INTRODUCTION

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Targeting treatment to the eosinophilic airway inflammatory response in asthma is now a major recommendation of current asthma management guidelines (1). Budesonide, a potent topical glucocorticosteroid, is effective therapy for asthma and is believed to act by reducing airway inflammation (2). Potential mechanisms of action include a reduction in the eosinophil infiltrate (3), inhibition of vascular exudation, and inhibition of mucous secretion. Whereas the maximal benefit from inhaled steroids may take 2 to 6 wk of regular therapy (4), corticosteroids may exert an effect in a shorter period of time. In vitro studies have shown effects of corticosteroids within several hours (2, 5). Studies of the acute effects of corticosteroids in asthma demonstrate that lung function may improve within 6 h of administration of a single dose of prednisolone 40 mg (6) or inhaled corticosteroid (ICS) (7). These studies indicate that there is an acute effect of ICS on airway function in subjects with asthma.

The mechanism of the acute steroid effect in asthma is as yet unclear. It is known that there is a prompt decline in circulating eosinophils in peripheral blood after a single dose of oral corticosteroid (11). A reduction in airway eosinophils is reported with 2 wk of corticosteroid therapy (12, 13). One possible mechanism of this effect is through apoptosis or programmed cell death. The induction of eosinophil apoptosis could reduce eosinophil survival in the airway. Eosinophils undergo apoptosis in vitro (14) and in vivo (13) after corticosteroid therapy. They are then removed without cellular disruption by macrophages, protecting the surrounding tissue from damage (15).

We hypothesized that the short-term improvement in airway function, which occurs in the first 6 h after inhalation of budesonide, results from reduction in inflammation of the airways caused by accelerated clearance of eosinophils from the lung by apoptosis. The primary aim of this study was to determine the effects of a single dose of inhaled budesonide on sputum eosinophils, mast cells, and airway responsiveness in adults with asthma. A secondary objective was to determine whether the mechanism of clearance of eosinophils was by apoptosis. This was investigated by examining eosinophils and apoptotic eosinophils from induced sputum after a single inhaled dose of 2,400 µg budesonide.

	METHODS

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Study Design

The study was a randomized, double-blind, placebo-controlled, crossover trial. Subjects attended for three visits. Adults with chronic asthma were asked to withhold ICS for 4 d, inhaled short-acting ₂- agonists for 6 h, and theophylline for 48 h before each visit. The first visit was a screening visit and consisted of a medical history, allergy skin tests, baseline spirometry, sputum induction, hypertonic saline challenge, and an asthma symptom score. Subjects with induced sputum eosinophils  7% proceeded to the randomized treatment phase.

At Visit 2 subjects in whom the induced sputum eosinophil count was  7% were randomized to receive a single dose of either inhaled budesonide 2,400 µg (Astra Draco, Lund, Sweden) or placebo. The study medication was administered, under supervision, at approximately 9:00 A.M. as six inhalations from a multidose dry powder inhaler (Turbuhaler; Astra Draco, Lund, Sweden). Spirometry was performed hourly for 6 h starting at 9:00 A.M. and then, at 6 h postdose, airway responsiveness was measured by inhalation of hypertonic (4.5%) saline. Sputum was induced during this procedure and analyzed. A period of approximately 7 ± 3 d separated Visits 2 and 3. No study medication was given to subjects between Visits 2 and 3. Normal medication was resumed with subjects withholding ICS for 4 d before Visit 3. At Visit 3 subjects were crossed over to the other treatment and the assessments of Visit 2 were repeated. Identical Turbuhaler devices were used to ensure blinding, and allocation was concealed by using sealed, coded envelopes. The randomization sequence was generated by Astra using a random number generator.

Subjects

Forty-one subjects attending the Respiratory Outpatient Clinic at the John Hunter Hospital were screened for the study. All subjects included were 18 yr of age or older and had asthma as defined by American Thoracic Society (ATS) criteria (16). Subjects were excluded if they had been treated with oral corticosteroids or antibiotics within the previous 4 wk, or were current smokers. Subjects withheld medications as described, and no other changes in asthma medication were allowed during the study. No subjects were using long-acting ₂-agonists, leukotriene antagonists, or antihistamines. Twenty-six subjects were randomized, and all subjects gave written informed consent for this study, which was approved by the Hunter Area Research Ethics Committee.

Clinical Assessment

Skin-prick tests were performed with extracts (Dome/Hollister-Steir, Bayer Pharmaceuticals, Sydney, Australia) for house dust mites (Dermatophagoides pteronyssinus and Dermatophagoides farinae), mold mix (Alternaria, Aspergillus mix, Hormodendrum, Penicillium mix), mixed grasses, cat fur (cat hair and epithelium), and cockroach, together with positive (histamine) and negative (glycerine) controls. A skin-prick test was defined as positive if the weal diameter was 3 mm or greater at 15 min. A subject was considered atopic if a positive skin-prick test was recorded for any allergen. Spirometry was performed on a Minato Autospiro AS-600 (Minato Medical Science Co., Ltd., Osaka, Japan), and the best of three FEV₁ and FVC maneuvers was recorded. Subjects were asked to rate their asthma symptom severity using the modified Borg scale (17) hourly from 9:00 A.M. for Visits 2 and 3, before performing spirometry maneuvers.

Hypertonic Saline Challenge and Sputum Induction

Hypertonic saline (4.5%) was inhaled for doubling time periods (30 s, 1 min, 2 min, 4 min up to a cumulative time of 15.5 min) from a Timeter MP500 ultrasonic nebulizer (Oregon, Pike, PA) as described (13, 18) in order to assess airway responsiveness and induce sputum for analysis. FEV₁ was measured 60 s after each saline dose. If a satisfactory sputum sample was not obtained at the time the FEV₁ had fallen  20%, nebulization with 4.5% saline was continued for 4-min periods once the FEV₁ had returned to within 10% of baseline value. The dose of 4.5% saline delivered to the mouth was assessed by weighing the nebulizer cup and tubing before and after the challenge.

Sputum Analysis

Sputum was processed as described (13, 18). Briefly, opaque, mucocellular sputum portions were selected and a 300-µl aliquot was aspirated from the Petri dish using a positive displacement pipette. The aliquot was added to 2,700 µl of 1:10 dithiothreitol (Sputolysin; Calbiochem, La Jolla, CA), mixed (30 min at 37° C), and filtered through 50-µm nylon gauze. A total cell count was performed and cytocentrifuge slides prepared (Shandon Cytospin III, Sewicky, PA). The quality of induced sputum samples was assessed as described (18) by evaluating the presence of pulmonary macrophages, the extent of squamous contamination, and an adequate number of cells for analysis. This gave a quality score ranging from zero (poor quality) to 6 (good quality sample).

Eosinophil counts were expressed as the percentage of 400 granulated (neutrophils and eosinophils) polymorphonuclear cells on each of two slides fixed with methanol and stained with Chromotrope 2R (BDH Chemicals, Poole, UK). A cellular differential of 400 nonsquamous cells was also calculated and absolute cell counts reported. Mast cells were identified in Carnoy's fixed slides stained with acidic toluidine blue by counting 1,500 nucleated cells on each slide. Cells staining positive for the secreted form of eosinophil cationic protein (ECP) were identified using a monoclonal antibody (EG2+; Pharmacia, Cambridge, MA) which was detected using the alkaline phosphatase-antialkaline phosphatase (APAAP) technique. Positive control slides (bronchoalveolar lavage [BAL] eosinophils), an isotype control, and substrate controls were included with each staining run. Apoptotic eosinophils were recognized at light microscopy based upon the following features: cell shrinkage, nuclear coalescence (shift from bilobed to monolobed nucleus), and nuclear condensation. Two hundred eosinophils were examined under oil and their morphology categorized as normal or apoptotic, and the number of apoptotic eosinophils was expressed as a percentage of the total eosinophils counted, using a previously validated scoring system (11).

Statistics

The SAS statistical package Version 6.07 (SAS Institute Inc., Cary, NC) was used to perform the statistical analysis. The study was analyzed using an "all patients treated" (APT) approach. The efficacy variables were analyzed using analysis of variance with patient, treatment, and period as factors, and presented as mean differences between treatments ± SEM with confidence intervals (CI) and p values given when the two treatment groups were compared. The level of significance was set at p  0.05.

The provocation dose causing a 20% decrease in FEV₁ (PD₂₀) was calculated by linear interpolation of the dose-response curve and log transformed for analysis. The fold difference was calculated by subtracting ln(PD₂₀) for placebo from ln(PD₂₀) during budesonide treatment. The exponential of the difference was then calculated to produce the fold difference. The predicted normal FEV₁, FVC, and FEF_25-75% were calculated for each subject (19) and the changes in percent predicted FEV₁, FVC, and FEF_25-75% over time were calculated at each time point. The four subjects requiring bronchodilator therapy during the study period were excluded from the analysis of spirometry and PD₂₀. The difference in symptom severity was analyzed by calculating the mean maximal change at any time over the 6-h period when subjects were treated with placebo and budesonide. Wilcoxon's signed rank test was used for this analysis.

	RESULTS

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Subject Description

Of the 41 subjects enrolled in the study, 15 subjects failed to meet randomization criteria. Thirteen had an eosinophil count < 7%, one took budesonide the evening before randomization, and one was lost to follow-up. Twenty-six subjects (Table 1) were randomly assigned to treatment with 24 completing the study. Two subjects failed to complete the study. Subject 4 (male, age 47) was unable to provide an adequate sputum sample, and he was removed from the study having only received budesonide treatment. In addition, profuse oral thrush was noted between Weeks 1 and 2. Subject 11 (female, age 33) was removed because she required antibiotic therapy for an intercurrent illness (an infected tooth) during the placebo treatment period.

All subjects received the study medication as described. The mean (SD) time between each of the treatment periods was 7.5 (2.2) d. The mean (SD) time of inhalation of study medication when subjects received budesonide and placebo was 9:08 h (0:25 h), range 8:00 h to 10:06 h and 9:04 (0:17 h), range 8:18 h to 9:32 h, respectively. Four subjects required bronchodilator therapy for symptom control during the 6-h period of pulmonary function testing, and their symptom and lung function data were excluded from that point. There was no effect of treatment order and no carry-over effect was detected on the outcomes assessed.

Sputum Analysis

Sputum was successfully obtained from all subjects completing the study and had a mean (SD) quality score of 4.5 (1.5). Budesonide treatment resulted in a significantly lower sputum eosinophil count (Table 2, Figure 1). The difference between treatments was 12.2% (95% CI 3.2 to 21.2; p = 0.01, placebo  budesonide) in sputum eosinophils. Absolute cell counts demonstrated a reduction in eosinophils from 0.53 to 0.21 × 10⁶/ml with budesonide, whereas there was little change in neutrophil counts (Table 3). For mast cells, no significant difference was found between budesonide and placebo treatments (95% CI 0.4 to 0.3; p = 0.69). The number of apoptotic eosinophils (expressed as a percentage of the total number of eosinophils counted) and the number of activated eosinophils were similar after budesonide and placebo treatment (Table 2). The number of macrophages that had ingested eosinophils was slightly lower after budesonide.

View larger version (8K): [in this window] [in a new window]   View larger version (8K): [in this window] [in a new window]   Figure 1.   Mean (± SEM) sputum eosinophils (A) and mast cells (B) examined at 6 h after a single dose of 2,400 µg inhaled budesonide. There was a significant reduction in eosinophils (p < 0.05) but not mast cells.

Airway Responsiveness and Lung Function

Budesonide resulted in a significant 2.2-fold (95% CI 1.45 to 3.33) improvement in airway responsiveness to hypertonic saline. After placebo, PD₂₀ saline was 1.4 (3.3) ml and improved to 3.0 (4.2) after budesonide treatment. The difference between treatment (placebo  budesonide) for ln(PD₂₀) was 0.79 (95% CI 1.22 to 0.36; p = 0.0020, Figure 2). At 6 h FEV₁ percentage of predicted tended to be higher after budesonide than placebo (difference 2.7%, SE 1.6, Table 4, Figure 3), but this failed to reach significance (p > 0.05).

View larger version (19K): [in this window] [in a new window]   Figure 2.   Airway responsiveness to hypertonic saline expressed as PD20, examined 6 h after a single dose of inhaled budesonide (p < 0.05).

View larger version (9K): [in this window] [in a new window]   Figure 3.   Changes in FEV1 (% predicted) over 6 h after a single dose of budesonide (squares) or placebo (circles).

Asthma Symptom Scores

Asthma symptoms were rated hourly over 6 h using the Borg scale. Before treatment, symptom severity was rated at a mean (SD) of 2.1 (1.2), corresponding to "slight discomfort" from asthma. The maximal change in asthma symptom score was not different after treatment (mean difference = 0.02; SD = 1.1; p = 0.5).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

This study has successfully demonstrated that a single dose of inhaled budesonide significantly reduced sputum eosinophils 6 h after administration. This was accompanied by a 2.2-fold improvement in airway responsiveness to 4.5% saline. No significant difference in sputum mast cells was seen between the two treatments. The secondary objective of the study was to determine whether the mechanism of clearance of eosinophils from the lung was by apoptosis. This was examined and no significant difference was found between the two treatments. These results suggest that the mechanism of the anti-inflammatory action demonstrated by budesonide is not due to accelerated clearance of eosinophils by apoptosis.

This finding is in contrast to the results of a previous study (11) which looked at apoptosis of airway eosinophils during the resolution of an asthma exacerbation using inhaled beclomethasone treatment over a 2-wk period. It was found that sputum eosinophils were significantly reduced (as in the present study) and additionally, that the proportion of apoptotic eosinophils significantly increased as the exacerbation resolved. It may be that a longer duration of therapy or multiple dosing is required to induce recognizable apoptosis in airway eosinophils. The mechanism of the reduced eosinophil count was not identified in the present study but may involve a reduction in eosinophil influx as a result of the important effects that corticosteroids have on vascular exudation and cell adhesion mechanisms. Changes to vascular permeability of macromolecules are unlikely to alter the cellular composition of sputum, but may affect fluid phase markers.

Previous studies have demonstrated that corticosteroids are effective in reducing airway eosinophils. Oral corticosteroids have been shown to reduce eosinophils from bronchial biopsies over a period of 2 to 6 wk (20). ICS have a similar effect in reducing airway eosinophils from bronchial biopsy over a period of 1 wk (21). Sputum eosinophils have been reduced using ICS over 2 wk (12, 13, 22), and with oral corticosteroids in 48 h (23, 24). In the present study, it was found that the anti-inflammatory effect of ICS on airway eosinophils could be seen as early as 6 h after a single dose.

This is the first report of an acute effect of ICS on airway hyperresponsiveness (AHR) to hypertonic saline. Long-term corticosteroid treatment, between 3 wk and 12 mo, and using 300 to 1,200 µg/d is associated with improved airway responsiveness to histamine, methacholine, and distilled water (4, 25, 26). One open study has reported a positive effect of ICS on AHR to hypertonic saline (27). The effects of a single dose of budesonide were examined in children and an improvement in methacholine AHR was found 6 h after the dose (28). Vanthenen and coworkers (4) also reported a 2-fold increase in methacholine airway responsiveness 6 h after budesonide 800 µg. The present study differs from previous work in that a higher dose of inhaled steroid was used (2,400 µg) and the outcome was assessed using the indirect stimulus, hypertonic saline. This dose was chosen to mimic the maximal recommended ICS dose for use in unstable asthma, and the results complement studies showing an effect of ICS in acute exacerbations of asthma (9, 10, 29).

The study demonstrated a slight increase in FEV₁ after acute budesonide therapy, as has previously been reported (6, 8). It is possible that this effect on FEV₁ could have contributed to the change in airway responsiveness. However, there was no correlation between the two parameters, and the change in airway responsiveness was relatively larger than the small change in lung function, suggesting that it was not merely due to an improvement in airway caliber. It seems likely that the increase in PD₂₀ with budesonide was caused by its anti-inflammatory effect on the airways involving a reduction in eosinophil numbers. No improvement in asthma symptoms (as measured using the Borg scale) was seen. This may have been due to a "floor" effect. Subjects reported few symptoms before treatment and were sedentary during the study visits, thereby allowing little room for symptoms to be further reduced by treatment.

Asthma treatment guidelines recommend that oral corticosteroids should be given to treat asthma exacerbations (1). Recent studies (29) suggest that high-dose ICS may also be beneficial in mild asthma exacerbations, probably because of their anti-inflammatory activity. Together, these results indicate a need to examine the effects of high-dose ICS in acute asthma in more detail.

In conclusion, this study demonstrates that a single dose of budesonide 2,400 µg can improve both airway inflammation and AHR as early as 6 h after the dose. Induced sputum eosinophils, but not mast cells, were significantly lower after treatment. There was no difference in apoptotic eosinophils after treatment, suggesting that the mechanism of clearance of eosinophils was not by apoptosis. The acute effects of ICS warrant further evaluation and may identify a role for this therapy in mild exacerbations of asthma.