INTRODUCTION

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Airway inflammation is an important characteristic in patients with asthma (1). Eosinophils, basophils, and mast cells have been the most consistent cell type found to be elevated in asthmatic airways, and appear to participate in symptomatic asthma (2, 3). These inflammatory cells are sources of the cysteinyl leukotrienes (LT) C₄, D₄, and E₄ (4), which are lipid mediators known to cause bronchoconstriction in human airways (5), and may play an important role in the pathogenesis of asthma (6).

There is accumulating evidence that the cysteinyl leukotrienes may mediate asthmatic airway inflammation. Allergen inhalation by sensitized patients with asthma results in acute bronchoconstriction, airway hyperresponsiveness, increases in airway eosinophils, basophils, and mast cells (1, 7), as well as leukotriene production (10). Also, inhalation of LTE₄ by patients with asthma causes acute bronchoconstriction, airway hyperresponsiveness (11), and airway inflammation (12). Leukotriene antagonists have been shown to inhibit the airway inflammatory response induced by instillation of allergen (15) and inhaled LTE₄ (14), suggesting interruption of the cysteinyl leukotriene pathway may regulate these inflammatory responses.

High-intensity exercise by patients with asthma can result in exercise-induced bronchoconstriction. There are some similarities between exercise-induced and allergen-induced bronchoconstriction, including elevated levels of cysteinyl leukotrienes (16, 17), and the efficacy of treatment with antileukotrienes in attenuating the bronchoconstriction caused by both stimuli (17). Based on these similarities it might be expected that exercise-induced bronchoconstriction, like allergen-induced bronchoconstriction, may result in airway inflammation and airway hyperresponsiveness. This has important implications for patients with asthma who develop exercise-induced bronchoconstriction.

The purpose of this study was to determine whether exercise-induced bronchoconstriction is associated with airway inflammation and hyperresponsiveness. The study was designed in a controlled, randomized crossover fashion, such that each subject served as his or her own control. Each subject underwent an exercise challenge resulting in exercise-induced bronchoconstriction, and a control challenge with inhaled methacholine as a bronchoconstrictor control challenge. Methacholine was used as a control, as it is known to cause bronchoconstriction without inducing airway inflammation (22, 23). Airway inflammation was assessed by measurements of inflammatory cells in induced sputum at baseline and 2, 4, 7, and 24 h after challenges.

	METHODS

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Subjects

Thirteen nonsmoking subjects, at least 18 yr old, with mild asthma were recruited for the study (Table 1). These subjects were selected based on a provocative concentration of methacholine causing a 20% fall in FEV₁ (PC₂₀) below 2 mg/ml, which is associated with an increased risk of exercise-induced bronchoconstriction (24), or a history of exercise-induced bronchoconstriction. The subjects had stable asthma with a forced expiratory volume in 1 s (FEV₁) greater than 70% of predicted. Subjects used no medication other than inhaled ₂-agonists in the last 4 wk prior to and during the study. Medication was withheld for at least 8 h before each visit, and subjects were instructed to refrain from rigorous exercise, tea, or coffee in the morning before visits to the laboratory. Subjects did not have asthma exacerbations or respiratory tract infections for at least 4 wk prior to entering the study. Two subjects were excluded from the study because they did not develop exercise-induced bronchoconstriction during the randomized part of the study. One subject did not complete the study due to asthma deterioration. Comparisons of exercise and methacholine challenges were carried out on the remaining 10 subjects. Eight of the subjects underwent allergen-inhalation challenge as a positive control for the ability to develop airway eosinophilia. The study was approved by the ethics committee of McMaster University Health Sciences Centre, and subjects gave signed consent.

Study Design

The study was carried out with a randomized, crossover design. Subjects attended the laboratory for two or more screening sessions. During the initial screening visit, subject characteristics and history were documented, and subjects completed methacholine inhalation to demonstrate airway hyperresponsiveness. Subjects in whom the PC₂₀ was less than 2 mg/ml, or had a history positive for exercise-induced bronchoconstriction, proceeded to the next stage of screening. On a separate day, a stage 1 incremental cycle ergometer exercise test was performed until subjective exhaustion. Subjects demonstrating an exercise-induced fall in FEV₁ returned on another day to undergo a dry air exercise challenge. If the fall in FEV₁ from baseline was > 15% after dry air exercise challenge, subjects were enrolled in the study. At least 24 h separated all screening visits.

Each subject then completed two study periods consisting of exercise challenge or methacholine challenge. Each study period consisted of three visits to the laboratory. Methacholine PC₂₀ and induced sputum differential and total cell counts were determined 1 d before challenge. Study challenges (exercise or methacholine) were carried out the following morning. Blood samples were collected immediately before challenge. FEV₁ was measured immediately before challenge, and at regular intervals after exercise until 120 min after challenge. Sputum and blood samples were obtained at 2, 4, and 7 h after challenge. At 24 h after challenge, methacholine PC₂₀ was measured, and sputum and blood samples were obtained. Each challenge was separated by a washout period of at least 7 d.

Allergen inhalation challenges were also carried out at least weeks before or 7 d after the exercise and methacholine challenges. Of the eight subjects who inhaled allergen, one subject completed the allergen challenge within 1 yr of exercise challenge, two subjects within 2 mo, and the remaining subjects within 1 mo. Samples of sputum were taken before and 7 and 24 h after allergen challenge. These challenges were included as a positive control, to demonstrate the potential of these subjects to develop airway eosinophilia after submaximal bronchoconstriction.

Spirometry

Spirometry was measured with a Collins water-sealed spirometer and kymograph. FEV₁ was measured in triplicate at baseline and single FEV₁ measurements were performed following challenges with exercise and methacholine. Volumes were recorded at body temperature, atmospheric pressure, saturated with water vapor.

Methacholine Challenge

Methacholine was purchased from the McMaster University Hospital Pharmacy in a stock concentration of 128 mg/ml. This was diluted in physiological saline into doubling concentrations ranging from 64 to 0.016 mg/ml. These working concentrations were stored at 4° C for up to 3 mo. Methacholine inhalation challenge was performed as described by Cockcroft (25). Subjects inhaled normal saline, then doubling concentrations of methacholine phosphate from a Wright nebulizer (Roxon Medi Tech, Montreal, PQ, Canada), operated by oxygen at 50 psi and at a flow rate that gave an output of 0.13 ml/min for 2 min. FEV₁ was measured with a water-sealed spirometer at 30, 90, 180, and 300 s after each inhalation. The test was terminated when at least a fall in FEV₁ of 20% of the baseline value occurred, and PC₂₀ was calculated. The maximum bronchoconstrictor response was taken to be the largest fall in FEV₁ measured within 2 h after inhalation.

Exercise Challenge

During the screening period, a stage 1 incremental cycle ergometer exercise test was performed as described by Jones (26). Subjects exercised on a stationary cycle ergometer and the workload was increased by 100 kpm each minute until subjective exhaustion. Subjects wore nose clips and breathed room air through a mouthpiece on a three-way Hans Rudolph valve. Minute ventilation was measured from the expiratory side of the valve (Sensor Medics, CA), and heart rate was recorded from ECG leads (Quinton Instrument Company, WA). FEV₁ was measured immediately before exercise and at regular intervals after exercise.

Dry Air Exercise Challenge

Dry air exercise challenge was carried out during the screening period at a work rate equal to 80% of the maximum achieved during the incremental test. If this resulted in a fall in FEV₁ from baseline of 15- 45%, this work rate was chosen for the study exercise challenge. If the percentage fall in FEV₁ was greater or less than this range, then further screening challenges were performed at correspondingly lesser or greater work rates to establish the workload required for exercise- induced bronchoconstriction. The subjects then repeated the dry air challenge at this chosen workload during the study period. The dry air exercise challenge was carried out on the stationary cycle ergometer, and subjects were instructed to cycle at the chosen work rate for 5 min. Subjects wore nose clips and breathed dry air (< 10% relative humidity) from a Douglas bag reservoir connected via the inspiratory port of the valve. Minute ventilation and heart rate were recorded as during the stage 1 exercise test. FEV₁ was measured immediately before exercise and then again at 1, 3, 5, 8, 10, 15, 20, 30, 45, 60, 90, and 120 min after challenge. The maximum fall in FEV₁ was taken to be the largest fall in FEV₁ within 2 h after challenge. The area under the curve (AUC) was determined by plotting the response using graphics software (Fig P.; Fig P Software Corporation, Durham, NC), which calculated the area of the FEV₁ time response.

Allergen Inhalation Challenge

Allergen extracts were stored at 70° C. Allergen for skin tests was diluted in phosphate-buffered saline with 1.5% benzyl alcohol and stored at 4° C, and allergen for inhalation was diluted in physiological saline on the day of use. The allergen extract selected for inhalation produced the largest skin test wheal, and was diluted for inhalation at the concentration determined from a formula described by Cockcroft and coworkers (27) using the results from the skin test and the methacholine PC₂₀. Allergen inhalation challenges were performed using a Wright nebulizer operated by oxygen at 50 psi and at a flow rate that gave an output of 0.13 ml/min, and aerodynamic mass median diameter of 1.0-1.5 µm, using the method as described by O'Byrne and coworkers (28). Doubling concentrations of allergen were inhaled by tidal breathing for 2 min, with FEV₁ measured 10 min after each inhalation. Inhalations were stopped when the FEV₁ had fallen by at least 15% from baseline, and was subsequently measured at 20, 30, 45, 60, 90, and 120 min and at hourly intervals up to 7 h postallergen inhalation. The early airway response, at least a 15% fall in FEV₁, was taken to be the largest fall in FEV₁ within 2 h after challenge, and the late response was taken to be the largest fall in FEV₁ between 3 and 7 h after allergen inhalation. Subjects were considered to be dual airway responders if the late fall in FEV₁ exceeded 15%.

Sputum Analysis

Sputum was induced and processed using the method described by Pizzichini and coworkers (29). Subjects inhaled 3, 4, then 5% saline for 7 min each. The induction was stopped when an adequate sample was obtained, or if the FEV₁ dropped 20% from baseline. Unsatisfactory samples were obtained three times during the study. Cell plugs with little or no squamous epithelial cells were selected from the sample using an inverted microscope, separated from saliva, and weighed. Samples were aspirated in four times their volume of 0.1% dithiothreitol (Sputolysin; Calbiochem Corp., San Diego, CA) and four times their volume of Dulbecco's phosphate-buffered saline (DPBS; Gibco BRL, Life Technologies, Grand Island, NY). The cell suspension was filtered through a 52-µm nylon gauze (BNSH Thompson, Scarborough, ON, Canada) to remove debris, then centrifuged at 1,500 rpm for 10 min. The total cell count was determined using a hemocytometer (Neubauer Chamber; Hausser Scientific, Blue Bell, PA) and expressed as the number of cells per milliliter sputum. Cells were resuspended in DPBS at 0.75-1.0 × 10⁶/ml. Cytospins were prepared on glass slides using 50 µl of cell suspension and a Shandon III Cytocentrifuge (Shandon Southern Instruments, Sewickly, PA), at 300 rpm for 5 min. Differential cell counts were obtained from the mean of two slides with 400 cells counted per slide stained with Diff-Quik (American Scientific Products, McGaw Park, IL). Metachromatic cell (mast cell and basophil) counts were obtained from slides stained with toluidine blue, from the mean of two slides with 1,500 cells observed on each slide.

Statistical Analysis

All summary statistics are expressed as mean and standard error of the mean (SEM) with the exception of methacholine PC₂₀ measurements and cells per milliliter sputum, which were log-transformed prior to statistical analysis and expressed as geometric mean and geometric standard error of the mean (GSEM). Two-tailed Student's t test for paired observations was used to compare bronchoconstriction after exercise and methacholine. Methacholine PC₂₀ and blood and sputum inflammatory cells were analyzed for the effects of challenge and time using a two factor repeated measures ANOVA (30). Statistical analyses were performed using computer software (Statistica 4.5; Stat Soft Inc., Tulsa, OK).

	RESULTS

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The maximum percentage fall in FEV₁ was greater after methacholine inhalation challenge than after exercise challenge, being 29.9 ± 1.5% and 21.3 ± 1.5% after methacholine and exercise, respectively (p = 0.002); however, there was no difference in the area of the FEV₁ time response, being 17.8 ± 1.6% FEV₁ × hours after methacholine and 17.2 ± 1.6% FEV₁ × hours after exercise (p = 0.73) (Figure 1). The maximum early percentage fall in FEV₁ after allergen inhalation was 28.9 ± 2.7%.

View larger version (16K): [in this window] [in a new window]   Figure 1.   Percentage change in FEV1 for 2 h after inhalation challenges with methacholine (solid squares) and exercise (open squares).

Neither exercise challenge nor methacholine challenge changed methacholine airway hyperresponsiveness. The methacholine PC₂₀ was 0.54 mg/ml (GSEM 1.4) before and 0.76 mg/ ml (GSEM 1.5) at 24 h after exercise challenge, and 0.59 mg/ ml (GSEM 1.4) before and 0.64 mg/ml (GSEM 1.4) at 24 h after methacholine inhalation challenge (p = 0.47). Furthermore, the change in methacholine PC₂₀ pre- to postchallenge was not significantly different between exercise and methacholine inhalation challenges (p = 0.79).

There was no effect of time or challenge on the total number of cells per milliliter sputum. Also, the percentage sputum eosinophils did not change following exercise challenge or methacholine challenge (p = 0.47), being 10.4 ± 2.2% before exercise and 12.3 ± 2.7% at 2 h, 12.0 ± 2.5% at 4 h, 12.4 ± 3.3% at 7 h, and 12.8 ± 3.0% at 24 h after exercise, and 9.6 ± 2.8% before methacholine inhalation and 17.0 ± 5.4% at 2 h, 13.8 ± 3.0% at 4 h, 14.2 ± 3.8% at 7 h, and 14.4 ± 2.6% at 24 h after methacholine inhalation (Figure 2). There was no effect of exercise or methacholine on the percentage sputum EG2-positive cells (p = 0.27). By contrast, after allergen inhalation, the percentage sputum eosinophils increased significantly from 6.6 ± 2.7% before allergen to 31.0 ± 6.5% at 7 h and 21.8 ± 4.3% at 24 h (p = 0.03) (Figure 2).

View larger version (18K): [in this window] [in a new window]   Figure 2.   Percentage sputum eosinophils at baseline (BL) and 2, 4, 7, and 24 h after inhalation challenge with methacholine (solid bars), exercise (open bars), and allergen (hatched bars). *p < 0.05 allergen- induced increase in sputum eosinophils.

The percentage sputum neutrophils was significantly elevated from baseline at 2, 4, 7, and 24 h after both exercise and methacholine challenges (p < 0.04) (Figure 3), but there were no significant differences between the challenges (p = 0.17). There was an associated significant decrease in the percentage sputum macrophages (p < 0.003) (Figure 3) at 2, 4, 7, and 24 h after challenges, again with no difference between challenges (p > 0.20) (Figure 3). There was no effect of time or challenge on the percentage sputum metachromatic cells (mast cells and basophils) (p > 0.45).

View larger version (22K): [in this window] [in a new window]   Figure 3.   Percentage sputum neutrophils (top panel ) and macrophages (bottom panel ) at baseline (BL) and 2, 4, 7, and 24 h after inhalation challenge with methacholine (solid bars) and exercise (open bars). *p < 0.05 change from baseline after exercise and methacholine challenges.

The total cell count in peripheral blood significantly increased at 2 h (p = 0.0008), 4 h (p = 0.005), and 7 h (p = 0.003), but not at 24 h (p = 0.43) after exercise and methacholine challenges. The number of neutrophils in peripheral blood significantly increased at all time points after both exercise and methacholine challenges (p < 0.01). The number of eosinophils in peripheral blood was significantly different between 4 and 24 h after exercise challenge (p < 0.004). However, there was no effect of time, exercise, or methacholine on the number of peripheral blood eosinophils (p > 0.78), monocytes (p > 0.18), or lymphocytes (p > 0.05) (Figure 4).

View larger version (26K): [in this window] [in a new window]   Figure 4.   Peripheral blood eosinophils (top panel ), neutrophils (middle panel ), and lymphocytes (bottom panel ) at baseline (BL) and 2, 4, 7, and 24 h after inhalation challenge with methacholine (solid bars) and exercise (open bars). *p < 0.05 change from baseline after exercise and methacholine challenges. p < 0.05 difference between 4 and 24 h after exercise challenge.

	DISCUSSION

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This study has demonstrated that exercise-induced bronchoconstriction does not worsen eosinophilic airway inflammation or airway hyperresponsiveness in individuals with asthma who develop airway eosinophilic inflammation after allergen-induced bronchoconstriction. If repeated exercise were to induce airway inflammatory events, as does repeated inhalation low doses of allergen (31), it would imply that regular exercise performed by patients with asthma may worsen asthma over time. This is unlikely, as results from a longitudinal study suggest that exercise-induced bronchoconstriction is not a predictor for subsequent development of symptomatic asthma (32).

Whether exercise causes airway inflammation has been investigated in a limited number of studies. The results, however, are controversial. Bronchoalveolar lavage (BAL) performed 3 h after exercise challenge was associated with a higher number of eosinophils than in BAL 3 h after methacholine challenge (33); however, in another study there was no change observed in BAL inflammatory cells at 1 or 24 h after exercise challenge (34). Interpretations of these results are complicated by the inability to sample BAL frequently after exercise to measure inflammatory cell kinetics.

In this study we measured inflammatory cells from induced sputum, which can be sampled safely and repeatedly (29) with high reproducibility after bronchial challenges such as allergen inhalation. With this method, we have observed transient changes in the composition of airway inflammatory cells after allergen inhalation challenge (8). In this study, neither of the challenges, except for allergen inhalation, caused a significant eosinophilic response in the airways. We did not observe increased sputum eosinophilia at 2 and 4 h after exercise challenge, at a time when an increase was previously reported in BAL (33) or at 7 and 24 h after exercise challenge, at a time when we measured an increase in eosinophils in the sputum of these same subjects after allergen inhalation challenge. Our observations are supported by Tateishi and coworkers who observed no change in Creola bodies in sputum 0-2 and 6-9 h after exercise challenge as compared to an increase after allergen inhalation challenge (35).

Allergen is a stimulus known to cause sputum eosinophilia in patients with atopic asthma. Allergen inhalation challenge was included in the current study to document the development and magnitude of sputum eosinophilia observed in these patients with mild, atopic asthma following allergen-induced bronchoconstriction of a magnitude similar to that after exercise. Allergen inhalation is associated with rapid release of leukotrienes into the airway (36), which are excreted in the urine where they can be measured. Release of leukotrienes into the airway is thought to play an important role in the development of allergen-induced airway inflammation, as pharmacological treatment with drugs interrupting the leukotriene pathway is effective in attenuating these allergen-induced inflammatory responses (15). Similarly, exercise-induced bronchoconstriction has been reported to increase urinary levels of leukotrienes (16, 17), through mast cell activation and subsequent release in the airway (37). Other laboratories have been unable to measure exercise-induced increases in urinary leukotriene levels (38, 39), suggesting that exercise-induced bronchoconstriction may increase the production of leukotrienes, but at very low levels. Inhaled concentrations of LTD₄ causing a fall in FEV₁ similar to that measured after exercise challenge does not increase sputum eosinophils 4, 7, or 24 h after inhalation. Acute increases in airway levels of leukotrienes effective in producing a submaximal bronchoconstrictor response may not be sufficient to cause airway inflammation, as submaximal bronchoconstriction by inhalation of the potent bronchoconstrictor LTD₄ does not induce airway inflammation (23), whereas submaximal bronchoconstriction induced by inhalation of higher concentrations of the less potent metabolite LTE₄ can induce airway inflammation (12). This suggests that high airway levels of leukotrienes may be critical for generation of an inflammatory response. Appropriate mediators or cofactors that are present after allergen inhalation, such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-5 (IL-5), may also be necessary to induce an eosinophilic inflammatory response following exercise challenge. Inhibitory mediators released after exercise may modulate the airway response to leukotrienes. Prostaglandin E₂ (PGE₂), for example, is released during exercise (40) and has been implicated as a bronchodilator and antiinflammatory mediator. Inhalation of exogenous PGE₂ has been shown to regulate exercise- and allergen-induced bronchoconstriction (41, 42), and allergen-induced airway inflammation (43). Mediators such as this may inhibit inflammatory effects of leukotrienes synthesized during exercise in patients with asthma, however, the role of bronchodilator mediators in controlling airway function during exercise has yet to be resolved.

The maximum percentage fall in FEV₁ was greater with methacholine than with exercise challenge, however, the AUC (0-2 h) was the same. We were satisfied that the control methacholine challenge provided a similar overall bronchoconstrictor response. The airway response to exercise has a rapid onset and recovery, similar to that after methacholine and LTD₄ inhalation (23), but different than the slow onset and prolonged recovery following allergen inhalation. This suggests that exercise-induced bronchoconstriction in these subjects with asthma operates through binding of mediators, such as leukotrienes, to specific receptors on airway smooth muscle, and is supported by studies demonstrating attenuation of exercise-induced bronchoconstriction with antileukotriene drugs (17, 20, 21). The mechanism of allergen challenge, in contrast, is cell mediated and can result in a late airway bronchoconstrictor response, airway hyperresponsiveness, and airway inflammation (8, 28).

We observed sputum neutrophilia after exercise challenge and after methacholine challenge. The increase in percentage sputum neutrophils observed after these challenges is likely a result of the sputum induction procedure, as repeated induction of sputum has been shown to cause elevations in the percentage of sputum neutrophils (44, 45). The sputum macrophages measured following challenges are most likely depressed as a direct result of the observed neutrophilia, as has been preciously shown in our laboratory (23).

We cannot dismiss the possibility that exercise can cause a small degree of airway inflammation, at levels below the detection limit of our sampling methods. Although there are a number of studies that demonstrate neither methacholine inhalation nor sputum induction is associated with changes in the level of airway eosinophilia (8, 22, 23, 44), it may be possible that these procedures could mask any small change in inflammatory cell differentials. We did observe a reduction in peripheral blood eosinophils at 4 h after exercise compared to the number measured 24 h postexercise. This is similar to, but less pronounced than the kinetics of peripheral blood eosinophilia following allergen challenge, where eosinophil levels are lower than diluent control initially, followed by a rebound in numbers at 24 h exceeding the preallergen baseline level (45). If eosinophils were being recruited from the blood into the airway, we would expect to be able to measure an increase in airway eosinophil levels. Furthermore, there was no increase in sputum metachromatic cells (mast cells and basophils) after exercise challenge. Although mast cells have been reported to become activated after exercise challenge (37), exercise does not appear to alter the kinetics of mast cell and basophil recruitment into the airway as does allergen challenge (9).

In summary, this study could not demonstrate any increase in airway eosinophils measured in sputum samples between 2 and 24 h after exercise. Further studies will be required to determine whether this is due to the relatively low levels of exercise-induced leukotriene release in the airways, a lack of cofactors that may be necessary for the development of airway inflammation, or the presence of antiinflammatory/inhibitory factors produced during exercise.