Exhaled Nitric Oxide in Difficult Childhood Asthma
More Light or Still Chasing Shadows?

Stephen M. Stick, M.D.

Department of Respiratory Medicine, Princess Margaret Hospital for Children, Perth, Australia

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Over the past decade there has been increasing interest in the potential for measurements of nitric oxide (NO) in exhaled breath (FE_NO) as an indicator of airway inflammation in asthma. Progress in this area has recently been reviewed comprehensively (1). Briefly, exhaled NO is elevated in asthma, during acute asthma attacks, and late asthmatic responses to inhaled allergens. Exhaled NO levels fall after treatment with corticosteroids and are significantly correlated with eosinophils in sputum (2). Increased FE_NO in asthma is argued to be due to upregulation of inducible nitric oxide synthase (iNOS or type 2 NOS). Inducible NOS is expressed to a greater extent in the airway epithelium of asthma patients than in healthy subjects, and there is a significant positive correlation between concentrations of FE_NO and iNOS expression (1). NO is reduced in the exhaled air of asthmatics after the administration of a specific iNOS inhibitor (3). Validated techniques are now available to measure lower respiratory NO by minimizing nasal contamination (4), thus making measurements of FE_NO a feasible laboratory tool.

In the present issue of the Journal (pp. 1376-1381), Payne and colleagues (5) provide further evidence in support of the utility of FE_NO measurements for monitoring asthma. This is one of few studies in children to use bronchial biopsy specimens to assess inflammation. They report an association between persistently raised FE_NO and biopsy-proven, persistent eosinophilic airway inflammation in children with difficult asthma treated with systemic corticosteroid. This observation might explain some of the different reported outcomes from studies that have examined relations between FE_NO, eosinophilic inflammation, and responses to corticosteroids. As pointed out by Payne and colleagues, a number of studies have demonstrated significant correlations between FE_NO and airway eosinophilia. However, these observations are most consistent in steroid-naïve asthmatics. The study described in this issue of the Journal presents data to suggest that a subgroup of asthmatic children with persistent symptoms that are unresponsive to treatment with corticosteroid have persistent eosinophilic mucosal inflammation. They argue quite reasonably that the persistence of raised levels of FE_NO is a useful indicator of this ongoing process. The degree of eosinophilia that constitutes persistence after treatment in the report by Payne and colleagues appears to have been defined post hoc relative to the elevation of FE_NO rather than any pathophysiologic criteria related to eosinophil number or state of activation. Due to the nature of the study, it was not possible to examine change in FE_NO relative to change in eosinophil count, and therefore comparison with other studies is not possible. However, a recent report has provided strong evidence that the responses of measures of eosinophilic inflammation and FE_NO to anti-inflammatory agents are discordant. Lim and colleagues (6) demonstrated that improvements in eosinophilic inflammation were not accompanied by reduced FE_NO in asthmatic patients treated with low-dose theophylline. Whereas corticosteroids reduce airway inflammation and downregulate iNOS, theophylline has no effect on iNOS activity directly and therefore is perhaps a better model to examine the relations between inflammation and FE_NO in asthma.

Another potentially important consideration is the apparent failure of corticosteroids in the study by Payne and colleagues to downregulate iNOS independent of any effects on inflammation. The investigators do not report change in FE_NO but use a cutoff value of 7 ppb based upon experience in their laboratory to indicate whether FE_NO is raised or not after administration of corticosteroids. Corticosteroids are potent inhibitors of iNOS (for example, the inhibitory concentration for dexamethasone is 6 µmol/L compared with 250 µmol/L for aminoguanidine). Is it possible that the source of FE_NO in the group of difficult-to-treat asthmatics with persistence of eosinophilia and FE_NO greater than 7 ppb is due to the activity of another NOS isoform or due to constitutive upregulation of iNOS unrelated to the presence of inflammation? There is as yet little direct evidence to support such suggestions but there are fragments of information from a variety of studies to suggest that these arguments should not be readily dismissed.

Recent studies have demonstrated differences in FE_NO in association with polymorphisms of the type 1 NOS gene (7). The presence of a polymorphism associated with relatively increased NO production, together with increased alveolar diffusion caused by inflammation (8), could result in significantly increased concentrations of airway NO. Studies have demonstrated raised levels of FE_NO in healthy atopics who might not have airway inflammation (9). Furthermore, in adults with asthma, atopy appears to a better predictor of FE_NO concentrations than airway responsiveness (10). These observations suggest that factors other than inflammation and possibly NOS isoforms other than iNOS might contribute significantly to airway NO concentrations in asthma.

Whatever the source of raised airway NO, could NO contribute to the persistence of eosinophils in the mucosa rather than just reflect airway inflammation? There are limited data from animal experiments using specific NOS inhibitors to support such a hypothesis. Tulic and colleagues demonstrated that pretreatment with the iNOS inhibitor aminoguanidine reduced eosinophil influx into the airways after allergen challenge in allergic rats (12). Ferreira and colleagues reported similar observations in allergen-challenged rats after nonspecific inhibition of NOS with N-nitro- L-arginine methyl ester (L-NAME) (13).

The report by Payne and colleagues provides an intriguing glimpse into the complexity of NO regulation in asthma. However, with each new study that investigates the potential for measurements of FE_NO as a diagnostic tool there appear to be more questions generated about the biology of NO in the airway than there are answers. Perhaps by focusing on some of these questions, new therapeutic opportunities might be realized based upon a better understanding of the factors that control NO production in the lung.

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