes within minutes to activate a host of proteolytic enzymes. 9, 10 DCs on the contrary, demonstrate a more subtle oxidation within their phagosomes over the course of several hours and a lower potency for degradation relative to macrophages.…”

Phagocytosis and antigen presentation in dendritic cells

“…Macrophages lack azurophil, specific granules, and secretory vesicles found in neutrophils (18,19). Macrophages also participate actively in the removal of dead cells.…”

“…After recognition, macrophages engulf their 'prey' into phagosomes, which mature to digestive organelles (phagolysosomes) by interchanging membrane and soluble material with different intracellular compartments. Macrophages lack azurophil, specific granules, and secretory vesicles found in neutrophils (18,19). Consequently, phagosome maturation in macrophages differs in quality and kinetics from that in neutrophils.…”

ESR detection of intraphagosomal superoxide in polymorphonuclear leukocytes using 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide

“…The localization of NADPH oxidase components in the phagosome membrane was also reported [11]. The localization of the cytosolic components of NADPH oxidase and the mechanisms of their assembly in the phagosome membrane have been extensively studied [12][13][14]. Recently, histochemical determination of 02-generation using DAB/Mn 2+ revealed that the soluble stimulants PMA and fMLP produced 02"-not only on the cell surface but also in intracellular nonphagocytic-vacuoles 115 '16].…”