title: Eosinophils are a major source of nitric oxide-derived oxidants in severe asthma: characterization of pathways available to eosinophils for generating reactive nitrogen species.
abstract: Eosinophil recruitment and enhanced production of NO are characteristic features of asthma. However, neither the ability of eosinophils to generate NO-derived oxidants nor their role in nitration of targets during asthma is established. Using gas chromatography-mass spectrometry we demonstrate a 10-fold increase in 3-nitrotyrosine (NO(2)Y) content, a global marker of protein modification by reactive nitrogen species, in proteins recovered from bronchoalveolar lavage of severe asthmatic patients (480 +/- 198 micromol/mol tyrosine; n = 11) compared with nonasthmatic subjects (52.5 +/- 40.7 micromol/mol tyrosine; n = 12). Parallel gas chromatography-mass spectrometry analyses of bronchoalveolar lavage proteins for 3-bromotyrosine (BrY) and 3-chlorotyrosine (ClY), selective markers of eosinophil peroxidase (EPO)- and myeloperoxidase-catalyzed oxidation, respectively, demonstrated a dramatic preferential formation of BrY in asthmatic (1093 +/- 457 micromol BrY/mol tyrosine; 161 +/- 88 micromol ClY/mol tyrosine; n = 11 each) compared with nonasthmatic subjects (13 +/- 14.5 micromol BrY/mol tyrosine; 65 +/- 69 micromol ClY/mol tyrosine; n = 12 each). Bronchial tissue from individuals who died of asthma demonstrated the most intense anti-NO(2)Y immunostaining in epitopes that colocalized with eosinophils. Although eosinophils from normal subjects failed to generate detectable levels of NO, NO(2-), NO(3-), or NO(2)Y, tyrosine nitration was promoted by eosinophils activated either in the presence of physiological levels of NO(2-) or an exogenous NO source. At low, but not high (e.g., >2 microM/min), rates of NO flux, EPO inhibitors and catalase markedly attenuated aromatic nitration. These results identify eosinophils as a major source of oxidants during asthma. They also demonstrate that eosinophils use distinct mechanisms for generating NO-derived oxidants and identify EPO as an enzymatic source of nitrating intermediates in eosinophils.
authors: Department of Cell Biology, Cleveland Clinic Foundation, Cleveland, OH 44195, USA.
authors: J C
authors: JC
authors: MacPherson
authors: S A
authors: SA
authors: Comhair
authors: S C
authors: SC
authors: Erzurum
authors: D F
authors: DF
authors: Klein
authors: M F
authors: MF
authors: Lipscomb
authors: M S
authors: MS
authors: Kavuru
authors: M K
authors: MK
authors: Samoszuk
authors: S L
authors: SL
authors: Hazen
mesh_terms: D047209:Eosinophil Peroxidase; D004804:Eosinophils / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D005609:Free Radicals / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D009566:Nitrates / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000378:metabolism; D009573:Nitrites / Q000378:metabolism; D016877:Oxidants / Q000378:metabolism*; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000378:metabolism; D010666:Phenylpropionates / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013224:Status Asthmaticus / Q000378:metabolism* / Q000473:pathology; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D005609:Free Radicals; D009566:Nitrates; D020030:Nitric Oxide Donors; D009573:Nitrites; D016877:Oxidants; D010666:Phenylpropionates; D011506:Proteins; D017382:Reactive Oxygen Species; C039642:peroxynitric acid; D009569:Nitric Oxide; C002744:3-nitrotyrosine; D014443:Tyrosine; C118327:3-bromotyrosine; C008869:phloretic acid; D047209:Eosinophil Peroxidase; D010544:Peroxidases; C087259:3-chlorotyrosine