Morphometric estimation of superoxide generation in allergen-induced airway hyperresponsiveness

BACKGROUND: We previously reported that the airway hyperresponsiveness (AHR) that follows antigen challenge is mediated, in part, by the generation of reactive oxygen species. The purpose of this study was to provide quantitative morphologic evidence of oxygen radical production in the airways during antigen-induced AHR. EXPERIMENTAL DESIGN: Allergic sheep with a history of early and late bronchial responses to inhaled Ascaris suum antigen were used. The sheep were either challenged with antigen (N = 5) or saline (N = 5) and then euthanized 24 hours later when AHR had been documented to occur. Complete transverse sections of the right cranial lobar bronchus were obtained from the animals and were washed three times in Tris buffer and then incubated for 20 minutes in an oxygenated solution of diaminobenzidine which, in the presence of superoxide and manganese, forms an insoluble amber reaction product. Superoxide dismutase (SOD, 2250 units/ml) and phorbol ester (phorbol myristate acetate, 0.5 μg/ml) were added to some tissues and used as negative and positive controls, respectively. The bronchial samples were then fixed and embedded in paraffin for light microscopy. The diaminobenzidine reaction product was quantified by determining the volume fraction of reaction product by point counting with a differential interference contrast microscope without counterstain. RESULTS: Diaminobenzidine reaction product increased 5-fold (p < 0.05) in challenged animals, and this response was blocked by SOD. The reaction product was localized in and around the airway epithelium. Antigen challenge also resulted in a 2.4- and 2.0-fold increase in eosinophils and metachromatic cells in the airway wall. There were no differences in the number of neutrophils between groups. Pretreatment of animals (N = 2) with the combination of the 5-lipoxygenase inhibitor (zileuton, 10 mg/kg, po) and the platelet-activating factor antagonist (WEB-2086, 3 mg/kg, iv) agents, which have been shown to block AHR and antigen-induced inflammation in the sheep model, also blocked the antigen-induced superoxide formation. CONCLUSIONS: These data suggest that superoxide and increased numbers of mediator- containing inflammatory cells are present in sheep airways 24 hours after antigen challenge. The interaction of this reactive oxygen species with these cells could contribute to the AHR seen at this time.