Role of peroxynitrite in macrophage microbicidal mechanisms in vivo revealed by protein nitration and hydroxylation |
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| Authors: | Linares E Giorgio S Mortara R A Santos C X Yamada A T Augusto O |
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| Institution: | 1. Departamento de Bioquı́mica, Instituto de Quı́mica, Universidade de São Paulo, São Paulo, Brazil;2. Departamento de Parasitologia, Instituto de Biologia, Universidade Estadual de Campinas, São Paulo, Brazil;3. Departamento de Histologia, Instituto de Biologia, Universidade Estadual de Campinas, São Paulo, Brazil;4. Departamento de Microbiologia, Imunologia, e Parasitologia da Universidade Federal de São Paulo, São Paulo, Brazil;1. Dept. of Mathematics, University of California, San Diego, La Jolla, CA 92093-0112, USA;2. Computer Science Department, Universidad Politécnica de Cataluña, Barcelona, Spain;1. Department of Radiology, University of Michigan Health System, Ann Arbor, Michigan, USA;2. Department of Surgery, University of Michigan Health System, Ann Arbor, Michigan, USA;1. Department of Anesthesiology, Medical College of Wisconsin, 8701 W Watertown Plank, Milwaukee, WI 53226, USA;2. Department of Pharmacology and Toxicology, Medical College of Wisconsin, USA;3. Zablocki VA Medical Center, 5000 W National Avenue, Milwaukee, WI 53295, USA;1. Hunan Provincial Key Laboratory of Materials Protection for Electric Power and Transportation, School of Chemistry and Biological Engineering, Changsha University of Science and Technology, Changsha 410114, China;2. State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China;1. Department of Radiology, University of Michigan Health System, Ann Arbor, Michigan, USA;2. Department of Surgery, University of Michigan Health System, Ann Arbor, Michigan, USA |
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| Abstract: | The cytotoxins produced by phagocytic cells lacking peroxidases such as macrophages remain elusive. To elucidate macrophage microbicidal mechanisms in vivo, we compared the lesion tissue responses of resistant (C57Bl/6) and susceptible (BALB/c) mice to Leishmania amazonensis infection. This comparison demonstrated that parasite control relied on lesion macrophage activation with inducible nitric oxide synthase expression (iNOS), nitric oxide synthesis, and extensive nitration of parasites inside macrophage phagolysosomes at an early infection stage. Nitration and iNOS expression were monitored by confocal microscopy; nitric oxide synthesis was monitored by EPR. The main macrophage nitrating agent was shown to be peroxynitrite derived because parasite nitration occurred in the virtual absence of polymorphonuclear cells (monitored as peroxidase activity) and was accompanied by protein hydroxylation (monitored as 3-hydroxytyrosine levels). In vitro studies confirmed that peroxynitrite is cytotoxic to parasites whereas nitric oxide is cytostatic. The results indicate that peroxynitrite is likely to be produced close to the parasites and most of it reacts with carbon dioxide to produce carbonate radical anion and nitrogen dioxide whose concerted action leads to parasite nitration. In parallel, some peroxynitrite decomposition to the hydroxyl radical should occur due to the detection of hydroxylated proteins in the healing tissues. Consequently, peroxynitrite and derived radicals are likely to be important macrophage-derived cytotoxins. |
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