• Electron paramagnetic resonance/spin trapping detection and mechanism of human neutrophil hydroxyl radical generation

      Ramos, Carroll Leslie; Rosen, Gerald M., Ph.D., J.D. (1994)
      Neutrophils are important components of the innate immune response to invading microorganisms and may also contribute to tissue damage in pathologies such as reperfusion injury and arthritis. A principle mechanism by which neutrophils damage microbes is the generation of a series of reactive oxygen intermediates. Although the production of superoxide, hydrogen peroxide and hypochlorous acid by neutrophils is well established, the endogenous capacity of these cells to generate hydroxyl radical without the addition of a supplemental iron catalyst has not been determined. Since hydroxyl radical reacts with biomolecules at diffusion controlled rates, it is important to determine if neutrophils generate this free radical and, if so, establish the mechanism by which it is formed. Kinetic and stability studies were designed to select an appropriate electron paramagnetic resonance/spin trapping system for the detection of hydroxyl radical under biological conditions. The spin trap 4-pyridyl 1-oxide-N-tert-butylnitrone (4-POBN) in combination with ethanol, in which hydroxyl radical is detected as the {dollar}\alpha{dollar}-hydroxyethyl spin adduct of 4-POBN (4-POBN-CH(CH{dollar}\sb3{dollar})OH), was found to be best suited for this application. The rate constant for the reaction of 4-POBN with {dollar}\alpha{dollar}-hydroxyethyl radical was ten to fifteen fold greater than that with other nitrone spin traps and the resulting spin adduct was stable in the presence of neutrophil secretory products. Using this technique, it was found that human neutrophils and monocytes stimulated with a phorbol ester generated hydroxyl radical. Detection did not require a transition metal catalyst and was abolished by superoxide dismutase, catalase and azide. Hydroxyl radical was not observed upon stimulation of monocyte-derived macrophages and myeloperoxidase-deficient neutrophils. However, myeloperoxidase-deficient cells supplemented with purified myeloperoxidase generated hydroxyl radical upon activation. Addition of purified myeloperoxidase to a model superoxide generating system resulted in the chloride-dependent detection of hydroxyl radical which was inhibited by superoxide dismutase, catalase and azide. Based on these findings, it was concluded that human neutrophils and monocytes generate hydroxyl radical through a myeloperoxidase-dependent mechanism which likely involves the reaction of superoxide and hypochlorous acid.