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-γ and lipopolysaccharide, and the reactive mixture released by single cells...

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Bibliographic Details
Published in:Chembiochem : a European journal of chemical biology 2008-06, Vol.9 (9), p.1472-1480
Main Authors: Amatore, Christian, Arbault, Stéphane, Bouton, Cécile, Drapier, Jean-Claude, Ghandour, Hala, Koh, Alaric C.W
Format: Article
Language:English
Subjects:
Animals
Cell Line
Cellular Biology
Electrochemistry
Fluorometry
Free Radical Scavengers - metabolism
Immunization
Interferon-gamma - metabolism
Life Sciences
Lipopolysaccharides - metabolism
macrophages
Macrophages - immunology
Macrophages - metabolism
Mice
microelectrode
Nitric Oxide - metabolism
Nitroprusside - analysis
Nitroprusside - metabolism
peroxynitrite
Peroxynitrous Acid - metabolism
reactive nitrogen species
Reactive Nitrogen Species - analysis
Reactive Nitrogen Species - metabolism
Reactive Oxygen Species - analysis
Reactive Oxygen Species - metabolism
Time Factors
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Summary: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-γ 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.
ISSN:1439-4227
1439-7633
DOI:10.1002/cbic.200700746