INTRODUCTION

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Asthma is a chronic inflammatory disorder of the airways characterized by infiltration of the airways with inflammatory cells such as eosinophils, and by the presence of nonspecific airway hyperresponsiveness (AHR) to a variety of stimuli. Although examination of bronchial mucosal biopsies and bronchoalveolar lavage (BAL) obtained by fiberoptic bronchoscopy remains the standard research procedure for the assessment of airway inflammation, less invasive methods have recently been developed to assess the inflammatory process. These noninvasive methods include the measurement of nitric oxide (NO) in exhaled breath (1) and the number of eosinophils in induced sputum (2). Levels of exhaled NO, and the number of eosinophils in induced sputum or eosinophils in bronchial biopsies and BAL have all been shown to be elevated in asthma (1). However there has been little work examining the interrelationships of these individual parameters. There are correlations between AHR and eosinophils in BAL and biopsies (3), and also between AHR and exhaled NO (4). Another important observation that links these markers with the inflammatory process in asthma relates to the effects of inhaled corticosteroid therapy on these various markers. Inhaled steroid therapy has been shown to decrease exhaled NO levels (5), and eosinophils in bronchial biopsies, BAL, and induced sputum in separate studies (6, 7). In order to further analyze the relationship between changes in eosinophil counts measured in the airways, exhaled NO, and AHR, we examined the effect of inhaled steroid therapy on these markers in the same patients, in order to determine the interrelationships between these various inflammatory parameters that can all be modulated with inhaled steroids.

	METHODS

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Patients

Fourteen patients with mild stable asthma (Table 1) receiving treatment with only the inhaled ₂-adrenergic agonist aerosol albuterol for intermittent relief of wheeze were recruited. All patients demonstrated a > 15% improvement in FEV₁ following 200 µg of albuterol and airway hyperresponsiveness to methacholine with a provocative concentration of methacholine producing a 20% fall in FEV₁ (PC₂₀) of < 4 mg/ml. All patients were atopic as defined by two or more positive skin prick tests to common allergens. None of the subjects studied had received oral or inhaled corticosteroids for the preceding 12 mo, or any other treatment apart from inhaled ₂-agonists. Current smokers or ex-smokers of more than 5 pack-years and patients with FEV₁ less than 80% predicted were excluded.

Study Design

The study was a 12-wk double-blind randomized cross-over study comparing the effects of the inhaled glucocorticosteroid budesonide (800 µg twice daily) via a multidose dry powder inhaler (Turbohaler; Astra, Draco, Sweden), with that of a matched placebo (Figure 1). This dose of inhaled steroid was chosen in order to observe a maximal effect on the various inflammatory parameters. Each treatment was administered for 4 wk, separated by a 4-wk washout period. Patients were asked to chart their peak flow rates twice daily and to chart their symptom scores and medication usage. Induced sputum was obtained at Day 1 and all patients were reviewed at Days 14 and 21 of each treatment limb, with repeat sputum induction and spirometry and airway responsiveness to methacholine performed at Day 26. At Day 28, fiberoptic bronchoscopy was performed. The study was approved by the Royal Brompton Hospital Ethics Committee, and all patients gave their informed consent.

View larger version (14K): [in this window] [in a new window]   Figure 1.   Schematic diagram of study design.

Lung Function and Challenge Tests

Baseline prebronchodilator spirometric parameters were recorded from the best of three attempts using a dry wedge spirometer (Vitalograph, Buckingham, UK), with measurements not varying by more than 5% or 0.2 L being acceptable. Spirometric measurements and provocation tests were performed at the beginning of each treatment period and at Day 26 of the treatment period. All patients abstained from using their inhaled ₂-agonists and from caffeine-containing foods for 12 h before the test. After completing a baseline spirometric measurement, a standardized bronchial provocation protocol was performed using nebulized buffered (pH: 6.0) isotonic methacholine solution. After an initial nebulized saline challenge, doubling doses of methacholine were administered starting at a dose of 0.06 mg/ml via a dosimeter (Mefar, Bovezzo, Italy). Spirometric values were recorded 2 min after administration until the first blow of a 20% fall in FEV₁ was achieved; the concentration of methacholine needed to cause a 20% fall in postsaline FEV₁ was calculated from concentration response curves by linear interpolation. Peak expiratory flow (PEF) measurements were undertaken by patients at home using a mini-Wright peak flow meter (Clement Clarke, Harlow, UK). Patients were instructed to record their peak flow rates and symptom scores in a diary card. Peak flow variability was calculated from the difference between the highest and lowest daily reading divided by the highest PEF reading multiplied by 100.

Nitric Oxide Measurements

Exhaled NO was measured by chemiluminescence analyzer (Model LR2000; Logan Research, Rochester, UK), with sensitivity from 1 ppb to 100 ppm of NO, accuracy ± 0.5 ppb, and response time of < 2 s to 90% of full scale. In addition, the analyzer also measured CO₂, expiration flow and pressure, and the exhaled volume in real time. The analyzer was fitted with a biofeedback display unit to provide visual guidance for the subject to maintain the pressure and exhalation flow within a certain range (3 ± 0.4 mm Hg and 5 to 6 L/min for end-exhaled NO measurements), hence improving test repeatability and enhancing patient cooperation. The sampling rate was 250 ml/min for all measurements. The analyzer was calibrated daily using NO-free certified compressed air to set absolute zero and then a certified concentration of NO in nitrogen of 90 ppb and 500 ppb (BOC Special Gases; Surrey Research Park, Guildford, UK), and certified 5% CO₂ (BOC). Ambient air NO levels were recorded and the absolute zero was adjusted prior to all measurements. For the exhaled measurements, subjects were exhaling slowly from total lung capacity over 20 to 30 s with exhalation flow 5 to 6 L/min, bypassing the analyzer and thus with minimal resistance to flow. NO was sampled from a side-arm attached to the mouthpiece. The mean value of the last 100 measurements, acquired with 0.04-s intervals, was taken from the point corresponding to the plateau of end-exhaled (CO₂ reading 5 to 6%), and representing the lower respiratory tract sample. Results of the analyses were computed and graphically displayed on a plot of NO and CO₂ concentrations, pressure, and flow against time.

Sputum Induction and Processing

The method of sputum induction and processing has been previously described (7). Spirometry was recorded before and 15 min after 200 µg of inhaled albuterol via a metered-dose inhaler. Subjects were instructed to mouthwash thoroughly with water, followed by inhalation of 3.5% saline nebulized via an ultrasonic nebulizer (DeVilbiss 99; DeVillbiss Co., Heston, UK) at maximal output. Subjects were encouraged to cough deeply after 5 min and at 3-min intervals thereafter until an adequate amount of sputum has been obtained. Mouthwashing before each cough was encouraged in order to minimize salivary contamination. The sample from the first cough was discarded as this is heavily contaminated with squamous epithelial cells (8). At least 2 ml of sputum was collected into a 50-ml polypropylene tube, kept at 4° C, and processed within 2 h. An adequate sample was defined if the patient was able to expectorate at least 2 ml of sputum and if the number of squamous epithelial cells in fresh sputum specimen was < 50% of the total number of inflammatory cells. Spirometry was repeated after the sputum induction. Any subject experiencing a decrease in FEV₁ of more than 15% of postalbuterol FEV₁ was observed until it had returned to the baseline.

Hanks' balanced salt solution (HBSS, 2 ml) containing 1% dithiothreitol (DTT; Sigma Chemicals, Poole, UK) was added to the sputum sample. This was vortexed and repeatedly aspirated many times at room temperature until the sputum was homogenized. The sputum volume was then recorded. Samples were further diluted with HBSS up to 10 ml, vortexed briefly and centrifuged at 400 × g for 10 min. The cell pellet was resuspended. Total cell counts were performed on a hemacytometer using Kimura stain. Cytospin slides were prepared and stained with May-Grunwald-Giemsa. Differential cell counts were performed by an observer blind to the clinical characteristics of the subjects. At least two slides were used for counting and 300 inflammatory cells were counted on each slide.

Fiberoptic Bronchoscopy

Subjects attended the bronchoscopy suite at 8:30 A.M. after having fasted from midnight and were pretreated with atropine (0.6 mg intravenously) to midazolam (5 to 10 mg intravenously). Oxygen (3 L/min) was administered via nasal prongs throughout the procedure and oxygen saturation was monitored with a digital oximeter. Using local anesthesia with lidocaine (4%) to the upper airways and larynx, a fiberoptic bronchoscope (Olympus BF10; Key-Med, Essex, UK) was passed through the nasal passages into the trachea. BAL was performed from the right middle lobe using 0.9% NaCl warmed to 37° C, with 4 successive aliquots of 60 ml. Four mucosal biopsies were taken from segmental and subsegmental bronchi. BAL cells were spun (500 × g for 10 min) and washed twice with HBSS. Cytospins were prepared and stained with May-Grunwald stain for differential cell counts. Cell viability was assessed by trypan blue exclusion.

Bronchial Biopsy and Processing of Tissues

Bronchial mucosal biopsies were immediately placed in optimal cutting temperature (OCT) embedding media, then snap frozen in isopentane precooled with liquid nitrogen and stored at 70° C. All biopsies were frozen within 20 min of collection. Six-micron sections cut on a cryostat were placed on poly-L-lysine coated microscope slides (Sigma), air dried for 30 min, then wrapped in aluminum foil and stored at 70° C before immunostaining.

In order to stain for the presence of inflammatory cells, a mouse monoclonal anti-human major basic protein (MBP) antibody (Monosam; Bradsure Biological, Loughborough, UK) was used for eosinophils, and a mouse monoclonal antibody to CD68 (Dakko, High Wycombe, Bucks, UK) for macrophages. Following the primary antibody, a biotinylated rabbit anti-mouse immunoglobulin (1:100) followed by peroxidase-conjugated avidin (1:200) was used. Chromogen fast diaminobenzidine was used for 5 min and the slides were counterstained in hematoxylin and mounted on mounting medium (dextropropoxyphene, DPX).

Cell Counts

Counts of positive immunoreactive cells were made on all sections and divided according to whether the immunoreactivity was in the airway epithelium or beneath the epithelium to a depth of at least 175 µm representing the subepithelium. For inflammatory cells, the number of positive cells were expressed as the number per field. At least four separate fields at ×400 magnification were examined. On each field, a length of epithelium 175 µm long, and an area of subepithelium of 175 × 175 µm² were counted. The use of 175 µm is based on the measuring graticule of the microscope at ×400 magnification (Zeiss, Germany). Immunoreactivity was graded from 0 to 5 depending on the presence of positive staining cells within one row of the counting grid. Zero was defined as no positive staining within any rows of the counting grid; 1 was defined as positive staining within one row of the counting grid; and 5 was defined as staining within five grid rows. All counts were made by an experienced observer unaware of the origin of the sections. The coefficient of variation between observations was less than 10% by two different observers.

Data Analysis

Data are presented as means ± SEM. The data sets were explored for normality of distribution, and differences between following treatment and between treatment periods were assessed with the appropriate paired sample tests (9). We examined for differences between the two treatment periods and found no significant treatment order and period effect.

	RESULTS

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Effect of Budesonide on PEF, PEF Variability, and Medication Usage

Following inhaled steroid treatment, there were significant improvements in morning and evening peak expiratory flow rates (PEFR) and a significant reduction in peak flow variability over the two treatment periods (Table 2). Morning and evening PEF improved significantly, and diurnal PEF variability was significantly less after inhaled steroid treatment. There was no significant difference in rescue ₂-agonist usage or symptom scores over the recorded periods, although baseline values were very low in these mild asthmatic subjects.

Effect of Budesonide on FEV₁ and PC₂₀

There was no significant difference in FEV₁ and PC₂₀ between the start and the end of the placebo period. However, mean FEV₁ improved from 3.52 ± 0.13 to 3.77 ± 0.19 L (p < 0.05) and PC₂₀ from 0.42 ± 0.14 to 1.74 ± 0.18 mg/ml methacholine (p < 0.005) after 1 mo of steroid treatment. There was a statistically significant improvement in FEV₁ comparing the end of the active treatment arm with the end of the placebo treatment arm (Table 2). There was a trend toward improvement in PC₂₀; however, the difference between log PC₂₀ at the end of the inhaled steroid and placebo periods did not achieve statistical significance (p = 0.07).

Effect of Budesonide on Exhaled NO and Sputum Eosinophils

There was no significant difference in exhaled NO concentrations between the start and the end of the placebo periods, but exhaled NO was significantly reduced with steroid treatment from 42.6 ± 6.2 to 19.0 ± 3.0 ppb (p < 0.001). There was a significant effect of inhaled steroids on exhaled NO comparing the end of the active treatment arm with the placebo treatment arm of the study (Table 2). Sputum cytospins of adequate quality in pairs before and after treatment with either placebo or inhaled budesonide were available in 10 patients. There was a significant reduction in the percentage of sputum eosinophils from 4.9 ± 1.8 to 1.4 ± 0.8% following steroid treatment (p < 0.05) (Table 2).

Effect of Budesonide on Eosinophils in BAL and Bronchial Biopsies

There were 11 pairs of BAL cytospins and 13 pairs of biopsies of adequate quality for examination. The effect of budesonide on BAL eosinophils and on eosinophil immunoreactivity in biopsies is shown in Table 3. There was no significant effect of steroid treatment on total BAL cells or BAL eosinophils. After treatment with inhaled steroids, there were significant reductions in epithelial and submucosal MBP-positive immunoreactivity scores from 1.11 ± 0.35 to 0.17 ± 0.09 (p < 0.01) in the epithelium, and 2.16 ± 0.32 to 0.94 ± 0.27 (p < 0.005) in the submucosa. There was no significant reduction in epithelial CD68 immunoreactivity (macrophages) but there was a significant reduction in submucosal CD68 immunoreactivity scores from 1.29 ± 0.16 to 0.67 ± 0.18 (p < 0.01).

Correlations

Correlation analyses were undertaken with measurements from patients taken at the end of each treatment period. The data were examined for distribution and the appropriate method of statistical correlation applied. All possible combinations were tried and significant correlations are summarized in Table 4.

There was a significant correlation between sputum eosinophils and FEV₁ (percentage of predicted) before commencing treatment with inhaled steroids (r = 0.63, p = 0.05), and between sputum eosinophils and PC₂₀ (r = 0.67, p < 0.05). Significant correlations were found between exhaled NO and PC₂₀ (r = 0.64, p < 0.05), and exhaled NO and peak flow variability (r = 0.65, p < 0.05) prior to treatment. No significant correlations between sputum eosinophils and biopsy MBP immunoreactivity or BAL eosinophils were found.

After treatment with inhaled steroids there was a significant correlation between log PC₂₀ and airway eosinophils immunoreactivity (r = 0.8, p < 0.05), and between PC₂₀ and BAL eosinophils (r = 0.74, p < 0.005). There was also a significant correlation between exhaled NO and BAL eosinophils after treatment with inhaled steroids (r = 0.64, p < 0.05).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

We have shown that inhaled budesonide in a group of mild asthmatics improved lung function, airway hyperresponsiveness, together with reduction of exhaled NO, and eosinophils measured in sputum and in airway biopsies. There is no currently accepted single marker of airway inflammation in asthma, and furthermore there are no studies exploring the interrelationship between all these parameters, which have been advocated as markers of airway inflammation in asthma. To this end we have performed this study to investigate the modulation of these parameters by inhaled steroids, and to compare direct measures of airway inflammation with markers that are not directly linked to airway inflammation but representative of clinical endpoints currently advocated for use in the assessment of asthma. Despite the mild asthma in our patients, we were able to demonstrate the effects of steroid action on various inflammatory parameters; among the markers studied, the inhibition of eosinophils in bronchial biopsies, which is the most direct marker, was the greatest and most consistent compared with the other parameters.

Physiological variables such as PEF, FEV₁, and AHR, which are presently used to assess asthma control, mirror the clinical severity of asthma (10). With respect to PC₂₀ for methacholine, conventional understanding of the relationship between responsiveness and airway inflammation suggests that airway responsiveness is increased in patients with more active airway inflammation and airway hyperresponsiveness is regarded as the best physiological index of asthma severity (11). Our results are consistent with others showing a trend toward improvements in these physiological variables after steroid treatment. However, all these variables which are used in the assessment of asthma treatment are not directly linked to airway inflammation and are affected by the use of bronchodilators. Therefore it is necessary to consider more direct measurements of airway inflammation.

Although exhaled NO has not been established as an inflammatory parameter, clinically exhaled NO is elevated in asthmatics compared with normal control subjects (12). NO is a free reactive radical synthesized by a diverse range of cells in the airways (13). NO synthases (NOS) catalyze the conversion of L-arginine to L-citrulline during which NO is produced. Functionally, NOS exists as constitutive and inducible forms. The inducible form of NOS (iNOS) is regulated at the level of transcription and can be activated by proinflammatory cytokines such as interleukin (IL)-1 and tumor necrosis factor- (TNF-), cytokines important in asthma (14). NO may have proinflammatory effects and the high amounts of NO formed by iNOS may be deleterious in asthma (15). Under circumstances of increased airway inflammation such as clinical exacerbations and late response after allergen change (16), exhaled NO levels are elevated, and the elevated NO can be modulated by steroids (17). Consistent with this and in keeping with previous studies, we observed a correlation between exhaled NO and log PC₂₀, and a correlation between peak flow variability and exhaled NO levels suggesting higher NO levels in patients with more severe asthma. Although the precise mechanism that links NO with airway inflammation and whether it reflects airway inflammation remain to be elucidated, our data suggest that exhaled NO may be a useful marker in the assessment of asthma control, which is noninvasive and easy to measure.

The examination of sputum induced by nebulization of hypertonic saline is widely advocated as a less invasive method to investigate airway inflammation. This technique is reproducible and enables the examination of cellular constituents, as well as quantitative measurements of cytokines important in inflammation (7, 18). The procedure is well tolerated by asthmatics and can be performed even in patients with severe airflow obstruction (19). Sputum eosinophils are higher in asthmatics compared with normal control subjects and higher in those individuals with poorly controlled asthma irrespective of treatment with inhaled steroids compared with steroid-naïve asthmatics. Moreover in patients who develop a late response after allergen challenge (8), sputum samples taken 24 h postchallenge had an eosinophilic response, increased amounts of eosinophil cationic protein (ECP), as well as increased concentrations of IL-5, which is probably the key cytokine responsible for this response. This suggests that the examination of sputum is representative of inflammation in the asthmatic airway. Our results are consistent with previous observations with a significant reduction in sputum eosinophils after treatment with steroids as well as a correlation between sputum eosinophils with baseline lung function and nonspecific reactivity.

The technique of BAL to obtain fluid and cells lining the airways has been important in establishing the importance of inflammation and various mediators in the pathogenesis of asthma (20). BAL findings have been related to spirometric measurements, bronchial hyperresponsiveness, and various clinical features of asthma (21), but it is poorly tolerated in severe asthma, which has limited its utility. Although the eosinophilic involvement in asthma is emphasized by several investigations of BAL, the increased number of eosinophil in the BAL seems to be less important than their activity (22). We were unable to observe a significant reduction in the number of BAL eosinophils, which is also in keeping with observations by Trigg and coworkers (6), suggesting that this may be a less sensitive parameter to monitor inflammation.

Examination of the number of inflammatory cells in airway biopsies may be the most direct marker of airway inflammation and has advanced the understanding of the inflammatory nature and immunopathology of asthma. Despite the importance of these findings, however, histology has not been advocated to monitor airway inflammation owing to the difficulty and invasive nature of the procedure and there is little reference data from disease groups. Common findings in relation to cellular inflammatory events in the airways of mild stable asthmatics are also present in the airway biopsies from fatal asthma, with most of the literature of bronchial histology comparing asthmatics with normal nonatopic control subjects (23). The importance of eosinophils in asthma is well recognized. However, there is little evidence in the literature that relates to the degree of airway inflammation to severity of asthma aside from exacerbations and fatal asthma (24). A link between eosinophilic inflammation and asthma severity has been suggested, but the correlation, although statistically significant, was poor (r = 0.42) (24). Furthermore, the assessment of airway biopsies is also subject to intrasubject variability in airway inflammation, which is related to anatomic site and time at which the biopsy is performed, for all inflammatory cell counts (25). Our results indicate a correlation between the degree of airway hyperresponsiveness and the number of activated mucosal eosinophils. This relationship is not evident during the placebo limb of the study, possibly due to a poor correlation between bronchial histology and asthma severity, which is amplified by the spectrum of asthma severity, even in mild asthmatics. There is evidence for the beneficial effects of inhaled steroids on airway inflammation and the current data are consistent with other studies, suggesting that the efficacy of inhaled steroids may well be in part related to reduction in inflammatory cells such as macrophages and eosinophils. These cells can produce products that mediate inflammation through the modulation of chemotactic cytokines, such as IL-5, granulocyte-macrophage colony-stimulating factor (GM-CSF), and RANTES (26, 27).

The inflammatory component of asthma is highly complex involving several different cell types and mediators released. It is likely to be of multifactorial pathophysiology. This study has shown the expected regulation of all the measured indices that reflect airway inflammation. Although possibly the most interesting aspects of this study were the correlation analyses, failure to find more significant relationships between the various inflammatory parameters and changes in the observed relationships after treatment with inhaled steroids may be due to the lack of power to detect a small difference within our group of steroid-naïve patients. However, it is difficult to find a more diverse group of patients not on anti-inflammatory agents, as current asthma management guidelines advocate the use of inhaled steroids in patients with daily symptoms (28). It is more likely due to the differential action of inhaled steroids on different indices and mechanisms that underlie airway inflammation. We found no internal correlation between the various methods of assessing eosinophils; this is consistent with previous studies (29). Although eosinophils may originate from different compartments within the respiratory tract, they remain sensitive to modulation with inhaled steroid therapy in sputum and mucosal biopsies, with a trend to reduction in BAL. There are no studies to date that examine the relationship between PC₂₀, sputum eosinophils, exhaled NO, BAL eosinophils, and bronchial histology in the composite assessment of airway inflammation and the effect of treatment on inflammatory indices. Our study suggests that inhaled steroids are likely to inhibit the various arms of the inflammatory process, which are not necessarily causally linked to each other. Despite the contribution of bronchial histology toward our understanding of asthma, it remains invasive, with limited utility in the assessment of airway inflammation. Hence there is a need for more studies to investigate less invasive measures of inflammation and its relation to asthma severity. The correlation data from our study suggest that NO, PC₂₀, and PEFR variability may be reasonable ways of assessing inflammation in our patients, however our patients may not have been the most suitable for an assessment of the most useful marker of inflammation in asthma because they had mild asthma that would not require inhaled corticosteroid therapy, according to published guidelines (28). In order to determine the most useful marker of inflammation in asthma, studies are required on a more diverse population of patients with more symptomatic asthma who require regular anti-inflammatory medication. The potential importance of using markers of airway inflammation is supported by the recent study by Sont and coworkers who reported improved outcome measures by using PC₂₀ to guide asthma management (30).

In summary, this study has shown that budesonide is an effective anti-inflammatory agent in mild asthma, capable of inducing a significant improvement in airway function, a trend toward improvement in airway reactivity, and a significant reduction of inflammatory parameters including exhaled NO, sputum eosinophils, and bronchial mucosal histology; however, BAL eosinophils were not significantly decreased. These inflammatory markers are all inhibited by inhaled steroids; however, the relatively weak associations between these changes suggest that they may represent different mechanisms that underlie the inflammatory response, which are all inhibited by inhaled steroids. The reduction in noninvasive inflammatory parameters, although not directly correlated with direct histologic reduction in inflammatory cell numbers within the airways, suggests these markers may be useful as parameters in the assessment of airway inflammation in asthma.