Am. J. Respir. Crit. Care Med., Volume 163, Number 5, April 2001, 1233-1240

Nitric Oxide and Protein Nitration are Eosinophil Dependent in Allergen-Challenged Mice

HIROAKI IIJIMA, ALEXANDRE DUGUET, SEOK-YONG EUM, QUTAYBA HAMID, and DAVID H. EIDELMAN

Meakins-Christie Laboratories, Respiratory Division, Department of Medicine, McGill University Health Centre, Montréal, Québec, Canada

To explore the possible role of eosinophils in NO-mediated tissue injury, we studied a murine model of allergic asthma. Male A/J mice were sensitized and challenged intranasally with ovalbumin (OVA). Following challenge, the number of eosinophils in bronchoalveolar lavage fluid (BALF) increased from 0.4% of total cells at baseline (0.02 × 10⁴ cells/ml) to 60.2% at 48 h after the challenge (9.34 × 10⁴ cells/ml). The rise in eosinophil count was accompanied by a 40.3% increase in total NO₂ plus NO₃ (NO_x) in BALF. This in turn was accompanied by expression of inducible NO synthase (NOS II) in airway epithelial and inflammatory cells, as well as by evidence of staining for 3-nitrotyrosine (3NT) in peribronchial inflammatory cells and at the epithelial surface. Both NO_x production and 3NT were significantly reduced by pretreatment of the challenged mice with the highly specific NOS II inhibitor N-3-aminomethyl-benzyl-acetamidine-dihydrochloride (1400W), as well as by the nonselective NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME). L-NAME and 1400W also reduced the number of BALF eosinophils (37.2% and 61.5%, respectively, as compared with the control value), suggesting that NO production by NOS II contributes to eosinophil recruitment. To further examine the role of eosinophils, we pretreated additional mice with an anti-interleukin (IL)-5 antibody, which reduced BALF eosinophilia following OVA challenge by 90.1%. In concert with the decrease in eosinophils, the anti-IL-5 antibody reduced NO_x in BALF almost to the baseline value, and decreased the number of 3NT-positive cells in the peribronchial region by 74.4%. Western blot analysis of protein extracted from whole lung confirmed the reduction in tyrosine nitration by anti-IL-5 antibody. These findings indicate that NO and eosinophilic inflammation are closely coupled, and suggest that eosinophils are an important source of tyrosine nitration.

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