Eosinophils are a major source of nitric oxide-derived oxidants in severe asthma: Characterization of pathways available to eosinophils for generating reactive nitrogen species

Eosinophil recruitment and enhanced production of NO are characteristic fea tures of asthma. However, neither the ability of eosinophils to generate NO -derived oxidants nor their role in nitration of targets during asthma is e stablished. Using gas chromatography-mass spectrometry we demonstrate a 10- fold increase in 3-nitrotyrosine (NO2Y) content, a global marker of protein modification by reactive nitrogen species, in proteins recovered from bron choalveolar lavage of severe asthmatic patients (480 +/- 198 mu mol/mol tyr osine; n = 11) compared with nonasthmatic subjects (52.5 +/- 40.7 mu mol/mo l tyrosine; n = 12). Parallel gas chromatography-mass spectrometry analyses of bronchoalveolar lavage proteins for 3-bromotyrosine (BrY) and 3-chlorot yrosine (ClY), selective markers of eosinophil peroxidase (EPO)- and myelop eroxidase-catalyzed oxidation, respectively, demonstrated a dramatic prefer ential formation of BrY in asthmatic (1093 +/- 457 mu mol BrY/mol tyrosine; 161 +/- 88 mu mol ClY/mol tyrosine; n = 11 each) compared with nonasthmati c subjects (13 +/- 14.5 mu mol BrY/mol tyrosine; 65 +/- 69 mu mol ClY/mol t yrosine; n = 12 each). Bronchial tissue from individuals who died of asthma demonstrated the most intense anti-NO2Y immunostaining in epitopes that co localized with eosinophils. Although eosinophils from normal subjects faile d to generate detectable levels of NO, NO2-, NO3-, or NO2Y, tyrosine nitrat ion was promoted by eosinophils activated either in the presence of physiol ogical levels of NO2- or an exogenous NO source. At low, but not high (e.g. , >2 muM/min), rates of NO flux, EPO inhibitors and catalase markedly atten uated aromatic nitration. These results identify eosinophils as a major sou rce of oxidants during asthma. They also demonstrate that eosinophils use d istinct mechanisms for generating NO-derived oxidants and identify EPO as a n enzymatic source of nitrating intermediates in eosinophils.