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The labyrinth of asthma phenotypes and exhaled NO
  1. Unit of Allergy and Respiratory Medicine
  2. Department of Pediatrics
  3. University of Padova
  4. 35128 Padova
  5. Italy
  6. eugi{at}

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In this issue of Thorax Payneet al 1 suggest that it is possible to distinguish different patterns of difficult childhood asthma by measuring exhaled NO (eNO). The knowledge that severe asthma may have different phenotypes with different types of airway inflammation is not new and has been clearly shown by bronchoalveolar lavage (BAL) and airway biopsy studies in adult patients.2The potential importance of these data lies in the classification of different patterns by using a non-invasive instantaneous measurement such as eNO.

Exhaled NO levels are known to be increased in atopic asthma, to increase during an exacerbation, to decrease with anti-inflammatory therapy,3 and to rise as the dose of inhaled steroids is reduced.4 In addition, eNO levels are correlated with eosinophils in induced sputum,5 bronchial hyperresponsiveness to AMP,6 and exercise,7and to increase in the late phase following allergen challenge.8 As a diagnostic tool, eNO levels discriminated asthmatics from non-asthmatics with a high sensitivity and specificity in a group of subjects with chronic cough.9 All the above evidence supports the contention that eNO may be considered a surrogate marker of airway inflammation in asthma.10 ,11

NO is a freely diffusible gas generated from l-arginine by NO synthases. These enzymes are found in many cells of the lung, including nerves, epithelial cells, alveolar macrophages, and other inflammatory cells. It has been suggested that the increased eNO levels found in asthmatic subjects result from an increase in the expression of inducible nitric oxide synthase 2 (NOS-2) in the respiratory tract induced by the action of proinflammatory cytokines.12Corticosteroids inhibit induction of NOS-212 ,13; however, eNO concentrations remain somewhat increased in asymptomatic patients with normal spirometric parameters, consistent with the presence of ongoing airway inflammation. Even though NOS-2 activity seems to be the principal cause of hypernitrosopnoea in asthmatic patients, there are additional determinants of eNO production such as airway acidification, S-nitrosothiol breakdown, and infections.10 Despite clear evidence of increased eNO levels in asthma, the function of NO as a toxic proinflammatory or protective agent is still unresolved.

How should eNO be measured in clinical practice? The technique for measuring eNO has now been standardised for cooperative subjects and the initial problems related to flow dependency and nasal contamination have been resolved. Recommended techniques have recently been published in an official statement of the American Thoracic Society14 and the utilisation of standardised methods is of utmost importance for allowing comparison between different laboratories. The recommended exhaled flow for online measurement is 50 ml/s in children and adults exhaling against a resistance to close the velum and avoid contamination from nasally derived NO.

Although bronchial eosinophilia is considered a hallmark of asthma, it is not necessarily specific to asthma and has also been found in biopsy specimens from atopic non-asthmatic subjects.15 There is mounting evidence that different inflammatory patterns may exist in asthmatic subjects.16In particular, the mechanisms associated with the development of severe corticosteroid dependent asthma seem to be heterogeneous. Wenzelet al 2 showed that severe asthma can be divided pathologically into two inflammatory groups based on the presence or absence of bronchial eosinophils. The type of inflammation appeared to be associated with different structural changes and physiological patterns. The eosinophil positive group had a thicker sub-basement membrane and had a much higher incidence of respiratory failure and mechanical ventilation than the eosinophil negative group. Even though the two groups shared very few inflammatory features, both had a persistent increase in neutrophils. There is accumulating evidence that neutrophils may play a role in severe refractory asthma.2 Neutrophil number and activation are increased in the airways of subjects with status asthmaticus and during exacerbations of asthma. The cause of neutrophilic inflammation has not been determined, but the high levels of interleukin (IL)-8 found in asthmatic patients may enhance neutrophil recruitment and activation.17 Since neutrophils are relatively steroid resistant, it was hypothesised that the neutrophilic inflammation may account for the poor response to corticosteroids seen in refractory asthma.

The finding of different patterns of airway inflammation could have a major impact on potential treatment options for difficult asthma. Assuming that uncontrolled inflammation may result in airway remodelling,18 every effort should be made to define the inflammatory phenotypes of asthma in more detail. Even though steroid resistant asthma is fortunately not common in children, a priority is to develop simple non-invasive tools that can reflect different types of inflammation. The study by Payne et al 1 suggests that different patterns of difficult asthma in children may be identified by measuring eNO concentrations. Of interest is the identification of two subgroups of children who had persistent symptoms after prednisolone treatment—those with raised eNO concentrations and those with normal concentrations of eNO before and after treatment. All patients were atopic and therefore the differences in eNO concentrations cannot be attributed to the influence of atopy.19 ,20 The authors speculate that raised eNO concentrations may reflect uncontrolled eosinophilic inflammation while low eNO concentrations may reflect either a non-eosinophilic inflammation or an absence of inflammation. Probably these latter patients need alternative treatments. In a recent studyinhaled NO was used as a life saving therapy in children with status asthmaticus.21 Whether inhaled NO therapy could be useful in patients with low eNO concentrations is not known.

Unfortunately Payne et al 1 do not report any data on bronchial histology from these children with severe asthma and cannot say whether NO is a reflection of a specific pattern of airway inflammation. In this regard, however, the same authors published preliminary data on bronchial biopsy specimens from children with severe asthma and, as in adult patients, found an absence of airway eosinophilia in a subgroup of children.22 The only distinguishing feature of these eosinophil negative patients was a lower level of eNO compared with patients with eosinophilic airway inflammation. In a recent article inThorax Çokuğraşet al reported a lack of eosinophilic inflammation in children with moderate asthma.23 More biopsy studies will be required in asthmatic children before the type of inflammation which occurs in childhood asthma can be characterised.

More than one asthma phenotype is now also apparent in young children with recurrent wheezing during preschool and early school years.24 In the first years of life there are asthma-like syndromes that are transient and early asthma that persists.25 Evidence suggests that measuring eosinophil infiltration in the airways may prove fruitful in distinguishing transient infantile wheezers from infants with early onset asthma. Studies based on BAL in young wheezing children26 ,27 have found different inflammatory patterns, with eosinophil mediated airway inflammation in those with atopy and persistent wheezing and neutrophil mediated inflammation in those with transient wheezing. Epidemiological studies have provided useful data and now we know that risk factors such as age, genetic background, and allergic sensitisation may be strong indicators for asthma risk.25 Unfortunately, these risk factors are difficult to apply to each single affected individual. For this and many other reasons it is universally recognised that the identification of asthma specific inflammatory markers would be very useful. In preliminary studies the measurement of eNO concentrations has been used successfully to distinguish infants with virus induced wheezing from those with early onset asthma,28 ,29 but further work is necessary to evaluate its predictive value in prospective population studies. To date there have been no studies of the relationship between bronchial histology/BAL profiles and eNO concentrations in wheezing infants.

Another intriguing phenomenon seen in different asthma phenotypes is the difference in eNO concentrations between atopic and non-atopic asthma. There is a subgroup of asthmatic patients who are not demonstratively atopic, the so-called “intrinsic variant” of the disease. However, this does not seem to be a distinct immunopathological entity.30 Bronchial biopsy studies have shown that both so-called intrinsic and extrinsic asthma are characterised by infiltration of eosinophils, high affinity IgE receptor bearing cells, and enhanced expression of Th2-type cytokines compared with controls. It has been suggested that, in intrinsic asthma, there might be local IgE production directed against unknown antigens, possibly of viral origin or even autoantigens,30whereas in extrinsic asthma the response is directed against environmental allergens. Recent studies have shown that adults and children who are atopic and asthmatic have higher eNO levels than non-atopic asthmatic subjects.19 ,20 ,31-33 Even if the relationship between atopy and airway inflammation remains unclear, a quantitative relationship between natural and laboratory exposure to allergens and eNO has been shown,8 ,34 with the highest levels of eNO being found in patients who were both sensitised and exposed to relevant allergens.35 Since the immunopathology of extrinsic and intrinsic asthma seems to be similar,30the atopic process itself may have an independent influence on eNO levels through genetic factors36 or mechanisms that are still unknown.

In the light of actual knowledge it can be concluded that the measurement of eNO may, by a non-invasive and rapid means, help in the understanding of the different underlying pathophysiological mechanisms of asthma phenotypes. Studies comparing direct measurement of airway inflammation (bronchial biopsy and BAL) and eNO concentrations are to be encouraged to address the issue of whether this exhaled biomarker could help the clinician to find an exit from the labyrinth of different patterns of childhood asthma.