Real-time amperometric analysis of reactive oxygen and nitrogen species released by single immunostimulated macrophages.
Macrophages are key cells of the immune system. Immunologically activated macrophages are known to release a cocktail of reactive oxygen and nitrogen species. In this work, RAW 264.7 macrophages were activated by interferon-gamma and lipopolysaccharide, and the reactive mixture released by single cells was analyzed, in real time, by amperometry at platinized carbon microelectrodes. In comparison with untreated macrophages, significant increases in amperometric responses were observed for activated macrophages. Nitric oxide (NO*), nitrite (NO2*-), and peroxynitrite (ONOO-) were the main reactive species detected. The amounts of these reactive species were quantified, and their average fluxes released by a single, activated macrophage were evaluated. The detection of ONOO- is of particular interest, as its role and implications in various physiological conditions have been widely debated. Herein, direct evidence for the formation of ONOO- in stimulated macrophages is presented. Finally, the presence of 1400W, a selective inducible nitric oxide synthase (iNOS) inhibitor, led to an almost complete attenuation of the amperometric response of activated RAW 264.7 cells. The majority of the reactive species released by a macrophage are thus likely to be derived from NO* and superoxide (O2*-) co-produced by iNOS.
Amatore
Christian
C
Laboratoire PASTEUR and LIA XiamENS, Ecole Normale Superieure, CNRS, UPMC Université Paris 06; Departement de Chimie, 24 rue Lhomond, 75005 Paris, France. Christian.Amatore@ens.fr
Arbault
Stéphane
S
Bouton
Cécile
C
Drapier
Jean-Claude
JC
Ghandour
Hala
H
Koh
Alaric C W
AC
D000818:Animals; D002460:Cell Line; D004563:Electrochemistry; D005470:Fluorometry; D016166:Free Radical Scavengers / Q000378:metabolism; D007114:Immunization; D007371:Interferon-gamma / Q000378:metabolism; D008070:Lipopolysaccharides / Q000378:metabolism; D008264:Macrophages / Q000276:immunology* / Q000378:metabolism*; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism; D009599:Nitroprusside / Q000032:analysis / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000378:metabolism; D026361:Reactive Nitrogen Species / Q000032:analysis / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000032:analysis / Q000378:metabolism*; D013997:Time Factors
D016166:Free Radical Scavengers; D008070:Lipopolysaccharides; D026361:Reactive Nitrogen Species; D017382:Reactive Oxygen Species; D030421:Peroxynitrous Acid; D009599:Nitroprusside; D009569:Nitric Oxide; D007371:Interferon-gamma