Electron microscopic identification of hydrogen peroxide detected in fixed human polymorphonuclear leukocytes during phagocytosis

Abstract

Summary: Polymorphonuclear leukocytes (PMNs) engaged in phagocytosis produce reactive oxygen species (ROS), such as those that occur in an activated NADPH oxidase reaction, to eliminate ingested microorganisms. The translocation of NADPH oxidase components to produce antimicrobial free radicals from the vesicles to the phagosomes may be important. Hydrogen peroxide (H 2 O 2 ) derived from O 2 -has been observed by electron microscopy using a cerium method. However, 2'-7'-dichlorofluorescin diacetate can also detect H 2 O 2 through fluorescence. The main objective of the present study was to measure the H 2 O 2 -dependent fluorescence of PMNs after opsonized zymosan A (OPZ) phagocytosis using a microplate reader under different fixation conditions, including 0.5, 1, and 10% glutaraldehyde (GA) individually for 1, 5, 10, or 30 min. An additional objective was to visualize, through the use of electron microscopic cytochemistry, the process of H 2 O 2 generation in OPZ phagocytic fixed PMNs. The fixed PMNs showed that the largest fluorescent value was produced by a concentration of 0.5% GA for all fixation times. This suggested that the fixation of PMNs with a high concentration of GA inhibited phagocytosis and produced ROS. In the fixed PMNs, electron microscopic results showed that after 1 min of mixing, some PMNs attached to particles and exhibited mild deposits in their secretory vesicles. When PMNs engulfed particles, free radical-producing vesicles had enhanced reaction deposits 10 min later and fused to the phagosomal membrane, releasing numerous free radicals into the lumen. Time-dependent H 2 O 2 production was enhanced in the secretory vesicles, some of which were fused exactly to the phagosome membranes.

Independent trafficking of flavocytochrome b558 and myeloperoxidase to phagosomes during phagocytosis visualised by energy‐filtering and energy‐dispersive spectroscopy‐scanning transmission electron microscopy

“…Moreover, immune–transmission electron and confocal laser scanning microscopy also supported the vesicular localisation of AIM2 in inflammasomes in human neutrophils. These data indicated that the secretory vesicles measured approximately 0.09–0.15 m in diameter and were in close agreement with values from our previous study (Moriguchi & Ohno, ) (Figs. ).…”

“…PMNs were mixed with opsonised zymosan particles (OPZ) for 10 min at 37°C. PMNs engulfing OPZ were fixed with pure 2% glutaraldehyde for 10 min Moriguchi & Ohno, 2014). Thereafter, Nox-mediated H 2 O 2 production was demonstrated in cerium (Ce) medium [1 mM CeCl 3 , 3 mM NADPH and 10 mM sodium azide in 0.1 M Tris-maleate buffer (pH 7.5)] for 20 min (Ohno et al, 1982;Hirai et al, 1991).…”

Fosfomycin enhances phagocyte-mediated killing of Staphylococcus aureus by extracellular traps and reactive oxygen species

“…Previous reports demonstrated that a major intracellular bactericidal mechanism used by phagocytes is the generation of ROS via oxidative burst 17 24 25 26 27 28 . Oxidative burst analysis showed that FOM induced much more ROS in S. aureus RBF or PLK cell-treated THP-1 cells or neutrophils than these phagocytes alone ( p < 0.05, Fig.…”

“…Physiological generation of ROS occurs either as by-products of (redox) reactions in various cell organelles, including mitochondria, peroxisomes, and endoplasmic reticulum, or by primary enzyme function, such as with oxidases and oxygenases. Plasma membrane-bound phagocyte NADPH oxidase was commonly thought to be the main source of ROS delivery into the extracellular space during respiratory bursts and into engulfed phagosomes for microbial killing 25 26 27 28 . The oxidative burst pumped electrons into the phagosome that was compensated by a flux of K + ions across the membrane in a pH dependent matter.…”

The Role of Reactive Oxygen Species (ROS) in the Formation of Extracellular Traps (ETs) in Humans

“…Due to a transient rise in oxygen demand, this behavior was originally named respiratory (or oxidative) burst (e.g., [ 40 , 78 ]). Plasma membrane-bound phagocyte NADPH oxidase is commonly thought to be the main source of ROS delivery to the extracellular space during respiratory bursts, and into engulfed phagosomes for microbial killing [ 65 , 79 , 80 ]. In addition, NADPH oxidase derived ● O 2 − has been shown to promote microbial killing in the phagosome also indirectly by enabling the activation of serin proteases (e.g., cathepsin G and neutrophil elastase, NE) in neutrophil azurophilic granules via modulation of ion influx and pH [ 14 , 81 , 82 ] (there is, however, evidence suggesting that the importance of this mechanism for the microbicidal capacity of the neutrophils is limited [ 83 ]).…”