Monitoring in real time with a microelectrode the release of reactive oxygen and nitrogen species by a single macrophage stimulated by its membrane mechanical depolarization.
Macrophages are key cells of the immune system. During phagocytosis, the macrophage engulfs a foreign bacterium, virus, or particle into a vacuole, the phagosome, wherein oxidants are produced to neutralize and decompose the threatening element. These oxidants derive from in situ production of superoxide and nitric oxide by specific enzymes. However, the chemical nature and sequence of release of these compounds is far from being completely determined. The aim of the present work was to study the fundamental mechanism of oxidant release by macrophages at the level of a single cell, in real time and quantitatively. The tip of a microelectrode was positioned at a micrometric distance from a macrophage in a culture to measure oxidative-burst release by the cell when it was submitted to physical stimulation. The ensuing release of electroactive reactive oxygen and nitrogen species was detected by amperometry and the exact nature of the compounds was characterized through comparison with in vitro electrochemical oxidation of H2O2, ONOO-, NO*, and NO2(-) solutions. These results enabled the calculation of time variations of emission flux for each species and the reconstruction of the original flux of production of primary species, O2*- and NO*, by the macrophage.
Amatore
Christian
C
ENS, Département de Chimie, UMR CNRS-ENS-UPMC 8640 Pasteur, 24 rue Lhomond, 75231 Paris cedex 05, France. Christian.Amatore@ens.fr
Arbault
Stéphane
S
Bouton
Cécile
C
Coffi
Karen
K
Drapier
Jean-Claude
JC
Ghandour
Hala
H
Tong
Yuehong
Y
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology; D002478:Cells, Cultured; D005609:Free Radicals / Q000032:analysis / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D066298:In Vitro Techniques; D008264:Macrophages / Q000502:physiology*; D008564:Membrane Potentials / Q000502:physiology; D051379:Mice; D008839:Microelectrodes; D009569:Nitric Oxide / Q000378:metabolism; D010084:Oxidation-Reduction; D030421:Peroxynitrous Acid / Q000378:metabolism; D010812:Physical Stimulation; D026361:Reactive Nitrogen Species / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013997:Time Factors
D005609:Free Radicals; D026361:Reactive Nitrogen Species; D017382:Reactive Oxygen Species; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; D006861:Hydrogen Peroxide