Exercise is a common stimulus for bronchoconstriction not only in subjects with clinically recognized asthma but also in elite athletes (10). Dehydration of the lower airways during exercise hyperpnea, associated with release of mast cell mediators, has been proposed as an important determining factor for the severity of exercise-induced bronchoconstriction (EIB) (2). The mast cell activation is thought to occur in response to a hyperosmolarity of the airway surface liquid that occurs in response to evaporative water loss in conditioning large volumes of ventilated air in a short period. The hypothesis is supported by in vitro evidence that human mast cells release their mediators in response to hyperosmolar challenge with mannitol (20). Moreover, inhalation of mannitol causes hyperosmolarity of the airways surface liquid and is a potent stimulus for induction of bronchoconstriction and release of mast cell mediators in subjects with asthma (7). The evidence for mast cell activation is particularly supported by measurements of urinary excretion of metabolites of PGD2 in urine because PGD2 in humans is almost exclusively biosynthesized in mast cells (14). Accordingly, EIB in subjects with asthma was followed by increased urinary excretion of the PGD2 metabolite 9α,11β-PGF2 (31). There is also evidence of histamine release after EIB. The mast cell is also one source of the potent bronchoconstrictors cysteinyl leukotrienes (CysLT) that mediate a component of EIB in subjects with asthma (12). However, CysLT are biosynthesized not only in mast cells but also in several other cells, such as eosinophils, that are activated in asthmatic reactions (13). In athletes, increased eosinophil count has so far only been found in induced sputum from swimmers and bronchial biopsies from cross-country skiers (5), suggesting that the increase in this cell type is influenced by environmental factors rather than exercise per se.
Although there is evidence for mast cell activation during EIB in asthmatic subjects (31), the role of mast cell mediators such as PGD2 and CysLT in EIB in athletes is unclear. In elite athletes, the observation of parallel inhibitory effects of dietary fish oil on urinary excretion of metabolites of PGD2 and CysLT and on the airway response to exercise suggests a direct link between mast cell activation and EIB (28). Further in nonasthmatic healthy subjects who did not bronchoconstrict to inhaled mannitol, there was also increased urinary excretion of 9α,11β-PGF2 (6). Likewise, a study in cyclists suggests that mediators are released because of the workload per se (9). The importance of mast cell activation for EIB therefore remains conjectural. Although mast cells release histamine in response to a hyperosmolar stimulus (18), the limited benefits of antihistamines on EIB in subjects with asthma (12) have caused some to question the role of this cell type in EIB. The well-established protective effect of the presumed mast cell inhibitor sodium cromoglycate (SCG) in EIB however provides circumstantial support for mast cell involvement (39), as does the measurements of mediators in sputum during EIB (21). Further, and in contrast to an antihistamine, SCG has the capacity to affect more than one of the mast cell mediators. In athletes with EIB, the severity of the bronchoconstriction after eucapnic voluntary hyperpnea (EVH) challenge has been shown to significantly correlate with increased concentrations of selected mediators-including CysLT, PGE2, histamine, LTB4, and thromboxane B2-in induced sputum (33).
To clarify the role of mast cell mediators, other than histamine, in the airway response to exercise in athletes, we studied the airway response to hyperpnea in two groups of athletes: one with EIB and one without, as determined by a screening EVH challenge (1,3). Under single-blind conditions, subjects were randomized to inhale placebo or 40 mg of SCG 15 min before repeated EVH challenges at least 2 d apart. The coprimary end points were lung function response and urinary excretion of the PGD2 metabolite 9α,11β-PGF2. The primary hypothesis of the study was that athletes with EIB (EIB+) would display mast cell activation after EVH as evidenced by increased urinary 9α,11β-PGF2. The secondary hypothesis was that the SCG treatment would protect against EVH-induced bronchoconstriction. The determination of the effect of the SCG treatment on the urinary excretion of 9α,11β-PGF2 would in addition provide an opportunity to assess the level in the stimulus-effect cascade that SCG possibly affected.
Finally, urinary excretion of leukotriene E4 (LTE4) was included as an exploratory end point because previous studies in subjects with asthma have produced conflicting data on whether or not SCG affects release of CysLT during bronchoconstrictive responses in humans (7,40). LTE4 is a primary stable urinary metabolite that serves as an index of endogenous biosynthesis of all CysLT (25). The reports on the effects of EIB on urinary LTE4 in subjects with asthma are also conflicting (31,36).
METHODS
Subjects
Twenty-two endurance-trained athletes, all nonsmokers, were recruited to take part in this study. Written informed consent was obtained, and all procedures were approved by the Central Sydney Area Health Service Ethics Committee (protocol No. X03-0164). The study conformed with the principles expressed in the Declaration of Helsinki.
Fourteen subjects considered running or cycling as their primary physical activity, five were triathletes and three were rowers. Most subjects competed either in local (n = 15) or in national races (n = 5) and trained on average 11 h·wk−1 (SEM = 1 h·wk−1). Subject baseline characteristics are displayed in Table 1. Of the 11 subjects in the EIB+ group, nine used short-acting β2-receptor agonists (SAB) on an as-needed basis, and three in addition were on regular therapy with the inhaled steroid fluticasone proprionate (FTP). During the study days, SAB were withheld for 6 h, and FTP was not used on the days of the study. All subjects were required to abstain from alcohol, caffeine, and niacin-containing drinks and food from 20:00 h the evening before the study, and no vigorous exercise was permitted for 24 h before each study day. None of the participants had a chest infection in the 4-wk period preceding the study.
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TABLE 1: Subject characteristics.
Study Design
Subjects attended the laboratory on three occasions; the visits were separated by at least 48 h.
Day 1 (screening visit).
Subjects were questioned on medical history of allergy and respiratory illness, family history of asthma, previous asthma or EIB diagnosis, current and previous medication use, and respiratory symptoms during or after exercise. Each subject had their forced expiratory volume in 1 s (FEV1) measured in triplicate, and this was repeated 10 min later to confirm stability. FEV1 had to be a least 80% of predicted (35) for the subject to be included in the study. An EVH challenge was then performed and used as a surrogate for exercise to detect EIB (1,3). After the challenge, maximal expiratory flow-volume curves (MEFV) were performed in duplicate at 1, 3, 5, 10, 15, and 20 min.
Assignment to the experimental (EIB+) or control group (EIB−) was made according to the postchallenge change in FEV1-which was used as an index of change in airway caliber-during the screening visit. If the fall in FEV1 was of 10% or more (1), the subject was considered as having EIB and assigned to the EIB+ group.
Day 2 or 3 (drug or placebo trials).
These two study days involved administration of either placebo or 40 mg of SCG (eight inhalations of Intal Forte; Sanofi Aventis, France) from a pressurized metered-dose inhaler, EVH test, spirometry measurements, and urine collection. Administration of interventions was done in a randomized single-blind manner. Each subject was studied at approximately the same time of the day for the placebo and drug trial and was asked to drink a glass of water every hour.
On each of the study days, after the first lung function measurements, subjects emptied their bladders and provided the first baseline urine sample. After a further 60 min, subjects were asked to provide another prechallenge urine sample and to perform another three MEFV curves. Then the active drug or its placebo was administrated. Subjects were instructed to take eight long, slow inhalations of the aerosol and to hold their breath for 10 s. Ten minutes after the first inhalation, MEFV curves were repeated. An EVH test was then performed approximately 15 min after drug or placebo. Post-EVH challenge measurements included MEFV at 1, 5, and 10 min after the challenge and thereafter at 10-min intervals for up to 90 min. Urine samples were collected at 30, 60, and 90 min after the challenge, and all the urine samples were stored without addition of preservatives at −80°C.
Lung Function Measurements
The MEFV curves were performed pre- and post-EVH in accordance with the ATS recommendations using a SpiroCard® (QRS Diagnostics, Plymouth, MN). FEV1 and forced vital capacity values measured at 10 min after active drug/placebo administrations were taken as the prechallenge values and used to calculate the postchallenge decrement in lung function.
EVH Challenge Test
The protocol used for the EVH test was based on the one originally described by Argyros et al. (3) requiring the subjects to breath at a target ventilation rate of 85% maximum voluntary ventilation (estimated from 30 times baseline FEV1) for 6 min. The setup used for the challenge was based on the one described by Anderson et al. (1) as follows. Briefly, a compressed dry gas mixture (∼5.0% CO2, 21% O2, and balance N2) at room temperature flowed from a cylinder through a rotameter (Fisher-Rosemount; Brooks Instruments, Hatfield, PA) via an open demand valve into a meteorological balloon (300 g, MFG No. 100 MRL; Kaysam Corp., Patterson, NJ) via a metal connector with tap (Morgan PKM 90750105) that allowed simultaneous filling and emptying. The gas was inhaled via the metal connector and breathing tube, via a two-way nonrebreathing valve (Hans Rudolph No. 2700) and a mouthpiece. The expired gas passed through a gas meter (American Dry Test Meters; American Meter Co., Horsham, PA), and the volume of air expired was then measured.
Measurements of Urinary Mediators
Enzyme immunoassay of 9α,11β-PGF2 was performed in serially diluted urine samples, using a rabbit polyclonal antiserum and acetylcholinesterase-linked tracer (Cayman Chemical Company, Ann Arbor, MI) essentially as described previously (6,7,31). The antibody cross-reacted with 2,3-dinor-9α-11β-PGF2 (10%), 13.14-dihydro-15-keto-prostaglandin F2α (0.5%). The cross-reactivity with other primary eicosanoid metabolites was below 0.01%. Analysis of urinary LTE4 was performed after a similar protocol (27) using a rabbit polyclonal antiserum directed against CysLT (Cayman Chemical Company) with acetylcholinesterase-linked LTE4 as tracer. The specificity of the antiserum was 100% for LTC4, 100% for LTD4, and 67% for LTE4. For both assays, the inter- and intraindividual assay variability was less than 15%. All data were normalized and presented as nanograms of excreted mediator per millimole of creatinine. Creatinine analyses were performed using a modification of Jaffe's (24) creatinine protocol.
Atopic Status
Skin prick tests were performed during the screening visit using standardized allergen extract against the following allergens: house dust mite, timothy grass, Alternaria, Aspergillus fumigatus, Aspergillus niger, cat dander, dog dander, perennial rye, and cockroach. A reaction with a wheal of ≥3 mm in diameter was considered a positive test. Total IgE and specific IgE to dust mites were measured on one of the study days in venous plasma using the UniCAP Analyser machine (Pharmacia, Stockholm, Sweden).
Statistical Analysis
All data are presented as mean ± SEM. The maximum fall in FEV1 is expressed as the percentage of the baseline value of FEV1. The area under the FEV1 time curve (FEV1-AUC1-90) was calculated from the percentage change from baseline FEV1 over the 90-min observation period by using the trapezoidal method. The percentage of protection to SCG was calculated by subtracting the percentage of fall or FEV1-AUC1-90 after SCG from the value after placebo and expressing it as a percentage of the placebo. For urinary excretion of 9α,11β-PGF2 and LTE4, the mean of the two samples collected in the hour before the EVH challenge was used as the baseline value. Values for the mediators are presented as absolute values or absolute change from baseline.
Normality of the data was checked using a Shapiro-Wilk test and homogeneity of variance using Levene's test. Group characteristics were compared using unpaired t-tests or Mann-Whitney tests. Lung function differences between groups and treatments were determined using repeated-measures ANOVA followed by, when required, Bonferroni post hoc analysis. To compare baseline and peak values for urinary mediators, we used paired t-tests. The association between lung function response to EVH and urinary mediator release was assessed via linear regression tests (Pearson correlation coefficients). The P < 0.05 level of significance was adopted for all tests. The statistical calculations were performed using the Statistical Package for the Social Sciences for Windows (Version 15.0; SPSS Inc., Chicago, IL).
RESULTS
Screening Day
Eleven of the 22 subjects recorded a decrease of ≥10% in their FEV1 values after EVH during the screening visit. The maximal fall in FEV1 in the responder group (EIB+) at the screening visit was 20.9% ± 2.9% compared with 4.0% ± 0.6% in nonresponders (EIB−). The subjects included in the EIB+ group had a normal but significantly lower baseline FEV1 than the EIB+ group (P = 0.041) and more were atopic (Table 1).
Study Days
Baseline values and workload.
There was no within-group difference in resting spirometry values between the placebo and the SCG study days (Table 2) nor any difference in the rate of ventilation achieved by either group on the placebo (EIB+ = 111 ± 4.7 L·min−1; EIB− = 120 ± 8.0 L·min−1) or the SCG (EIB+ = 114 ± 6.5 L·min−1; EIB− = 124 ± 7.2 L·min−1) study days. Furthermore, for both groups and both study days, the rate of ventilation was close to 80% of predicted indirect maximal voluntary ventilation (MVV), defined as 35 times FEV1 (EIB+ = 85% ± 2.4% and 88% ± 3.2% MVV after placebo and active drug, respectively; EIB− = 79% ± 4.3% and 83% ± 2.8% MVV after placebo and active drug, respectively).
TABLE 2: Data obtained from endurance-trained athletes with (EIB+) and without exercise-induced bronchoconstriction (EIB−) during the placebo and the experimental day (i.e., after administration of 40 mg of sodium cromoglycate (SCG)).
Lung function response to EVH.
The postchallenge fall in FEV1 observed during screening (see above) was reproduced during the placebo session in the EIB+ group and amounted to 20.3% ± 3.4%. In contrast, the postchallenge maximal fall in FEV1 was 11.5% ± 1.9% after SCG treatment (P = 0.003; Fig. 1, Table 2). Also, FEV1-AUC1-90 was significantly reduced on the SCG day (P = 0.004; Table 2). In the EIB+ group, the mean percentage protection afforded by SCG on the maximum fall in FEV1 and on FEV1-AUC90 was 39.3% and 57.2%, respectively.
FIGURE 1: Mean ± SEM percentage change in forced expiratory volume in 1 s (FEV1) before (pre) and after (post) inhalation of placebo (circles) and SCG (triangles) and for 90 min after eucapnic voluntary hyperpnea (EVH) in athletes with exercise-induced bronchoconstriction (EIB) (closed symbols) and athletes without EIB (open symbols). -, baseline values.
In the EIB− group, although there was no significant bronchoconstriction after either EVH challenge (Fig 1), there was in fact a statistical difference between treatments for the postchallenge FEV1 response (4.1% ± 0.6% after placebo compared with 2.7% ± 0.6% after SCG, P = 0.008; Table 2). However, FEV1-AUC1-90 was not significantly different between the placebo and the SCG study day in this group.
Urinary excretion of mediators.
Baseline urinary levels of 9α-11β,PGF2 were higher in the EIB+ group compared with the EIB− group on both experimental days (P < 0.05), whereas baseline LTE4 levels were lower (P < 0.05; Table 2). However, the levels of both mediators for both groups were within normal variability for each mediator (14,25).
In the EIB+ group, urinary 9α,11β-PGF2 excretion increased after EVH challenge during the placebo session (P = 0.006; Fig. 2A, Table 2). On the placebo day, there was also an increase in urinary 9α-11β,PGF2 after EVH challenge in the EIB− group (P = 0.036; Table 2). The peak postchallenge levels on the placebo day were, however, significantly higher in the EIB+ group compared with the EIB− group (P = 0.043; Table 2).
FIGURE 2: Urinary mediators before and after eucapnic voluntary hyperpnea (EVH) testing in 11 athletes with EIB. Individual values indicated by filled circles connected by line and group mean data displayed by line with variability (SEM) shown by vertical bar. All data normalized in relation to urinary excretion of creatinine. A. The prostaglandin D2 metabolite 9α-11β,PGF2 is a marker of mast cell activation. B, The CysLT metabolite leukotriene E4 is a marker of whole-body production of this class of potent bronchoconstrictors.
During the SCG session, there was no significant increase in urinary 9α-11β,PGF2 in the EIB+ group (Fig. 2A, Table 2). Although numerically lower than during placebo challenge, the 9α-11β,PGF2 values on the SCG day in the EIB− group were not significantly different from the prechallenge baseline (Table 2). There were no significant differences in postchallenge levels of 9α-11β,PGF2 between the two study groups on the SCG day.
For the EIB+ group, the values for LTE4 after EVH were increased above baseline values after placebo treatment (P < 0.001) but not after SCG treatment (Fig. 2B, Table 2). For the EIB− group, there was no change in urinary LTE4 postchallenge on the placebo day, whereas on the SCG day, the peak value was significantly higher than the baseline (P = 0.030). The EIB− group demonstrated higher peak values compared with the EIB+ group on the SCG day (P = 0.009) but not on the placebo day (P = 0.057, Table 2).
Relationship between airway response to EVH and excretion of urinary mediators.
In the EIB+ group, a significant correlation was found between the fall in FEV1 and the increase in urinary 9α,11β-PGF2 excretion after EVH (r = 0.582, P = 0.004; Fig 3). No similar association existed between FEV1 and LTE4 (not shown).
FIGURE 3: Correlation between the degree of bronchoconstriction after eucapnic voluntary hyperpnea (EVH) and excretion of urinary 9α,11β-PGF2 in athletes with EIB after administration of placebo (circles) or 4 mg of SCG (triangles).
DISCUSSION
The primary and the secondary study hypotheses were both confirmed, namely, that athletes with EIB had increased release of the mast cell marker 9α,11β-PGF2 in the urine after EVH and that the bronchoconstriction was substantially inhibited by pretreatment with the mast cell stabilizer SCG (32). Furthermore, as SCG also blocked the postchallenge increase in 9α,11β-PGF2 excretion, the data taken together lend strong support to the concept that mast cell mediators are true mediators of EIB in athletes. In this context, it should be acknowledged that PGD2 (22) as well as its metabolite 9α,11β-PGF2 (4) are also potent bronchoconstrictors and thus may contribute to the EVH-induced bronchoconstriction. Our finding of a correlation between the fall in FEV1 and the urinary excretion of 9α,11β-PGF2 further supports the role of the mast cells in the EIB.
Interestingly, the EIB− subjects also displayed increased urinary excretion of 9α,11β-PGF2 after the EVH challenge, and the small effect on lung function that the challenge had in these subjects was in fact significantly attenuated by SCG. The finding is consistent with our previous observations using the exercise-mimetic mannitol (7). In that study, healthy control subjects who did not bronchoconstrict in response to inhalation of mannitol did show significantly increased levels of urinary 9α,11β-PGF2 after the challenge. The data from the two studies support the conclusion that bronchoconstriction after an indirect challenge requires both release of bronchoconstrictive mediators and sensitivity of the airway smooth muscle to the specific mediators released. The previous findings with mannitol challenge and the present data using EVH challenge support the notion that both these indirect challenges cause mast cell activation in subjects without asthma, but it is only individuals with asthma and/or EIB who present significant bronchoconstriction because their airway smooth muscle is hyperresponsive to the specific mediators released.
The level of ventilation achieved by both groups of athletes was similar, and it was high, exceeding 110 L·min−1 on average. This level of ventilation is sufficient to recruit the small airways beyond generation 9 (17). This is relevant to our findings because it is in these smaller airways that the concentration of mast cells is highest for both healthy subjects and asthmatics (11).
In keeping with previous studies, we found, with one exception, that all the athletes with EIB were atopic (91%), whereas only four athletes without EIB (36%) were atopic. The relative risk of asthma has been shown to increase by approximately 75-fold in atopic endurance athletes compared with nonatopic controls (38). Presumably, increased exposure to environmental allergens causing enhanced airway inflammation is one factor that makes atopic athletes more prone to develop EIB. In atopic summer athletes, we previously proposed that hyperpnea-induced injury to the airway epithelium also might make the airway smooth muscle more sensitive and contribute to the pathogenesis of EIB (2).
In a study of subjects with asthma, Brannan et al. (7) were able to block the airway response to mannitol and to significantly reduce the postchallenge increase in urinary excretion of 9α,11β-PGF2 by inhalation of 40 mg of SCG. Interestingly, however, the peak versus baseline levels of urinary excretion of release of 9α-11β,PGF2 was two to three times greater after mannitol than after EVH in the present study. The differences may relate to differences in study populations and/or procedures of bronchial provocation. However, although testing with EVH may cause a transient hyperosmolar environment and activation of the mast cells, the mannitol challenge protocol with inhalation of progressively increasing doses (7) is likely to produce a much more sustained osmotic effect on mast cells with consequent greater release of mediators. For challenge of asthmatic subjects with allergen, it has been shown that cumulative challenge produces a greater increase in urinary LTE4 than a single-dose provocation (26). Taken together, the dose-response nature of the mannitol challenge for both bronchoconstriction and mediator excretion may contribute to make mannitol challenge more useful for research purposes than the EVH test. Whether this also translates to the diagnostic use of the two tests remains to be established.
The subjects with EIB in our study also displayed increased release of urinary LTE4 after the EVH challenge, and SCG inhibited that response as well. Because leukotriene antagonists have been shown to attenuate EIB (12), our observation suggests that the mast cell activation in athletes also somehow triggers release of CysLT that contribute to the bronchoconstriction induced by EVH. The results of the present study are consistent with leukotrienes playing a role in athletes with EIB and confirm the findings of Rundell et al. (37) who showed beneficial effects of montelukast in skaters. In subjects with asthma, it has indeed been shown that bronchoconstriction induced by dry, cold air is in part mediated by the release of CysLT (23).
The present finding that SCG inhibited the increase in urinary LTE4 after EVH seems to be in conflict with our previous study of another exercise-mimetic mannitol (7). In that particular study, the mast cell stabilizer SCG did not block mannitol-induced urinary excretion of LTE4, suggesting that CysLT are generated by cells other than mast cells that are stimulated by hyperosmolarity in the local environment, for example, eosinophils (29). It may be speculated that EVH is a less effective and a more transient stimulus for leukotriene formation in non-mast cells in comparison with mannitol. Because the increase in urinary mediator excretion was smaller and of shorter duration in the present EVH study (see above), it seems most likely that it was easier for SCG to inhibit this less vigorous response.
That EVH or exercise are less potent triggers of mast cell activation than allergens is consistent with previous studies measuring histamine release. Although allergen inhalation has been shown to cause a clear increase in the urinary excretion of 9α,11β-PGF2 and Nτ-methylhistamine (30), no such increase was noticed for Nτ-methylhistamine after 5 min of exercise on a stationary ergocycle at 80% maximum workload (31). This prompted the authors to suggests that 9α,11β-PGF2 is more sensitive than Nτ-methylhistamine for monitoring mild and transient episodes of mast cell activation, such as explored in this study.
With regard to different strengths of trigger factors for mediator release and provocation of EIB, a recent study interestingly showed that some athletes negative to both EVH and exercise test in the laboratory were positive for EIB after a sport-specific field-based challenge (34). This suggests that sometimes standardized laboratory-based bronchial provocation tests such as exercise or EVH incompletely mimic the stress athletes experience on the field. However, a large follow-up study in a general pulmonary practice supports the usefulness of EVH for diagnosis of EIB (8).
In conclusion, this study has documented that hyperpnea with dry air is associated with mast cell activation and release of mediators of bronchoconstriction in athletes, both with and without EIB. The known potent bronchoconstrictive effects of PGD2 (22) and CysLT (15), the protective effects of SCG on airway narrowing, and the excretion of 9α-11β,PGF2 and LTE4 all support a role for PGD2 and CysLT in the bronchoconstriction induced by the hyperpnea with dry air in athletes. These findings support the concept that the mechanism of EIB in athletes is linked to the release of inflammatory mediators after desiccation of the airway lining fluid within the airways (16,17,19). The protective effect of antileukotriene drugs on EIB in athletes and in subjects with asthma has already been shown (12,37). The present study introduces a rationale for future investigations to define the benefits on EIB of intervention with agents that affect either the formation or the action of PGD2 in athletes as well as in asthmatics.
The authors are grateful to Ingrid Delin and Clare Perry for skilful technical support. The study was supported by grants from the National Health and Medical Research Council (MRC) of Australia and by the Swedish MRC, Swedish Heart Lung Foundation, Stockholm County Council (ALF), Vinnova, and Karolinska Institutet.
None of the authors have a conflict of interest to declare.
The results of the present study do not constitute endorsement by the American College of Sports Medicine.
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