Abstract
Using the electron spin resonance/spin trapping system, 4-pyridyl 1-oxide N-tert-butylnitrone (4-POBN)/ethanol, hydroxyl radical was detected as the alpha-hydroxyethyl spin trapped adduct of 4-POBN, 4-POBN-CH(CH3)OH, from phorbol 12-myristate 13-acetate-stimulated human neutrophils and monocytes without the addition of supplemental iron. 4-POBN-CH(CH3)OH was stable in the presence of a neutrophil-derived superoxide flux. Hydroxyl radical formation was inhibited by treatment with superoxide dismutase, catalase, and azide. Treatment with a series of transition metal chelators did not appreciably alter 4-POBN-CH(CH3)OH, which suggested that hydroxyl radical generation was mediated by a mechanism independent of the transition metal-catalyzed Haber-Weiss reaction. Kinetic differences between transition metal-dependent and -independent mechanisms of hydroxyl radical generation by stimulated neutrophils were demonstrated by a greater rate of 4-POBN-CH(CH3)-OH accumulation in the presence of supplemental iron. Detection of hydroxyl radical from stimulated monocyte-derived macrophages, which lack myeloperoxidase, required the addition of supplemental iron. The addition of purified myeloperoxidase to an enzymatic superoxide generating system resulted in the detection of hydroxyl radical that was dependent upon the presence of chloride and was inhibited by superoxide dismutase, catalase, and azide. These findings implicated the reaction of hypochlorous acid and superoxide to produce hydroxyl radical. 4-POBN-CH(CH3)OH was not observed upon stimulation of myeloperoxidase-deficient neutrophils, whereas addition of myeloperoxidase to the reaction mixture resulted in the detection of hydroxyl radical. These results support the ability of human neutrophils and monocytes to generate hydroxyl radical through a myeloperoxidase-dependent mechanism.
Highlights
11Departments of Medicine, Microbiology, and Immunology, University of North Carolina, Chapel Hill,North Carolina 27599, and the **VeteransAdministration Medical Center, Baltimore, Maryland 21218
Using the electron spin resonance/spintrapping sys- A major component of the neutrophil and mononuclear tem, 4-pyridyl l-oxide N-tert-butylnitrone (4-POBN)/ phagocyte response to a microbial challenge is the generation ethanol, hydroxyl radical was detected as the a-hy- of reactive oxygen species [1].The cascade of events associdroxyethyl spin trapped adductof 4-POBN, 4-POBN- ated with oxygen-dependent microbicidal activity begins with
Since reactive oxygen species have been pendentand-independent mechanisms of hydroxyl implicated in thepathology of tissue injury, resulting from a radicalgenerationbystimulatedneutrophilswere number of ischemic and inflammatory events (9, lo), it is demonstrated by a greater rate of 4-POBN-CH(CH3)- important to clarify the role of 'OH in phagocyte biology as OH accumulation in thepresence of supplemental iron. a basis for rational therapeutic design
Summary
4-POBN-CH(CH3)0H was not observed upon stimulation of myeloperoxidase-deficient neutrophils, whereas addition of myeloperoxidase to the reaction mixture resulted in the detection of hydroxyl radical. In several studies using the spin trapping system DMPO/ dimethyl sulfoxide, generation of 'OH by stimulated human neutrophils was not observed unless supplemental iron was provided [20,21,22,23].
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