INTRODUCTION

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Asthma is considered a chronic inflammatory disorder of the airways with a phlogistic phenotype characterized by activation of mast cells, macrophages, and by infiltration of eosinophils (1). The generation of cytokines from macrophages and other inflammatory cells is increasingly considered to be important in the amplification of the inflammatory response, and it is presumed that the worsening of airways inflammation could be responsible for asthma exacerbation (2). Bronchial inflammation and bronchial hyperresponsiveness are present also in patients with seasonal allergic asthma (3). There is good evidence that the striking increase of both clinical manifestations and bronchial hyperresponsiveness in allergic asthmatic subjects during the pollen season is the consequence of the worsening of the airway inflammation (3). Recently, Djukanovic and coworkers (4), performing bronchoalveolar lavage (BAL) and endobronchial biopsy before and during the grass pollen season, have demonstrated that allergen exposure induces an inflammatory response involving T cells, mast cells, and eosinophils. Because of the invasivity of these procedures no direct observations regarding the cellular environment of the lungs of pollen-sensitive asthmatic children are available. Unfortunately at the moment, we do not have simple means to monitor bronchial inflammation (1). There is therefore an increasing need of objective noninvasive markers to assess the underlying inflammation of the airway. Recent evidence suggests that the measurements of exhaled nitric oxide (NO) may represent a noninvasive approach for assessing the degree of ongoing airway inflammation (6). NO is an important mediator in numerous physiological processes in the respiratory system and it is involved in the pathophysiology of asthma (7, 10, 11). It is produced by many cells within the respiratory tract after stimulation by a variety of proinflammatory cytokines and can be easily detected in the exhaled air (7, 11).

The concentration of exhaled NO is increased in patients with airway inflammation such as asthma: this could be consistent with induction of inducible nitric oxide synthase (iNOS), an enzyme responsible for the synthesis of NO, in association with activation of mucosal mast cells, eosinophils, and T lymphocytes (7). This hypothesis is also supported by the increased expression of iNOS-like immunoreactivity observed in the airway epithelium of patients with asthma, suggesting an upregulated expression of iNOS in the lungs of asthmatics (10).

It is known that natural allergen exposure to grass pollen induces airways inflammation in asthmatic patients (4, 5) and recent insights suggest that measurement of exhaled NO may be a sensitive marker of disease activity (6). The aim of this study is therefore to evaluate the relationship between natural allergen exposure to grass pollen and the changes of exhaled NO in atopic asthmatic children in and out of the grass pollen season.

	METHODS

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Asthmatic Patients

Twenty-one Caucasian atopic children (16 males and 5 females, age 6 to 16 yr, mean 10.9 ± 3.1 yr) with a seasonal allergic asthma were studied (Table 1). They were recruited from the patients attending the Pediatric Pulmonology/Allergy outpatient clinic of the Department of Pediatrics of Padova in February and March 1996. All children had to have a history of atopic seasonal asthma with no or minimal asthma symptoms outside the pollen season and a positive skin prick test to grasses. There was no history of infections of the respiratory tract for at least 3 wk before the measurements and no subjects had received antibiotics in the previous week. None of the patients were smokers.

The diagnosis of asthma was based on clinical history and examination, pulmonary function parameters, and pulmonary function response to inhaled -adrenergic agents. All the subjects underwent skin prick testing by use of a panel of common inhalant allergens: mixed grass pollen, Parietaria, Artemisia vulgaris, Dermatophagoides pteronyssinus and D. farinae, Alternaria, and cat (Bayropharm; Bayer, Milano, Italy). The skin tests, done on the palmar surface of both forearms, were accepted as being positive if they resulted in a wheal reaction greater than 3 mm. All patients were shown to be highly positive to grass pollen, although 3 of 21 were also sensitive to D. pteronyssinus and D. farinae (Table 1). As therapy they used inhaled ₂-agonists occasionally, mainly during the pollen season and 8 of 21 patients used inhaled steroids (beclomethasone dipropionate [BCD] 400 to 500 µg/d) during the pollen season. Two of them used BCD over the entire period of the study and did not change the dose; the remaining six patients started inhaled BCD 5 to 6 wk before the in-season measurements and always used the same dose.

Healthy Controls

Twenty-one Caucasian healthy children, age- and sex-matched with asthmatics (16 males, 5 females, age 6 to 14 yr, mean 10.3 ± 2.5 yr) were recruited in two public schools of Padova (Table 1). In all healthy children there was neither history of significant airway disease and atopy nor upper respiratory infection in the previous 3 wk. They were not taking any medication and none was a smoker.

Protocol

All the patients underwent physical examination, spirometry, and measurements of exhaled NO on three occasions: before (March), during (May), and after (November) the grass pollen season. On each occasion, measurements were performed in the afternoon. In addition the subjects recorded their daily asthma symptoms (nocturnal wheeze, nocturnal cough, morning chest tightness, daytime wheeze, and subjective worsening of asthma due to exercise) on a scale 0 to 3 where 0 is no symptom, 1 mild, 2 moderate, 3 severe. Means were obtained for an average daily score. The patients recorded their symptoms during the week of the valuations performed in March and in November (out of the pollen season) and between mid-April and mid-June which is the period of grass pollination in our area (12). Before the pollen season the children were instructed to contact one of the investigators as soon as an increase in respiratory symptoms was noted.

The study was approved by the medical committee of our University Hospital and all patients gave informed consent.

Spirometry

Pulmonary function parameters (FEV₁, FVC, and FEF_25-75) were measured by means of a 10-L bell spirometer (Biomedin, Padova, Italy) and the best of three maneuvers was expressed as a percentage (%) of predicted reference values according to Polgar and Promadhat (13). Accuracy of the spirometer was regularly verified with a calibrated syringe.

Determination of Exhaled NO Levels

Exhaled NO was measured by a tidal breathing method with a chemiluminescence analyzer (CLD 700 Al-Med; Ecophysics, Durnten, Switzerland), modifying the original experimental setup proposed by Alving and colleagues (14). The analyzer is sensitive to NO concentrations from 1 to 1,000 parts per billion (ppb). Children were in a seated position wearing a nose clip and were asked to breathe at tidal volume through a mouthpiece directly connected by a Teflon tube to the analyzer via a two-way valve to avoid rebreathing. To exclude the effect of NO in ambient air (the ambient NO levels were always recorded), the children breathed NO-free air obtained by passing ambient air through a Purafill converter (Maihak Italia, Milan, Italy). NO-free air was accumulated in a 14-L collapsible reservoir maintained inflated to approximately 70 to 80%. NO levels were continuously analyzed by the chemiluminescence analyzer sampling at a constant flow of 0.7 L/ min. During mouth breathing NO reached a steady-state level after 1 to 2 min without further fluctuations. NO concentrations were recorded between the third and the sixth minute of mouth breathing. The subjects were asked to breathe without speaking or swallowing. The excess of exhaled air was scavenged through a tube with a final one-way valve to prevent contamination with ambient air. To keep the soft palate closed (15), the breathing circuit is provided with an internal restrictor that allows the exhalation with a low resistance providing a pressure of 3 to 4 cm H₂O at the mouthpiece. The range of expiratory flow was 180 to 230 ml/s. To prevent contamination, in-line filters were used. The accuracy of our method of measuring exhaled NO has been previously reported (9).

The chemiluminescence analyzer operates over a wide range of ambient temperatures (5-33° C) and humidity (0 to 95%) without an adverse effect on measurement accuracy. Before each study, the chemiluminescence analyzer was calibrated with a certified calibration mixture of NO in nitrogen (NO = 300 ppb, nitric oxide species [NO_X] = 308 ppb) (SIAD, Bergamo, Italy) with guaranteed stability. The mixtures are prepared with a gravimetric method using NO which has a purity > 99.9% certified both by wet analysis and gas chromatographic analysis. The mixtures are contained in aluminium cylinders whose internal surfaces are treated to reduce porosity and improve stability.

Pollen Count

The atmospheric pollen counts were done throughout the year by a volumetric spore trap (VPPS 2000; Lanzoni, Bologna, Italy) and the daily mean concentration was recorded and expressed as pollen grains per m³ of air per 24 h (12). In this study-area the grass pollen season is rather short and well-defined with a peaking trend in May. The decision about the timing of measurements of exhaled NO and spirometry during the season was based on the pollen count being more than 50/m³.

Statistical Analysis

All mean exhaled NO concentrations are reported in ppb. Results (NO, FEV₁, FEF_25-75) are expressed as means ± standard deviation (SD) of the mean. For the comparison of NO measurements between children with asthma and healthy children, a t test was used. Analysis of variance (ANOVA) was used for comparison of exhaled NO and spirometric values before, during, and after the pollen season. A p value lower than 0.05 was considered as significant.

	RESULTS

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Exhaled NO Determinations

The mean value of exhaled NO (steady-state levels) before the grass pollen season (March) was 12.7 ± 5.1 ppb and was significantly higher when compared with that of healthy control subjects (7.8 ± 2.7 ppb; p < 0.001). In the pollen season (May) there was a significant (p < 0.001) twofold increase in exhaled NO (21.4 ± 7.6 ppb) with respect to preseason baseline values. After the season (November) exhaled NO returned to preseasonal values (12.8 ± 5.8 ppb). The time courses of individual values of exhaled NO are shown in Figure 1. No significant relationship was found between the changes in exhaled NO and the changes in symptom scores (r = 0.22, p = NS).

View larger version (18K): [in this window] [in a new window]   Figure 1.   Time courses of individual values of exhaled NO in asthmatic children with grass pollen allergy. During the pollen season there was a significant (p < 0.001) increase in exhaled NO with respect to preseason baseline values. After the season exhaled NO returned to preseasonal values. Baseline values of exhaled NO of asthmatics were significantly higher (p < 0.001) than values of healthy control subjects.

Compared with children who were not treated with inhaled steroids (n = 13), children taking inhaled steroids (n = 8) presented a lower percentage increase in exhaled NO during the grass pollen season (+62% versus +98%, compared with preseasonal values) but the difference between the two groups was not significant (p = 0.3).

Pollen Count and Clinical Data

The pollen count was relatively low compared with previous seasons, with a peak of 73.5 grains/m³ counted on May 6. Before the first examination, out of season, all the subjects were asymptomatic. During the season, probably because the pollen counts were low, only 57% of the patients (12 of 21) experienced mild asthma when pollen started to appear in the air. A decline in asthma symptoms was then seen when the pollen count decreased. Symptoms score during the pollen season (mean 0.75 ± 0.71) was greater (p < 0.01) than out of season (0.15 ± 0.36 before and 0.16 ± 0.40 after pollen season, respectively).

Spirometry

The mean (± SD) baseline value of FEV₁ and FEF_25-75 was respectively 98.6 ± 9% and 107 ± 21.9% predicted before the pollen season and there were no significant changes in these parameters during and after the pollen season. The time courses of individual values of FEV₁ are shown in Figure 2. No discernible relationship (p = NS) was found between the values of exhaled NO and FEV₁ in each of the three sessions of measurements. The percentage change of FEV₁, comparing the pre- and in-season measurements was similar in children treated and not treated with inhaled steroids (+4.7% versus +2.9%, p = 0.5).

View larger version (21K): [in this window] [in a new window]   Figure 2.   Time courses of individual values of FEV1 (percentage of predicted) in asthmatic children with grass pollen allergy over the period of the study. There were no significant changes in FEV1 before, during, and after the pollen season (p = NS).

	DISCUSSION

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In the present study the primary objective was to investigate the time course of exhaled NO out of and during the grass pollen season in asthmatic children sensitized to these airborne allergens. We found that natural exposure to pollen led to an increase in exhaled NO concentrations during the season that returned to preseasonal values thereafter. In addition we found that, even out of the pollen season during symptom-free intervals, the values of exhaled NO of asthmatics were higher than those of healthy control subjects. To our knowledge no other data are yet available on the effect of exposure to pollen on exhaled NO in asthmatic patients. Probably because of the low pollen load in the year of the study, with respect to previous years, the increase in exhaled NO during the season was not accompanied by any change in standard measures of airflow limitation (i.e., FEV₁ and FEF_25-75) although a slight increase in asthma symptoms was noted in 57% of patients. This suggests that measurement of exhaled NO may be the earliest change reflecting loss of disease control before deterioration of pulmonary function. Similar findings have been reported in asthmatics treated with decreasing doses of inhaled glucocorticoids in which a progressive increase in exhaled NO was noted without significant changes in FEV₁ (8).

The measurement of exhaled NO in response to an allergen challenge has been recently evaluated by Kharitonov and coworkers (16). They reported that the late asthmatic response is associated with elevated exhaled NO concentrations whereas no change of NO was noted in subjects who showed an isolated early response. These findings suggest that the increased NO associated with the late inflammatory response may be a reflection of iNOS expression in the airways in response to inflammatory cytokines. In vitro, in fact, proinflammatory cytokines increase the expression of iNOS in cultured human airways epithelial cells (17) and this probably explains the raised values of exhaled NO we have found during the grass pollen season.

Grass pollen-induced asthma is an interesting model of allergic inflammation and a series of studies have demonstrated that increases of both airway inflammation and nonspecific airway responsiveness occur during the pollen season (4, 5, 18). Recently grass pollen-sensitive asthmatic subjects underwent BAL and bronchial biopsies before and during the pollen season (4). The investigators (4) showed that natural allergen exposure to grass pollen was associated with an inflammatory response involving T cells, mast cells, and eosinophils.

At present, there are no reliable noninvasive markers to assess underlying airway inflammation and, until now, the classic approach has involved the invasive technique of BAL and/ or bronchial biopsy (1). Recent evidence suggests that measurement of NO in exhaled air may give information on the degree of airway inflammation in asthmatic patients (6, 14, 22, 23). Studies in a large number of patients have shown that exhaled NO concentrations are increased in chronic asthmatics and decreased during effective anti-inflammatory treatment (11, 22, 24). Furthermore airway epithelial cells taken from patients with asthma and other forms of airway inflammation immunostain strongly for iNOS, the inducibile isoform of NO synthase (10). Raised values of NO have been also described during exacerbation of asthma with a rapid reduction after systemic glucocorticoids treatment (9, 24). This is probably a result of the effect of the corticosteroids on the iNOS gene, leading to a block of the increased expression of the enzyme and a reduction of exhaled NO (7). A dose dependency of exhaled NO on inhaled steroids has been reported in stable asthmatic children (22) and raised concentrations of NO have been described even when airflow limitation has resolved (24), suggesting that inflammation may persist longer.

In our study we unexpectedly found that, also out of the grass pollen season, asthmatic children had a higher release of exhaled NO than healthy control subjects. This finding is consistent with the hypothesis that asthmatic subjects may present mild airway inflammation even in the absence of symptoms and overt airflow limitation. For asthmatic adults who underwent BAL studies it is now recognized that airway inflammation is present in virtually all patients with asthma (1, 25, 26). It remains to be seen whether these observations are also true for children, in whom the bronchoscopic studies are limited because the procedures are invasive. However lung-biopsy specimens from two children with asthma in remission had pathological findings similar to the findings in two children who died of status asthmaticus (27).

In conclusion, this study shows that natural allergen exposure during the grass season is related to an increase of exhaled NO in pollen-allergic asthmatic children even in the absence of significant changes in airway function. This increase in exhaled NO may be the earliest change reflecting loss of disease control, suggesting that the measurement of this expired gas could be used as a sensitive noninvasive marker for monitoring disease activity in children with asthma.