Spin trapping evidence for myeloperoxidase-dependent hydroxyl radical formation by human neutrophils and monocytes

• Published in: The Journal of biological chemistry 1992-04, Vol.267 (12), p.8307-8312
• Main Authors: RAMOS, C. L, POU, S, BRITIGAN, B. E, COHEN, M. S, ROSEN, G. M
• Format: Article
• Language: English
• Subjects: ANIMAL CELLS; ANIMALS; Biological and medical sciences; BIOLOGICAL EFFECTS; BIOLOGICAL MATERIALS; BIOSYNTHESIS; BLOOD; BLOOD CELLS; BODY FLUIDS; CARCINOGENS; CATALASE; CHALCOGENIDES; CONNECTIVE TISSUE CELLS; E.S.R; ELECTRON SPIN RESONANCE; Electron Spin Resonance Spectroscopy; ELEMENTS; enzymatic activity; ENZYMES; ESTERS; formation; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Humans; Hydroxides; Hydroxyl Radical; HYDROXYL RADICALS; Immunobiology; IRON; Iron - physiology; LEUKOCYTES; leukocytes (neutrophilic); MACROPHAGES; MAGNETIC RESONANCE; MAMMALS; MAN; MATERIALS; METALS; MONOCYTES; Monocytes - drug effects; Monocytes - metabolism; Monocytes, macrophages; Myeloid cells: ontogeny, maturation, markers, receptors; myeloperoxidase; NEUTROPHILS; Neutrophils - drug effects; Neutrophils - metabolism; NITROGEN COMPOUNDS; NITROGEN OXIDES; Nitrogen Oxides - chemistry; ORGANIC COMPOUNDS; OXIDES; OXIDOREDUCTASES; OXYGEN COMPOUNDS; Peroxidase - metabolism; PEROXIDASES; PHAGOCYTES; PHORBOL ESTERS; PRIMATES; PROTEINS; Pyridines; RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT; RADICALS; RESONANCE; SOMATIC CELLS; Spin Labels; SUPEROXIDE DISMUTASE; SYNTHESIS; Tetradecanoylphorbol Acetate - pharmacology; TRANSITION ELEMENTS; VERTEBRATES 560300 > Chemicals Metabolism & Toxicology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(18)42443-x

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.