Nitric oxide and related enzymes in asthma: relation to severity, enzyme function and inflammation

BACKGROUND: Exhaled nitric oxide (FeNO) associates with asthma and eosinophilic inflammation. However, relationships between nitric oxide synthases, arginase, FeNO, asthma severity and inflammation remain poorly understood. OBJECTIVES: To determine the relationships of iNOS expression/activation and arginase 2 expression with asthma severity, FeNO, nitrotyrosine (NT) and eosinophilic inflammation. METHODS: Bronchial brushings and sputum were obtained from 25 normal controls, eight mild/no inhaled corticosteroids (ICS), 16 mild-moderate/with ICS and 35 severe asthmatics. The FeNO was measured the same day by ATS/ERS standards. The iNOS, arginase2 mRNA/protein and NT protein were measured in lysates from bronchial brushings by quantitative real-time PCR and Western blot. Induced sputum differentials were obtained. RESULTS: Severe asthma was associated with the highest levels of iNOS protein and mRNA, although the index of iNOS mRNA to arginase2 mRNA most strongly differentiated severe from milder asthma. When evaluating NO-related enzyme functionality, iNOS mRNA/protein expression both strongly predicted FeNO (r = 0.61, P < 0.0001 for both). Only iNOS protein predicted NT levels (r = 0.48, P = 0.003) with the strongest relationship in severe asthma (r = 0.61, P = 0.009). The iNOS protein, FeNO and NT, all correlated with sputum eosinophils, but the relationships were again strongest in severe asthma. Controlling for arginase 2 mRNA/protein did not impact any functional outcome. CONCLUSIONS AND CLINICAL RELEVANCE: These data suggest that while iNOS expression from epithelial brushings is highest in severe asthma, factors controlling arginase2 mRNA expression significantly improve differentiation of severity. In contrast, functionality of the NO pathway as measured by FeNO, NT and eosinophilic inflammation, is strongly associated with iNOS expression alone, particularly in severe asthma.