Abstract
Cysteinyl leukotrienes (cys-LTs; LTC4, LTD4, and LTE4) are generated predominantly by mast cells and eosinophils and induce airway smooth muscle contraction, microvascular leakage, and mucous hypersecretion whereas leukotriene B4 (LTB4) is a potent chemoattractant of neutrophils. We measured cys-LTs and LTB4 in exhaled breath condensate from children aged 7–14 years including healthy nonatopic children (n = 11) and children with mild intermittent asthma (steroid naive, n = 11), mild persistent asthma (low-dose inhaled steroid treatment, n = 13), or moderate to severe persistent asthma (high-dose inhaled steroid treatment, n = 13). Exhaled LTB4 levels were increased in patients with mild and moderate to severe persistent asthma compared with patients with mild intermittent asthma (126.0 ± 8.8 and 131.9 ± 7.1 versus 52.7 ± 3.8 pg/ml, p < 0.001 and p < 0.0001) and normal subjects (126.0 ± 8.8 and 131.9 ± 7.1 versus 47.9 ± 4.1 pg/ml, p < 0.0001). Elevated exhaled cys-LT levels were found in patients with mild and moderate to severe persistent asthma compared with normal subjects (27.9 ± 2.8 and 31.5 ± 4.5 versus 18.5 ± 0.5 pg/ml, p < 0.01 and p < 0.05). There was an inverse correlation between exhaled cys-LTs and LTB4 in patients with mild persistent asthma. We conclude that exhaled cys-LTs and LTB4 may be noninvasive markers of airway inflammation in pediatric asthma.
Airway inflammation is a characteristic of asthma but its role in childhood asthma is still not well defined, despite modern treatment approaches recommending potent antiinflammatory therapy for an increasing number of children. Investigation of the mechanisms and features of airway inflammation has helped to define the pathogenesis of asthma in adults and there is a need to extend this research into childhood asthma. Our lack of knowledge about the pathogenesis of pediatric asthma is largely because invasive methods of monitoring airway inflammation, such as flexible bronchoscopy and bronchoalveolar lavage (BAL), can be performed in children only when there is a clear indication to do so, and peripheral measurements of inflammation from blood or urine samples provide only indirect information about the airways. Noninvasive methods such as measurement of exhaled nitric oxide (NO), induced sputum, or exhaled breath condensate are better suited to investigating airway inflammation in childhood asthma (1, 2).
The inflammation in asthmatic airways is characterized by mucosal edema, shedding of the epithelium, fibrosis beneath the basement membrane, and infiltration by eosinophils and T lymphocytes and increased number of mast cells (3, 4), but there is increasing evidence that neutrophils may play a role in more severe asthma (5).
Increased numbers of eosinophils and mast cells have been found in bronchial biopsies (6), bronchoalveolar lavage (7), and sputum (8) of adults with current asthma, compared with normal subjects. Increased eosinophil numbers in induced sputum of asthmatic children are linked to current asthma symptoms and airway hyper-responsiveness (9) and eosinophilic airway inflammation may persist even in those children who were receiving inhaled corticosteroids, with normal lung function and good symptom control (10).
Neutrophil numbers and activation are increased in the airways of adult subjects with more severe asthma and during exacerbation of the disease (5, 11). Airway inflammation is present during acute exacerbations of childhood asthma and is characterized by infiltration and activation of both eosinophils and neutrophils (12). A significant increase in the number of neutrophils is also reported in BAL fluid from wheezing infants (13).
The cysteinyl leukotrienes (cys-LTs: LTC4, LTD4, and LTE4) are generated predominantly by mast cells and eosinophils and potently induce airway smooth muscle contraction (14), increase vascular permeability (15) and mucous hypersecretion, and alter mucociliary clearance (16). Furthermore, cys-LTs may recruit eosinophils into the lung, which in turn leads to the production of additional cys-LTs as well as other inflammatory mediators. Inhalation of LTE4 increases the number of eosinophils and neutrophils in airway mucosa (17) and in induced sputum (18). Cys-LTs receptor antagonists have clinical efficacy in the treatment of childhood asthma (19, 20), confirming the importance of cys-LTs in pediatric asthma.
Neutrophils release leukotriene B4 (LTB4) in response to various activating stimuli, and also have a high density of cell surface LTB4 receptors. LTB4 is a potent chemoattractant and activator of neutrophils, without any significant effect on airway muscle (21). LTB4 has no significant effect on airway function when given by inhalation to patients with asthma (22), but increases the number of neutrophils in BAL fluid (23). Furthermore, an LTB4 receptor antagonist reduces allergen-induced neutrophilia in BAL fluid in patients with asthma (24). Although LTB4 also increases microvascular permeability (25), the plasma leakage is slow in onset and a consequence of leukocyte activation and/or diapedesis (26). LTB4 may be important as a neutrophil chemoattractant in asthma as increased levels are found in BAL fluid from adult patients with asthma (27) and in children with acute asthma exacerbation and up to 1 month after recovery (28).
The aim of the study was to examine whether LTB4 and cys-LTs are detectable in exhaled breath condensates in children with asthma and to investigate the effect of steroid treatment on these mediators.
Four groups of children aged 7–14 years were studied: 11 healthy nonatopic children, 11 patients with mild intermittent asthma, 13 with mild persistent asthma, and 13 with moderate to severe persistent asthma (Table 1)
Subjects with Asthma | ||||||
|---|---|---|---|---|---|---|
| Control Subjects | Mild
Intermittent Asthma | Mild
Persistent Asthma | Moderate to Severe
Persistent Asthma | |||
| Number | 11 | 11 | 13 | 13 | ||
| Age, yr | 11.7 ± 0.4* | 11.2 ± 0.6 | 11.7 ± 0.6 | 11.5 ± 0.5 | ||
| Sex, M/F | 5/6 | 5/6 | 5/8 | 10/3 | ||
| FEV1, % predicted | 100 ± 5.1 | 96 ± 4.5 | 84 ± 5.7 | 73 ±4.4† | ||
| Atopy | 0 | 13 | 9 | 13 | ||
| Therapy | ||||||
| Inhaled steroids | ||||||
| < 400 μg/d | 0 | 0 | 13 | 0 | ||
| > 400 μg/d | 0 | 0 | 0 | 13 | ||
| ⩾ 1,000 μg/d | 0 | 0 | 0 | 7 | ||
Healthy control subjects were recruited from siblings of children attending the Pediatric Asthma Clinic or from children of staff. A parent was interviewed to confirm the lack of past or present lower respiratory disease. Children with nonasthmatic atopy were excluded.
The protocol was approved by the Ethics Committee of the Royal Brompton Hospital, and informed consent was obtained from all parents and children recruited into the study.
Subject details were obtained and then baseline spirometry and exhaled NO were measured, followed by collection of exhaled breath condensate.
FVC FEV1 were measured with a dry spirometer (Vitalograph, Buckingham, UK) and the best value of three maneuvers was expressed as a percentage of the predicted value.
Fractional exhaled NO was measured according to the 1999 American Thoracic Society Guidelines (31), using an NO analyzer (NIOX; Aerocrine, Stockholm, Sweden). Any exhalation that did not meet these requirements was discarded and the subject was asked to perform a new exhalation maneuver. At each session three correctly executed exhalations were recorded. It was calibrated with certified NO mixtures (200 ppb) in nitrogen (AGA Gas BV, Amsterdam, The Netherlands).
Exhaled breath condensate was collected by using a condenser, which allowed the noninvasive collection of nongaseous components of the expiratory air (EcoScreen; Jaeger, Würzburg, Germany). After rinsing their mouths, subjects breathed through a mouthpiece and a two-way nonrebreathing valve, which also served as a saliva trap. They were asked to breathe at a normal frequency and tidal volume, wearing a noseclip, for a period of 8 minutes. The condensate, at least 500 μl, was collected as ice at –20°C and immediately stored at –70°C.
Cys-LTs (LTC4, LTD4, and LTE4) and LTB4 concentrations were also measured with a specific enzyme immunoassay (Cayman Chemical, Ann Arbor, MI). The lower limit of detection for these assays was 15.0 pg/ml for cys-LTs and 4.4 pg/ml for LTB4.
Data were expressed as means ± SEM. One-way analysis of variance (Kruskal–Wallis) with the Mann–Whitney test for multiple comparisons was used to compare groups. The correlation between cys-LT and LTB4 levels, LT levels, and exhaled NO, as well as between LT levels and lung function (FEV1), was determined by nonparametric Spearman correlation analysis. Significance was defined as a value of p < 0.05.
Cys-LT concentrations were detectable in exhaled breath condensate of normal subjects (18.5 ± 0.5 pg/ml) and were increased significantly in children with mild persistent and moderate to severe persistent asthma (27.9 ± 2.8 and 31.5 ± 4.5 pg/ml, p < 0.01 and p < 0.05, respectively) (Figure 1)
Figure 1. Concentrations of cysteinyl leukotrienes (cys-LTs) in exhaled breath condensate from normal subjects and patients with mild intermittent, mild persistent, and moderate to severe persistent asthma. Individual data and means are shown. *p < 0.05; **p = 0.01; ***p < 0.01 compared with normal subjects.
[More] [Minimize]LTB4 was increased significantly in exhaled breath condensate collected from children with mild persistent and moderate to severe persistent asthma compared with children with mild intermittent asthma (126.0 ± 8.8 and 131.9 ± 7.1 pg/ml, respectively, versus 52.7 ± 3.8 pg/ml, p < 0.001 and p < 0.0001) and control subjects (126.0 ± 8.8 and 131.9 ± 7.1 pg/ml, respectively, versus 47.9 ± 4.1 pg/ml, p < 0.0001) (Figure 2)
Figure 2. Concentrations of leukotriene B4 (LTB4) in exhaled breath condensate from normal subjects and patients with mild intermittent, mild persistent, and moderate to severe persistent asthma. Individual data and means are shown. **p < 0.001; ***p < 0.0001 compared with normal subjects.
[More] [Minimize]In patients with mild persistent asthma, there was an inverse correlation between levels of cys-LTs and LTB4 in exhaled condensate (r = −0.76, p < 0.01) (Figure 3)
Figure 3. Correlations between cysteinyl leukotrienes (cys-LTs) and leukotriene B4 (LTB4) levels in exhaled breath condensate from patients with mild persistent asthma.
[More] [Minimize]In this study we investigated whether leukotrienes in exhaled breath condensate were increased in children with asthma. Our results demonstrate for the first time that cys-LTs and LTB4 in exhaled breath condensate are significantly increased in children with mild and moderate to severe persistent asthma.
The immunopathology of allergic asthma is complex and involves a range of cell types and mediators. Nevertheless, it is well recognized that eosinophils are prominent cells in adult and childhood asthma compared with nonasthmatic subjects. The role of cys-LTs is well established in induced bronchoconstriction in pediatric asthma (19), but our information about their participation in chronic inflammation in asthmatic airways is based mainly on indirect evidence from the efficiency of leukotriene receptor antagonists in asthma treatment (20).
Our findings demonstrate that cys-LTs may be involved in chronic airway inflammation of children with mild and moderate to severe persistent asthma, despite treatment with low or high doses of inhaled corticosteroids, which is consistent with previous data concerning adults with asthma (32, 33).
Although corticosteroids have been shown to increase eosinophil apoptosis in vivo (34) and there is evidence that steroid treatment reduces eosinophil number in induced sputum from children with asthma (35), airway eosinophilia may persist in children whose asthma is controlled by high-dose inhaled corticosteroid therapy, albeit at a lower level than in pediatric patients with symptomatic asthma (10). Furthermore, cys-LT levels in exhaled breath condensate may be related to more than the eosinophil number in the airways, as activated mast cells may be a major source of these mediators. Laviolette and coworkers demonstrated that blood eosinophils from patients with moderate asthma have an increased capacity to release LTC4 in response to in vitro stimuli compared with cells from subjects with mild or severe asthma. This is probably due to the different severity of disease and the higher dose of steroid treatment given to patients with severe asthma, causing a reduction in LTC4 production by blood eosinophils compared with cells from patients with moderate asthma (36). Airway inflammatory cells appear to be less sensitive to corticosteroids than blood cells (37) and thus airway cells from subjects with severe asthma may have a greater capacity to release cys-LTs, explaining our finding of elevated cys-LT levels in exhaled condensate of children treated with high doses of steroid.
On the other hand, the elevated cys-LT levels in exhaled breath condensate from steroid-treated children may indicate that the dose of inhaled corticosteroids used to treat the children in our study was sufficient to control clinical symptoms but not sufficient to completely suppress the inflammatory processes. Exhaled NO, which is a sensitive marker of airway inflammation in adult and pediatric asthma (38, 39), also reflects the ongoing airway inflammation in each of our asthmatic groups, with higher levels in patients with steroid-treated, mild and moderate to severe persistent asthma, in spite of the fact that corticosteroids decrease exhaled NO levels in both adult (38) and childhood asthma (39).
Nevertheless, lung function measurements of our patients did not show severe airway obstruction, even in the children with moderate to severe persistent asthma, which supports the view that exhaled NO may be a more sensitive marker of asthma control.
Although eosinophilic airway inflammation has been reported to correlate with exhaled NO in childhood asthma (40), we did not find any correlation between cys-LT levels in exhaled breath condensate and exhaled NO levels in any of the asthmatic groups. These results suggest that NO and cys-LTs are at least partly independent mechanisms of airway inflammation, despite the fact that cys-LTs have been reported to activate NO release from human polymorphonuclear cells in vitro (41), although this effect has not been demonstrated in vivo (42).
LTC4 and LTD4 may directly increase eosinophil survival (43), and thus increased eosinophil inflammation and greater cys-LT release in the airways of patients with more severe disease may lead to prolonged eosinophil survival, resulting in yet further cys-LT production and eosinophil recruitment. Cys-LT levels in exhaled condensate from currently asymptomatic children with mild intermittent asthma were not elevated, suggesting their role is greater in more severe disease.
We also demonstrated that LTB4 levels were elevated in exhaled breath condensate from children with mild and moderate to severe persistent asthma with regular steroid treatment, but not in children with mild intermittent asthma. These findings suggest that LTB4 may play a role in the pathogenesis of more severe pediatric asthma or that the elevated LTB4 levels in exhaled breath condensate may, at least in part, be the consequence of corticosteroid treatment, because corticosteroids increase neutrophil survival by reducing apoptosis. Although LTB4 has not been closely linked to asthma, in contrast to cys-LTs, there is increasing evidence that neutrophils may play a role in the pathogenesis of both adult and childhood asthma, during either acute exacerbations or in more severe disease. LTB4 is a potent neutrophil chemoattractant factor and may attract neutrophils into the airways (44). LTB4 has a role in mediating neutrophil survival in vitro by inhibiting neutrophil apoptosis, and may also be involved in corticosteroid-induced neutrophil survival (45).
Previous studies have reported that neutrophils from patients with asthma are in an activated state, as demonstrated by increased complement receptor expression (46) and increased activity of the 5-lipoxygenase pathway (47). Activation of neutrophils in asthma may result from their failure to appropriately terminate their responses when exposed to negative feedback signals (48), which might lead to further production of LTB4 resulting in prolonged neutrophil survival and further neutrophil recruitment.
Steroids have been shown to prolong neutrophil survival in vitro by reducing apoptosis (49), and therefore corticosteroid therapy may enhance neutrophilic inflammation in vivo. Furthermore, there is evidence that corticosteroids inhibit calcium ionophore-induced synthesis of cyclo-oxygenase products to a greater extent than of leukotrienes in stimulated cultured alveolar macrophages in vitro (50).
Taken together, chronic inhaled corticosteroid treatment might prolong neutrophil survival without evident inhibition of leukotriene synthesis, leading to further release of proinflammatory mediators that may contribute to recruitment of inflammatory cells and maintenance of chronic airway inflammation in asthma. The potential influence of steroids on the pattern of airway inflammation in childhood asthma is supported by the finding that neutrophil predominance during acute asthma exacerbations is greater with the use of inhaled corticosteroids, and even higher with oral steroid use (51).
We did not find elevated LTB4 levels in exhaled breath condensate collected from subjects with mild intermittent asthma, suggesting a role in airway inflammation in more severe, steroid-treated asthma than in mild asymptomatic disease. However, in previous studies elevated LTB4 levels were found in BAL fluid from atopic subjects with mild asthma who were symptomatic (27). Plasma LTB4 levels are also higher in steroid-naive children with asthma during acute asthma exacerbations and even 1 month after recovery (10). Our different results might be explained with the fact that patients in our study with mild intermittent asthma were currently asymptomatic and had not suffered any recent acute exacerbations.
There was no positive correlation between LTB4 and cys-LTs in any asthmatic group in our study, despite the reported effects of LTB4 on eosinophil survival and chemotaxis (44, 17) and previous findings that eosinophil and neutrophil numbers correlate in BAL fluid from steroid-naive subjects with asthma (52). Moreover, there was an inverse correlation between LTB4 and cys-LT levels in exhaled breath condensates in the mild persistent asthmatic study group. This result may reflect the contrary effect of steroids on eosinophil and neutrophil survival (34, 49).
In summary, we have provided evidence of increased formation of cys-LTs and LTB4 in the airways of children with mild and moderate to severe persistent asthma who were treated with inhaled steroids, compared with normal control subjects, which supports the involvement of cys-LTs and LTB4 in chronic airway inflammation. A 5-lipoxygenase inhibitor might, therefore, be more effective than LT receptor antagonists, as it inhibits synthesis of both LTB4 and cys-LTs. This study shows that exhaled breath condensate may be useful in assessing inflammation in the airways of children as young as 6–7 years.
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