title: Human neutrophils use the myeloperoxidase-hydrogen peroxide-chloride system to chlorinate but not nitrate bacterial proteins during phagocytosis.
abstract: The generation of extracellular oxidants by neutrophils has been widely investigated, but knowledge about the chemical reactions that occur in the phagolysosome, the cellular compartment that kills pathogens, is more limited. One important pathway may involve the production of potent halogenating agents such as hypochlorous acid (HOCl) by the myeloperoxidase-hydrogen peroxide-halide system. However, explorations of the oxidation chemistry of phagolysosomes have been hampered by the organelle's inaccessibility. To overcome this limitation, we recovered Escherichia coli that had been internalized by human neutrophils. We then analyzed the bacterial proteins for 3-chlorotyrosine, a stable marker of damage by HOCl. Mass spectrometric analysis revealed that levels of 3-chlorotyrosine in E. coli proteins increased markedly after the bacteria were internalized by human neutrophils. This increase failed to occur in E. coli exposed to neutrophils deficient in NADPH oxidase or myeloperoxidase, implicating H(2)O(2) and myeloperoxidase in the halogenation reaction. The extent of protein chlorination by normal neutrophils paralleled bacterial killing. Our observations support the view that the phagolysosome of human neutrophils uses the myeloperoxidase-hydrogen peroxide-chloride system to chlorinate bacterial proteins. In striking contrast, human neutrophils failed to nitrate bacterial proteins unless the medium was supplemented with 1 mm nitrite, and the level of nitration was low. Protein chlorination associated with bacterial killing was unaffected by the presence of nitrite in the medium. Nitration required NADPH oxidase but appeared to be independent of myeloperoxidase, suggesting that neutrophils can nitrate proteins through a pathway that requires nitrite but is independent of myeloperoxidase.
authors.affiliation: Department of Medicine, University of Washington, Seattle, WA 98195, USA. hqr@u.washington.edu
authors.forename: Henry
authors.initials: H
authors.lastname: Rosen
authors.forename: Jan R
authors.initials: JR
authors.lastname: Crowley
authors.forename: Jay W
authors.initials: JW
authors.lastname: Heinecke
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D006997:Hypochlorous Acid / Q000378:metabolism; D009504:Neutrophils / Q000276:immunology / Q000378:metabolism*; D009573:Nitrites / Q000378:metabolism*; D009195:Peroxidase / Q000378:metabolism*; D010587:Phagocytosis; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D002712:Chlorides; D009573:Nitrites; D014443:Tyrosine; D006997:Hypochlorous Acid; D006861:Hydrogen Peroxide; D009195:Peroxidase; C087259:3-chlorotyrosine