title: Pathways for intracellular generation of oxidants and tyrosine nitration by a macrophage cell line.
abstract: Two transformed murine macrophage cell lines (RAW 264.7 ATCC TIB-71 and CRL-2278) were examined for oxidant production at various times following activation by using a set of fluorescence and ESR-active probes. Stimulation with a soluble agonist or activation with bacterial lipopolysaccharide plus gamma-interferon caused only very small initial increases in O2 consumption above basal rates; however, at 2-4 h post-activation, respiration increased to 2-3-fold and remained at these elevated levels over the subsequent lifetime of the cell (20-30 h). Oxidation reactions were confined primarily within the cell, as was demonstrated by using phagocytosable dichlorodihydrofluorescein-conjugated latex beads and cyclic hydroxylamines with differing membrane permeabilities. From the intrinsic reactivities of these probes and the time course of their oxidations, one infers the induction of apparent peroxidase activity beginning at approximately 2 h post-activation coinciding with the increase in overall respiratory rate; this acquired capability was accompanied by accumulation of a stable horseradish peroxidase-reactive oxidant, presumably H2O2, in the extracellular medium. Nitrite ion rapidly accumulated in the extracellular medium over a period of 5-8 h post-activation in both cell lines, indicating the presence of active nitric oxide synthase (iNOS) during that period. Prostaglandin endoperoxide H synthase (COX-2) activity was detected at 15-20 h post-activation by the use of a sensitive peroxide assay in conjunction with a COX-2 specific inhibitor (DuP-697). Superoxide formation was detected by reaction with hydroethidine within the first hour following activation, but not thereafter. Consistent with the absence of significant respiratory stimulation, the amount of O2*- formed was very small; comparative reactions of cyclic hydroxylamine probes indicated that virtually none of the O2*- was discharged into the external medium. Myeloperoxidase (MPO) activity was probed at various times post-activation by using fluorescein-conjugated polyacrylamide beads, which efficiently trap MPO-generated HOCl in neutrophils to give stable chlorofluorescein products. However, chlorination of the dye was not detected under any conditions in RAW cells, virtually precluding MPO involvement in their intracellular reactions. This same probe was used to determine changes in intraphagosomal pH, which increased slowly from approximately 6.5 to approximately 8.2 over a 20 h post-phagocytosis period. The cumulative data suggest that activation is followed by sequential induction of an endogenous peroxidase, iNOS, and COX-2, with NADPH oxidase-derived O2*- playing a minimal role in the direct generation of intracellular oxidants. To account for reported observations of intracellular tyrosine nitration late in the life cycles of macrophages, we propose a novel mechanism wherein iNOS-generated NO2- is used by COX-2 to produce NO2* as a terminal microbicidal oxidant and nitrating agent.
authors.affiliation: Department of Chemistry, Washington State University, Pullman, Washington 99164-4630, USA.
authors.forename: Amy M
authors.initials: AM
authors.lastname: Palazzolo-Ballance
authors.forename: Christine
authors.initials: C
authors.lastname: Suquet
authors.forename: James K
authors.initials: JK
authors.lastname: Hurst
mesh_terms: D000818:Animals; D002460:Cell Line; D051546:Cyclooxygenase 2 / Q000378:metabolism; D052246:Cyclooxygenase 2 Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008070:Lipopolysaccharides / Q000494:pharmacology; D008262:Macrophage Activation; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D015335:Molecular Probes; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D009573:Nitrites / Q000378:metabolism; D009584:Nitrogen / Q000378:metabolism*; D016877:Oxidants / Q000096:biosynthesis*; D010101:Oxygen Consumption; D009195:Peroxidase / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D013997:Time Factors; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D052246:Cyclooxygenase 2 Inhibitors; D005456:Fluorescent Dyes; D008070:Lipopolysaccharides; D015335:Molecular Probes; D009573:Nitrites; D016877:Oxidants; D013481:Superoxides; D014443:Tyrosine; D009195:Peroxidase; D052247:Nitric Oxide Synthase Type II; C077623:Ptgs2 protein, mouse; D051546:Cyclooxygenase 2; D009584:Nitrogen