Exercise-induced bronchoconstriction (EIB) is characterized by a transient narrowing of the airways during or shortly after strenuous exercise. EIB is highly prevalent both in asthmatic patients (10) and in otherwise healthy elite athletes (12). The key triggering factor is believed to be dehydration of the airways as a result of warming and humidification of large volumes of inadequately hydrated air (1). This leads to an increase in airway osmolality (18) with the release of mediators, such as histamine and the eicosanoids (leukotrienes and prostaglandins), and subsequent contraction of the bronchial smooth muscle (1). The cascade of events leading to this release of bronchoactive agents and bronchoconstriction remains unclear.
Dehydration injury of the small airways is believed to be an essential component of the pathology of EIB (2,3). Airway epithelial injury has been reported at rest (21) and following exercise (20) in asthmatic patients with EIB. The mechanism underlying EIB is, however, thought to be different in athletes and asthmatic patients (1-3), and contradictory results have recently been obtained regarding the occurrence of bronchial epithelial damage in athletes. Although signs of injury to the airway epithelium have been observed at rest in swimmers (8) and after half-marathon races in nonasthmatic runners (15), no such evidence was obtained at rest in cold air athletes (8) or after a marathon in nonasthmatic runners (7). Moreover, in swimmers and cold weather athletes, no association was found between baseline epithelial cell count in sputum and airway hyperresponsiveness (8). It therefore remains to be determined whether airway epithelium contributes to EIB in athletes.
CC16 is a short-lived plasma protein that is secreted mainly within the terminal bronchioles by the nonciliated Clara cells (36). From there, it is believed to passively diffuse into the serum and then is rapidly cleared by glomerular filtration (22). The biological function of CC16 remains incompletely understood, but evidence suggests that it is involved in the inflammatory cascade (13,23,27).
Short-term changes of the concentration of CC16 in extrapulmonary fluids are known to occur in conditions where the airway epithelium integrity is compromised. In rats, a marked increase in serum CC16 has been found after acute lung injury and inflammatory changes (6). In healthy cyclists, a postexercise increase of serum CC16 was observed after exposure to ozone, a lung irritant that is well known to increase the epithelial permeability (9). These findings support the idea of a leakage of the small protein into the bloodstream during acute episodes of epithelial injury. CC16 has therefore been proposed as a novel noninvasive marker to evaluate the integrity of the bronchoalveolar-blood barrier (22).
The aim of the study was to establish whether transient disruption of the airway epithelial barrier occurs after eucapnic voluntary hyperpnea (EVH)-a surrogate for exercise-in athletes with EIB. The primary hypothesis was that a larger increase in urinary CC16 would be noticed after EVH in athletes and asthmatic patients with EIB compared with healthy trained and untrained controls. Fitness level of the individuals has been proposed as a confounder in the CC16 response to exercise (30), possibly because of an adaptation to repeated airway epithelial stress with training. Therefore, our secondary hypothesis was that the increase in CC16 would be less pronounced in trained than in untrained individuals.
The study population consisted of 50 female individuals aged 18-50 yr, nonsmokers, and with no history of respiratory infection in the 4-wk period preceding the challenge. All were free of any chronic medical condition apart from asthma or EIB. Twenty-eight subjects were summer sports athletes (runners n = 16, ball sports players n = 3, gym attendees n = 2, mountain bikers n = 2, swimmers n = 2, triathlete n = 1, kayaker n = 1, and highland dancer n = 1) with an aerobic training volume of 9 ± 1 h·wk−1 for 11 ± 1 yr; the remaining 22 subjects were untrained. Baseline forced expiratory volume in the first second (FEV1) was ≥80% of predicted (33) in all subjects and the FEV1-over-forced vital capacity (FVC) ratio was ≥70%. Fifteen subjects had a previous medical diagnosis of intermittent or mild, persistent asthma (19). Of these, six were prescribed inhaled short-acting β2-agonists alone, four short- and long-acting β2-agonists, four inhaled corticosteroids plus short-acting β2-agonists, and one ipratropium bromide.
The study was approved by the Grampian Local Research Ethics Committee (no. 07/S0802/5). Written informed consent was obtained from all participants after the purpose and nature of the study and the risk of all procedures were explained.
Subjects were asked to attend between 8:00 and 9:30 a.m. and on days 19-23 of the menstrual cycle when possible (37). They were required to abstain from caffeine and alcohol from 8:00 p.m. on the evening before and from vigorous exercise on the study day. Short-acting β2-agonists, inhaled corticosteroids, and ipratropium bromide were withheld for 8 h, and long-acting β2-agonists and antihistamines were withheld for 48 h before the study day.
Three forced expiratory maneuvers were performed on arrival, after 10 min of rest, and again at 60 min. Urine samples were collected on arrival and at 60 min. The sample at 60 min was defined as the baseline sample. Subjects were asked to drink 250 mL of water approximately 1 h before arrival and after each urine sample.
An EVH challenge was then performed using the Eucapsys system (SMTEC SA, Nyon, Switzerland). Subjects were required to breathe a compressed dry gas mixture of approximately 5% CO2, 20% O2, and balance N2 at a target ventilation rate of 30 times the baseline FEV1 (i.e., 85% of predicted maximal voluntary ventilation, MVV) for 8 min. Postchallenge procedures were forced expiratory maneuvers at 2, 5, 10, 15, 20, 30, 60, and 90 min and collection of urine samples at 30, 60, and 90 min. To check the atopic status of the subjects, a skin prick test was performed at 70 min after challenge. Urine samples were stored without the addition of preservatives at −60°C in the United Kingdom and shipped to Sweden on dry ice for analysis within 2 months (4).
Study groups were constituted a posteriori according to the response of each individual to the EVH challenge. A positive response to the test was defined as a fall in FEV1 from baseline ≥10% sustained for at least two consecutive recording time points.
Clara cell protein.
The urine samples were analyzed for Clara Cell protein (CC16) using the Human Clara Cell Protein ELISA kit from BioVendor (Modrice, Czech Republic) according to the manufacturer's protocol. The detection limit for CC16 was 20 pg·mL−1. All urine samples were analyzed for creatinine, and the results expressed as nanograms of excreted mediator per micromole of creatinine. Because creatinine values in the urine sample collected on arrival suggested a high variability in hydration status of the individuals, the second resting sample collected an hour after arrival and after standardized water ingestion was defined as the baseline.
Lung function measurements.
Forced expiratory maneuvers were carried out in accordance with ERS/ATS guidelines (29) on the CPX-Ultima (MedGraphics, St. Paul, MN). FEV1 and FVC were measured in triplicate at baseline and in duplicate after the EVH challenge. At each time point, the best FEV1 and best FVC readings were kept for analysis. Mean forced expiratory flow between 25% and 75% of the FVC (FEF25-75) and peak expiratory flow (PEF) values were taken from the blow with the largest sum of FEV1 and FVC.
Skin prick tests were carried out using standardized allergen extract (ALK, Abello, Hungerford, UK) of house dust mite, timothy grass, and cat hair, together with a positive and negative control. A reaction with a wheal of ≥3 mm in diameter was considered a positive test.
The maximum fall in FEV1 was the lowest FEV1 recorded after EVH expressed as a percentage of the baseline value recorded immediately before EVH. The area-under-the-FEV1-time curve (FEV1-AUC1-90) was calculated from the percentage change from baseline FEV1 during the 90-min observation period by using the trapezoidal method.
All data were checked for normality using a Shapiro-Wilk test. Subjects' characteristics and minute ventilation (expressed as absolute value and percent of predicted MVV) were analyzed using two-way between-subjects ANOVA (with "athletic status" and "EVH response" as fixed factors). When appropriate, pairwise comparisons were carried out post hoc using the LSD test. FEV1-AUC1-90 data were not normally distributed and compared between the four a posteriori groups using the Kruskal-Wallis test.
CC16 data did not follow a Gaussian distribution and were compared between groups and times using nonparametric tests. Between-group differences were determined using the Mann-Whitney test (for EVH− vs EVH+ and trained vs untrained comparisons) and the Kruskal-Wallis test (for the four groups comparison). Within-group differences were analyzed using the Wilcoxon test (for baseline vs peak) or the Friedman test (for baseline vs the three recovery time points).
A two-way χ2 analysis was used to compare the distribution of atopy among the different groups. When >20% of cells had an expected frequency of less than five, results were reported as a Fisher exact test.
A 0.05 level of significance was adopted for all tests. The statistical calculations were performed using the computer software SPSS 17.0 for Windows (SPSS, Inc., Chicago, IL).
Response to EVH challenge.
EVH challenge was positive in 19 subjects (38%). Of these, 10 (36%) were athletes and 9 (41%) were untrained subjects. The minute ventilation in absolute value and relative to MVV was not significantly different between EVH-positive and EVH-negative subjects and was not dependent on the training status of the study participants (Table 1). The mean ± SE fall in FEV1 in positive and negative subjects was 23.4% ± 2.6% and 5.9% ± 0.6%, respectively (Table 1). The percent decline in FEV1 after EVH challenge in the four groups is shown in Figure 1. FEV1-AUC1-90 was significantly larger in the EVH+ groups compared with the EVH− groups (P < 0.001; Table 1).
Three subjects (one untrained EVH+ and two untrained EVH−) had urinary CC16 levels below the detection limit and were excluded from the statistical analysis. Baseline values were not significantly different between the four study groups. After the EVH challenge, all subjects, with the exception of one EVH− athlete, showed an increase in urinary excretion of CC16. Peak postchallenge CC16 values were significantly increased from baseline in EVH-positive and EVH-negative subjects (P < 0.001) with no group difference (Fig. 2). Baseline, peak post-EVH, and ΔCC16 values were not significantly different between the four study groups (Table 1).
FIGURE 2-Urinary con...Image Tools
Urinary CC16 values rose immediately after the challenge in all four groups. However, the kinetic of recovery was different between athletes and untrained individuals. At 90 min of recovery, CC16 concentrations were back to baseline in the two athletic groups (EVH−, P = 0.170; EVH+, P = 0.114) but remained above baseline in the two untrained groups (EVH−, P = 0.008; EVH+, P = 0.036; Fig. 3).
FIGURE 3-Urinary con...Image Tools
Athletes were significantly older than untrained subjects (31.1 ± 1.7 yr for the two athletic groups vs 23.3 ± 1.4 yr for the two untrained groups, P = 0.003). The four groups were matched for height and weight (athletes EVH+ = 165 ± 2 cm and 59 ± 2 kg, athletes EVH− = 167 ± 2 cm and 59 ± 2 kg, untrained EVH+ = 161 ± 2 cm and 64 ± 4 kg, untrained EVH− = 165 ± 2 cm and 63 ± 3 kg).
Sensitization to timothy grass was more frequent in EVH+ compared with EVH− individuals: 42% (8/19) versus 10% (3/31), respectively (P = 0.014). No other allergen tested was associated with athletic status or EVH response.
Baseline lung function.
Lung function was normal in EVH-positive and EVH-negative subjects, irrespective of training status. Of the lung function parameters, only FEV1/FVC ratio, FEF25-75 and FEF25-75 (% predicted) were significantly lower in EVH-positive subjects. FVC (% predicted) was significantly higher in the athletes compared with the untrained (P = 0.024), whereas FEV1/FVC was lower (P = 0.049; Table 2).
This study demonstrates that 8 min of intense hyperpnea of dry air is associated with increased urinary excretion of CC16 in trained and untrained subjects, with and without EIB. Therefore, the increase in urinary CC16 excretion after bronchial provocation with dry air is not a unique feature of EIB. In the young females tested, the increase in urinary CC16 was sustained for at least 60 min after the end of the EVH test. The postchallenge increase in CC16 was, however, prolonged in untrained individuals. This supports the hypothesis of an adaptation of the airways to dehydration stress with training. Because the urinary concentration of CC16 is regarded as a new sensitive marker to detect an increased permeability (4,22), the increase in urinary CC16 provides evidence that dehydration of the small airways during short-term hyperpnea acutely affects the lung epithelium in humans.
A transient increase in the concentration of CC16 in urine was consistently observed in the females tested and occurred after only 8 min of intense hyperpnea of dry air. The duration of the stimulus required to elicit the CC16 response was shorter compared with that previously reported in humans after acute exercise in the absence of a concomitant exposure to noxious agents (exercise duration = 45-100 min [11,15,30]). This would suggest that intense hyperpnea in combination with inhalation of dry air is a very potent factor leading to the release of CC16.
The increase in CC16 that is seen in extrapulmonary fluids after acute exercise (11,15,30) and now after EVH could be a marker of a loss of integrity of the alveolar-capillary barrier, with increased leakage of this small protein into the bloodstream. Alternatively, it could be due to a combination of increased production of CC16, leading to an increased transepithelial diffusion gradient and an increase in pulmonary epithelial permeability. Increased ventilation per se and lung inflation are known to increase epithelial permeability in both animals and humans (17,38). We therefore believe that exercise, through dehydration and/or mechanical stress, perturbs the barrier function of the airway epithelium. Whether these perturbations have a long-term effect on the respiratory health of individuals remains unclear.
We previously suggested that recurrent injury repair of the epithelial barrier could, over time, lead to changes of the contractile properties of the airway smooth muscle and to bronchial hyperresponsiveness (2,3). On the other hand, CC16 is believed to play a protective role against airway inflammation (13,23), most likely through the inhibition of phospholipase-A2 activity (23). Phospholipase-A2 initiates prostaglandins and leukotrienes synthesis. Both exercise (28,34) and EVH (24,25) promote the release of prostaglandin D2 and cysteinyl-leukotrienes in subjects with asthma and in athletes. The transient increase of urinary CC16 in our study could thus partly be due to an increased secretion of CC16 by the Clara cells in an attempt to downregulate the inflammatory response associated with the dry air challenge. This may be of clinical relevance in that it suggests a common airway inflammatory response to dry air challenge in all individuals; the occurrence of a bronchoconstriction being therefore likely determined by the reactivity of the airway smooth muscle to the mediators released (24).
Urinary CC16 normalized in our athletes within 90 min, whereas a more sustained response was observed in the untrained subjects. Repeated exposures to ozone have been shown to reduce the transepithelial escape of lung CC16 and may be related to tolerance of the pulmonary response to ozone in rats (5). The quicker return of urinary CC16 to baseline levels in athletes might therefore indicate adaptation of the airways to dehydration stress after repeated exposure to hyperventilation during chronic exercise. Such an adaptive response has been demonstrated in mice with the promotion of active repair of bronchial epithelium damaged after mild- to moderate-intensity endurance training (14).
EVH was used in this study as a surrogate for exercise because dehydration of the small airways is believed to be a key triggering factor of EIB (1). After a high-intensity swimming test of similar duration (6-8 min), Romberg et al. (35) recently noticed an increase in urinary CC16 in young swimmers (35% with airway responsiveness to exercise and/or mannitol). Unlike our study, the short-term changes in concentration of CC16 in extrapulmonary fluids after swimming can be attributed to both hyperventilation-induced and nitrogen trichloride-induced epithelial injury (11). Reduced serum levels of CC16 were previously recorded at rest and after outdoor exercise in children regularly visiting indoor chlorinated pools; a result that was attributed to the negative effect of chlorination byproducts on Clara cell function (26). In our study, only 3 of the 28 athletes specifically trained in swimming (one EVH+), and their baseline CC16 values were highly variable (0.009, 0.087, and 0.011 ng·μmol−1 of creatinine). It is therefore unlikely that chronic exposure to nitrogen trichloride interfered with our results.
Several methodological precautions were taken while collecting and analyzing our urine samples for CC16. First, as done in the past (4) and as routinely used for other serum/urinary biomarkers, CC16 values were adjusted for creatinine levels. This is critical because the rate of clearance of CC16 is directly dependent on the glomerular filtration rate. Moreover, only female participants were recruited to avoid possible contamination of the samples by CC16 in prostate secretions (4,39). Finally, to account for diurnal variations of urinary CC16 levels (4), all subjects were tested in the morning. Despite all those precautions, but in line with previous works (4,35), large interindividual variability in baseline urinary levels was observed (0.001-11.598 ng·μmol−1 creatinine); divergence most likely to be attributed to differences in renal clearance.
Careful sampling and quantifying suggest that the increase in urinary CC16 noticed in our subjects after EVH is a genuine phenomenon that reflects epithelial stress and perhaps stimulation of the Clara cells. There is considerable direct evidence regarding stress of the airway epithelium after hyperpnea of dry air (16,32). What still remains unclear is (i) whether an osmotic change of the airway lining fluid directly causes disruption of the epithelial tight junctions (31), (ii) whether the mechanical stress associated with deep breathing exacerbates the dehydration effect, (iii) how the Clara cells respond in vivo to these two osmotic and mechanical stressors, and (iv) the role of the Clara cells in the inflammatory response associated with dry air hyperpnea.
In summary, this study shows that hyperpnea of dry air causes an increase in urinary CC16 excretion in trained and untrained subjects, with and without EIB. This suggests that disruption of the distal respiratory epithelium occurs in all individuals, whatever their bronchial response or fitness level, when large volumes of unconditioned air are inhaled during a short period. Considering the large proportion of athletes who develop breathing disorders during their career (12), the long-term effects of repetitive injury to the airway epithelium need to be established. If the implication of epithelial injury in the pathogenesis of EIB in athletes is proven, possible forms of prevention/management will then need to be implemented.
The study was supported by the Grampian National Health Service (endowment grant 07/45).
There is no conflict of interest to declare by any of the authors.
The results of the present study do not constitute endorsement by the American College of Sports Medicine.
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