The eosinophilic inflammatory response in
asthma is associated with
protein nitration, detected as immunostaining for
3-nitrotyrosine (3NT). As the presence of 3NT is strongly correlated with upregulation of the inducible form of
nitric oxide synthase (
NOS II), it has been hypothesized that 3NT formation results from the action of
peroxynitrite (ONOO-), a highly reactive NO derivative produced from the reaction of molecular NO and O2-. However, recent observations have suggested that the action of
peroxidases, including
eosinophil peroxidase (EPO), may be responsible for
protein nitration. In this study, we used murine models of allergic
asthma to address the relative contribution of EPO and
NOS II to
protein nitration. We studied EPO-deficient New Zealand White (NZW) mice, which were sensitized and challenged intranasally with
ovalbumin (OVA). Despite comparable levels of
eosinophilia, NO, and
superoxide production, NZW mice exhibited markedly decreased 3NT staining around the airways after OVA challenge when compared with two other strains (A/J and C57BL/6J). Immunocytochemical analysis of bronchoalveolar lavage (BAL) cells and lung sections suggested that 3NT staining was largely confined to eosinophils. This was confirmed by Western Blot analysis of
proteins from different subsets of BAL cells that demonstrated a marked decrease in 3NT formation in eosinophils from NZW mice. These results contrast with those obtained in OVA-sensitized and -challenged
NOS II deficient mice, which despite decreased NO production, exhibited similar 3NT staining in the airways after OVA challenge as in wild-type control mice. In this model,
protein nitration was thus not a function of NO production by
NOS II. We conclude that in the mouse, 3NT formation after specific
allergen challenge is dependent on EPO activity, particularly in eosinophils themselves. In contrast, 3NT formation is not driven by upregulation of
NOS II expression in this model and does not appear to depend on increases in the level of NO production.