American Journal of Respiratory and Critical Care Medicine

In asthma, the acute increment of airway responsiveness caused by exposure to allergen is associated with influx of eosinophils into the airways. The relationship between chronic airway hyperresponsiveness and airway inflammation is unclear, as they do not change consistently following long-term anti-inflammatory treatments. We studied 71 patients with chronic asthma and allergic sensitizization to perennial allergens. Airway responsiveness was determined by inhalation of methacholine, and airway inflammation was quantified by induced sputum (n = 28) or bronchoalveolar lavage (n = 43) and bronchial biopsy (n = 20). The relationships between airway responsiveness and the numbers of different inflammatory cells were assessed by multiple regression analysis. No significant correlations were found between the degree of airway responsiveness and the numbers of inflammatory cells in sputum or bronchoalveolar lavage or bronchial biopsy. By contrast, baseline lung function was inversely related to the numbers of eosinophils and directly related to the numbers of macrophages. The eosinophil cationic protein contents of either sputum or bronchoalveolar lavage were significantly correlated with the percentages of eosinophils but not with airway responsiveness. We suggest that other factors (e.g., airway wall remodeling or autonomic dysfunction) may be responsible for most of the interindividual variability of airway responsiveness in asthma.

Although airway inflammation and hyperresponsiveness are recognized as major characteristics of bronchial asthma (1), their relationship is still poorly understood. Acute exposure to allergen causes an increase of airway responsiveness that is consistently associated with an influx of inflammatory cells in the airways (2), which may suggest a causal relationship between airway inflammation and hyperresponsiveness (3, 4). This conclusion, however, is questionable as treatment with inhaled steroids does not cause consistent decrements in airway hyperresponsiveness and numbers of inflammatory cells in the airways (5-9). Furthermore, recent morphologic and functional studies have shown that airway hyperresponsiveness may be sustained by airway wall remodeling (10) and inability to dilate constricted airways (11). If these inferences are correct, a close relationship between hyperresponsiveness and numbers of inflammatory cells in the airways should not be expected. Indeed, the results of correlation studies on baseline airway responsiveness and airway inflammation have been largely inconsistent, with an equal number of positive and negative reports (2-4, 6, 12-30). Small numbers of patients or inclusion of healthy subjects in some studies may in part explain this inconsistency. Moreover, in all previous studies, simple regression analysis was used, which is not appropriate when the dependency of a variable (e.g., degree of airway responsiveness) on a series of independent variables (e.g., inflammatory cells) is sought.

In this study, the relationships between the baseline lung function and the degree of airway responsiveness and the numbers of inflammatory cells in sputum, bronchoalveolar lavage (BAL), and bronchial biopsy were investigated in a fairly large sample of asthmatic patients by using multiple regression analysis. In this way, the effect of each cell type on airway responsiveness was evaluated independent of the effects of all other cell types using a combination of methods of evaluating airway inflammation.

Subjects

A total of 71 asthmatic patients were included in the study. Airway inflammation was evaluated in 28 patients (Group 1) by induced sputum and in 43 patients (Group 2) by BAL with (n = 20) or without bronchial biopsy. Nineteen patients of Group 2 were the object of previous reports (2, 31). All patients had a history of mild to moderate asthma of at least 2 yr in duration and were sensitized to perennial allergens (house dust mite or pet dandruff). None of the patients had suffered from infections of the upper respiratory tract or exacerbations of asthma in the previous month before the study. Subjects sensitized also to pollen were studied out of the relevant season.

The patients were informed on the methodology and the aim of the study, and only those who gave written consent were included.

All patients of Group 1 were studied at the Hammersmith Hospital in London and all those of Group 2 at the University of Genoa. Ethical permission was obtained from each institutional committee.

Study Protocol

Patients were screened by history, physical examination, spirometry, and skin prick testing. They were asked to return to the clinic after they had refrained from taking short-acting β2-stimulants for at least 12 h. Cromones had to be discontinued 1 wk before the study. None of the patients was on long-acting β2-stimulants or theophylline. None of the patients had received oral or inhaled steroids in the previous month at least. Only three patients of Group 1 and two of Group 2 had received inhaled steroids in the year preceding the study.

Subjects of Group 1 attended the laboratory on two different occasions within 1 wk: the first for methacholine challenge, the second for induced sputum. Subjects of Group 2 attended the laboratory on a single occasion to undergo methacholine challenge and, 1 to 3 h later, bronchoscopy.

Methacholine Challenges

Methacholine (Sigma Chemical Co., St. Louis, MO) was dissolved in distilled water and delivered by an ampul-dosimeter device (Mefar, Brescia, Italy) driven by compressed air at a pressure of 1.5 kg/m2 with 1-s actuations and 5-s intervals between breaths. Aerosols were inhaled during quiet tidal breathing.

For Group 1, a standardized challenge modified from that described by Dixon and Ind (32) was used. The output of the dosimeter was 9 μl per puff. Subjects took five inhalations of isotonic saline as control, which were followed by five inhalations each of doubling methacholine concentrations from 0.25 to 64 mg/ml corresponding to 0.01- and 2.8-mg doses. A 3-min interval was allowed before each concentration increment. FEV1 was measured by a dry wedge spirometer (Vitalograph, Buckinghamshire, UK) 1.5 min after each concentration and the highest of three acceptable measurements within 100 ml was retained to create dose-response curves.

For Group 2, a standardized dosimetric challenge (33) was used. After 20 inhalations of isotonic saline as a control, doubling doses of methacholine were inhaled from 0.02 to 5 mg. Incremental doses were obtained using three different methacholine solutions (1, 10, and 50 mg/ml) and varying the number of breaths. A 3-min interval was allowed before each dose increment. FEV1 was measured 1 min after each dose by a turbine spirometer (Micro Spirometer; Micro Medical Ltd, Rochester, UK), and the best of three acceptable measurements was retained to create dose-response curves.

The noncumulative doses causing a 15% fall of FEV1 from control (PD15) were calculated by interpolation between two adjacent points of the log dose-response curves.

Sputum Collection and Analysis

FEV1 was measured before and 10 min after inhalation of albuterol (200 μg by metered-dose inhaler). Then, ultrasonically nebulized (DeVilbiss 65; DeVilbiss Co., Somerset, PA) hypertonic (4.5%) saline was inhaled for 1, 2, 4, 8, and 16 min. FEV1 was measured 1 min after each inhalation period. Subjects were instructed to rinse their mouth with water and to cough and produce sputum after each inhalation period. The whole sputum sample was examined by inverted microscopy and portions were selected to minimize salivary contamination. Sputum specimens were examined within 2 h. Dithiothreitol (Sputolysin; Calbiochem Co., San Diego, CA) diluted (1/10) in distilled water was added in a volume corresponding to twice the weight of the selected sputum portion. After shaking for 20 min in a water bath at 37° C, the sample was further diluted with phosphate-buffered saline (PBS) in a volume equal to that of sputum plus dithiothreitol and PBS. The suspension was filtered through sterile gauze to remove mucus and centrifuged at 1,000 × g for 5 min. The supernatants were aspirated and stored at −70° C. The cell pellet was resuspended in a volume of PBS equal to that of the filtered suspension. The total cell count was determined by a Burkers chamber hemocytometer. The cell suspension was then centrifuged at 450 rpm for 6 min (Shandon 3 Cytocentrifuge; Shandon Southern Instruments, Sewickley, PA). Two cytospin slides were fixed by methanol and stained by May-Grunwald-Giemsa for differential count of 500 nucleated non-epithelial cells. Two further slides were fixed in Carnoy solution and stained with 0.5% toluidine blue at pH 0.1 for quantitation of metachromatic cell count on 1,500 cells. Only counts from cytospins with less than 20% squamous epithelial cells and cell viability exceeding 50% were retained.

Bronchoalveolar Lavage

Bronchoscopy was started when the FEV1 had returned within 10% of pre-methacholine challenge. Atropine (0.5 mg intramuscularly) and diazepam (10 mg intramuscularly) were given as a premedication. A fiberoptic bronchoscope (Olympus BF, type P10) was passed through the nose after local anesthesia (lidocaine, 2% solution) of the nostrils and instillation of adrenaline (0.1/1,000 solution, 1 ml each side). After local anesthesia of pharnyx and airways, the bronchoscope was wedged into a subsegmental branch of the right middle lobe. Five 20-ml aliquots of sterile saline were instilled and then aspirated at a negative pressure of 50 to 120 mm Hg.

The fluid recovered was filtered through two layers of sterile gauze and centrifuged at 500 × g for 5 min. The cell pellet was washed once and resuspended in Hanks' balanced salt solution without Ca2+ and Mg2+, at a concentration of 106 cells/ml. A small sample of the cell suspension was centrifuged (Cytospin; Shandon Southern Instruments), spinning approximately 100,000 cells at 500 rpm for 5 min onto a glass slide. Cells were air-dried and stained with Diff-Quik (Merz & Dade A.G., Dudingen, Switzerland) for differential count of 300 cells per slide, by light microscopy. Epithelial cells were not included in differential count.

Bronchial Biopsy

In each subject, four biopsies were taken from the tracheal carina and the right upper lobe bifurcation immediately after completion of BAL. After fixation in 10% buffered formalin solution at room temperature, the specimens were embedded in paraffin, cut at 5 μm with a rotative microtome, and stained with hematoxylin-eosin and toluidine blue. Microscopic examination was performed by two independent observers unaware of the precise aim of the studies. Too small and incorrectly oriented biopsies were discarded. Metachromatic cells, granulocytes, and lymphomonocytes lying within 200 μm from the basement membrane were counted by means of an eyepiece graticule at ×500 magnification over five fields. Endothelial cells, pericytes, and Schwann cells were not included in the count.

Eosinophil Cationic Protein Assay

The cell-free supernatants from 14 BAL and 13 sputum samples were assayed for eosinophil cationic protein (ECP) by fluoroimmunoassay (Pharmacia CAP System ECP FEIA; Kabi Pharmacia Diagnostic AB, Uppsala, Sweden). Briefly, anti-ECP covalently coupled to ImmunoCAP reacted with the ECP present in the supernatant. After washing, enzyme-labeled antibodies against ECP were added to form a complex. After incubation, unbound enzyme–anti-ECP was washed out and the bound complex was incubated with a developing agent. After stopping the reaction, the fluorescence of the eluate was measured in FluoroCount 96. The fluorescence was directly proportional to the concentration of ECP in the sample and expressed as nanograms per milliliter. The albumin concentration in the supernatant of BAL was determined by nephelometry and used to normalize ECP values for dilution. The sensitivity of ECP assay was 2 ng/ml.

Statistical Analysis

The PD15 values were log-transformed for statistical analysis and are presented as geometric means. All other data are presented as mean ± SEM. The relationships between airway responsiveness or baseline lung function and airway inflammation were assessed by multiple regression analysis with stepwise selection of the independent variables. The dependent variables were PD15 or FEV1; the independent variables were the absolute numbers of each inflammatory cell type in sputum, or BAL, or bronchial biopsy. Pearson's single correlation coefficients were also calculated. A value of p < 0.05 was considered statistically significant.

Mean total and differential cell counts in sputum, BAL, and bronchial biopsy for the two groups are presented in Table 1.

Table 1. CHARACTERISTICS AND CELL COUNT DATA OF THE TWO GROUPS

Group 1 (Sputum)Group 2 (BAL)Group 2 (BB)
Sex, male/female12/1639/416/4
Age, yr        40 ± 3      24 ± 125 ± 2
FEV1, % pred*         97 ± 3      88 ± 280 ± 2
MCh PD15, mg 0.0990.1230.073
Total cells, ml−1 or mm−2   7.6 ± 7.5 × 105  8.8 ± 1.6 × 104 892 ± 122
Macrophages, % 51.9 ± 3.887.9 ± 1.4
Lymphocytes, % or mm−2   0.5 ± 0.1 8.7 ± 1.1570 ± 91
Neutrophils, % or mm−2  38.1 ± 4.0 1.1 ± 0.2Absent
Eosinophils, % or mm−2  8.5 ± 1.9 2.0 ± 0.451 ± 19
Metachromatic cells, % or mm−2 < 0.1Absent2.5 ± 0.8
Epithelial cells, %  1.0 ± 0.3

Definition of abbreviations: BAL = bronchoalveolar lavage; BB = bronchial biopsy; MCh PD15 = dose (mg) of methacholine causing a 15% decrease of FEV1 from control.

*From reference 37.

Presented as geometric mean.

The results of multiple regression analysis of airway responsiveness (PD15) and baseline lung function (FEV1) against inflammatory cells in sputum or BAL or bronchial biopsy are summarized in Table 2. Neither in Group 1 nor in Group 2 was a significant proportion of the variability of PD15 explained by the multiple regression model including inflammatory cells in sputum or BAL or bronchial biopsy. By contrast, approximately one third of the variability of baseline lung function was explained by the presence of inflammatory cells in sputum or BAL. The FEV1 was inversely related (negative regression coefficients) to the numbers of eosinophils in sputum (p < 0.001) or BAL (p < 0.05) but directly related (positive regression coefficients) to the numbers of macrophages in sputum (p < 0.005) or BAL (p < 0.001). The relationships between FEV1 and neutrophils were inconsistent (direct in Group 1 but inverse in Group 2; p < 0.05 for both).

Table 2. RESULTS OF MULTIPLE REGRESSION ANALYSIS

MCh PD15 FEV1 (% pred )
r2 βr2 β
Sputum0.22 (p = NS)0.41 (p < 0.05)
 Macrophages0.560.76
 Lymphocytes−0.12−0.13
 Neutrophils0.380.44
 Eosinophils−0.58−1.00
 Epithelial cells0.08−0.05
BAL0.19 (p = NS)0.32 (p < 0.01)
 Macrophages0.590.72
 Lymphocytes−0.070.20
 Neutrophils−0.21−0.38
 Eosinophils−0.35−0.49
BB0.09 (p = NS)0.08 (p = NS)
 Lymphocytes−0.09−0.94
 Eosinophils−0.130.17
 Metachromatic cells−0.270.23

Definition of abbreviations: β = partial correlation coefficient; other abbreviations as in Table .

For comparison with previous studies, the simple regression plots of PD15 and FEV1 (% pred) against the percentages of eosinophils in sputum and BAL are shown in Figures 1 and 2. The large proportion of patients with high degrees of airway hyperresponsiveness (PD15 < 0.1 mg) despite low percentages of eosinophils, particularly in BAL, should be noted.

The mean ECP concentration in sputum was 505 ± 322 ng/ ml, and the mean ECP/albumin ratio in BAL was 316 ± 174 ng/ mg. In six supernatants of BAL, ECP was below the detectable limit. Both in sputum and in BAL, the ECP level was significantly correlated (r = 0.72 and r = 0.85, respectively; p < 0.005 for both) with the percentage of eosinophils but not with PD15.

This study shows that airway hyperresponsiveness in perennial allergic asthma is not closely associated with the presence of inflammatory cells (eosinophils, neutrophils, lymphocytes, or macrophages) in the airways. A weak relationship was demonstrated between baseline lung function and airway inflammation.

Comments on Methodology

This study was devised to investigate the association between baseline airway hyperresponsiveness and the numbers of inflammatory cells present in the airway lumen or mucosa. We cannot exclude that inflammatory cells deeper than 200 μm below the basement membrane may be related to airway responsiveness or airway obstruction. No attempt was made to measure inflammatory cell–derived products other than ECP. Furthermore, completely degranulated mast cells could not be counted and mast cells surrounded by granules, suggesting ongoing degranulation, were observed in only six bronchial biopsies. Therefore, the effect of ongoing mediator release from inflammatory cells cannot be evaluated.

No immunochemistry was done to study inflammatory cell activation. Eosinophil activation is suggested by the highly significant correlations between ECP levels and percentages of eosinophils in either sputum or BAL. As no significant correlations were found between ECP level and airway responsiveness, it seems unlikely that enumeration of EG2+ cells would have yielded different conclusions.

Patients of Group 1 underwent the methacholine challenge and sputum collection on different days within 1 wk. We are confident that no exacerbations occurred as there were no significant changes in baseline FEV1 (mean differences 0.8%; 95% upper confidence limit: 1.4%), no increase in peak expiratory flow variability (< 15% in all subjects), and no increase in bronchodilator consumption. Patients of Group 2 were challenged with methacholine shortly (1 h at least) before BAL and bronchial biopsy. There is no evidence that inhalation of methacholine alters the airway cellularity (34).

Finally, the bronchoconstrictor stimulus was methacholine, which acts directly on airway smooth muscle. A relationship between airway responsiveness to stimuli acting through mediator release and airway inflammation cannot be excluded.

Comments on Results

Chronic airway inflammation with influx of activated eosinophils (3, 4, 12, 15, 21, 22, 29) and mast cell degranulation (35) has been suggested as a mechanism responsible for airway hyperresponsiveness in asthma. Were airway responsiveness closely related to airway inflammation, it could be used for monitoring the severity of the disease and the efficacy of anti—inflammatory treatments. The results of the present study indicate that the degree of airway responsiveness to inhaled methacholine is not a predictor of the numbers of inflammatory cells in the asthmatic airways. Our results are straightened by the use of three different techniques for assessing airway inflammation.

No clear evidence of a close relationship between airway hyperresponsiveness and airway inflammation emerges from previous studies. In some studies, no significant relationships between airway hyperresponsiveness and eosinophilic inflammation could be demonstrated by using either BAL (2, 6, 8, 13, 14, 20) or bronchial biopsy (9, 16, 19, 23) or induced sputum (28, 30). Surprisingly, Jeffery and colleagues (7) found the highest numbers of eosinophils and mast cells in the bronchial mucosa of allergic normoreactive rather than hyperreactive individuals. In other studies, weakly significant correlations were reported between baseline airway responsiveness and eosinophils in BAL (3, 12, 15, 17, 21, 29) or bronchial biopsy (4, 22, 29) or sputum (24-27).

In some studies (12, 15, 29), healthy control subjects or subjects with airway hyperresponsiveness but no symptoms of asthma (24) were included. This may have biased the results, as healthy and asthmatic subjects cannot be considered as random samples from the same population. Reanalysis of the data of Pliss and associates (15) after exclusion of healthy subjects yielded no significant correlation between airway responsiveness and the percentage of eosinophils in BAL (r = 0.03, p = 0.3 compared with r = −0.48, p = 0.03 reported in the original paper). On the other hand, the use of simple regression analysis might have also biased the results because of the confounding effect of the other inflammatory cells present. Reanalysis of the data of Kirby and coworkers (3) by multiple regression analysis showed airway responsiveness to be significantly related to metachromatic cells but not to eosinophils in BAL.

The results of studies looking at the effects of anti-inflammatory treatments are consistent with a lack of close relationships between airway inflammation and hyperresponsiveness in asthma. Inhaled steroids caused a decrease of airway responsiveness that was paralleled by a decrease of eosinophils and other inflammatory cells in bronchial mucosa in only one uncontrolled study (9). In contrast, the majority of studies demonstrate a relative dissociation between the decrease of inflammatory cells and the decrease of airway hyperresponsiveness (5-8).

After experimental inhalation of allergen, there is an influx of eosinophils into the airways, preceding the development of the late-phase response and correlated with the increase of airway responsiveness to methacholine (2). This may appear at variance with the dissociation between airway inflammation and hyperresponsiveness found in this study. Modeling studies based on morphologic observations suggest that chronic airway hyperresponsiveness may be sustained by an increased thickness of the airway wall or hypertrophy of airway smooth muscle (10). In this scenario, the presence of particular inflammatory cell types in the airway is not a prerequisite for baseline airway hyperresponsiveness. This view is supported by the observation that in a large proportion of patients with high degree of airway hyperresponsiveness the number of eosinophils in BAL was less than 2%, which is the average usually reported in asthmatic subjects (36). Our finding and this discussion do not negate the wider view of some kind of general association, within the whole asthma population, between airway responsiveness and airway inflammation. In patients with more severe asthma, both measurements are likely to be increased compared to patients with milder disease. In addition, the influx of inflammatory cells following acute exposure to allergen is likely to cause a relatively small increment of airway responsiveness, which may be appreciated within but not between subjects.

We found significant inverse relationships between baseline FEV1 and eosinophilic airway inflammation consistent with previous studies (3, 15, 26, 27). We speculate that the baseline lung function is sensitive to differences in natural exposure to allergen to a greater extent than the airway responsiveness to methacholine. Moreover, there was a direct relationship between baseline FEV1 and the numbers of macrophages in either sputum or BAL. The role of macrophages in asthma is not clear and merits further investigation.

Conclusions

This study in a large sample of asthmatic patients indicates that chronic airway hyperresponsiveness is independent of the numbers of inflammatory cells in the airway lumen or mucosa. We suggest that other factors, e.g., airway wall remodeling or autonomic dysfunction, may be the major determinants of the interindividual variability of airway responsiveness in asthma (11). The clinical implications are that single measurements of airway responsiveness to pharmacologic stimuli cannot provide information on airway inflammation and vice versa.

Supported in part by a grant from the University of Genoa.

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