Abstract
It has been suggested in cross-sectional studies that provocation with adenosine 5 ′ -monophosphate (AMP) more closely reflects the inflammatory process in asthma than does provocation with methacholine or histamine. We investigated whether the steroid-induced improvement in the provocative concentration of AMP producing a 20% decline in FEV1 (PC20 AMP) is more closely associated with the concomitant reduction in airway inflammation than is the improvement in PC20 methacholine. In 120 asthmatic patients, we measured PC20 methacholine and PC20 AMP as well as sputum induction and nitric oxide (NO) in exhaled air before and after 2 weeks of treatment with corticosteroids. Improvement in PC20 AMP was solely related to reduction in airway inflammation (i.e., change in the number of sputum eosinophils, lymphocytes, epithelial cells, and concentration of NO in exhaled air). In contrast, improvement in PC20 methacholine was related to both reduction in airway inflammation (i.e., change in the number of sputum eosinophils and lymphocytes) and increase in FEV1 %predicted. The total explained variance of the improvement in bronchial hyperresponsiveness was greater for AMP than for methacholine (36% versus 22%, respectively). We conclude that PC20 AMP is more sensitive to changes in acute airway inflammation than is PC20 methacholine, further reinforcing the notion that PC20 AMP can be a useful tool for monitoring the effects of antiinflammatory therapy.
Keywords: adenosine 5′-monophosphate; hyperresponsiveness; inflammation; asthma; sputum
Both bronchial hyperresponsiveness (BHR) and airway inflammation are well-established features of asthma. Thus far, BHR has generally been measured with methacholine or histamine. Both of these substances act as direct stimuli, since they exert their effect directly on airway smooth muscle. Another stimulus with which to measure BHR is adenosine 5′-monophosphate (AMP). AMP is an indirect stimulus, since it has little effect on airway smooth muscle contraction in vitro (1). A major action of AMP appears to involve the release of histamine and other preformed mediators from immunologically primed mast cells, since AMP-induced bronchoconstriction is associated with an increase in the histamine level in plasma and bronchoalveolar lavage fluid (BALF) (2, 3). Moreover, the provocative concentration of AMP causing a 20% decrease in FEV1 (PC20 AMP) is inhibited by up to 80% by pretreatment with antihistamines (4). AMP may have some additional actions on neural pathways as well, since the airway response to it is partly attenuated by atropine and ipratropium bromide (5, 6).
We have recently demonstrated that PC20 AMP is more closely associated with airway inflammation than is PC20 methacholine (7). It has been suggested that PC20 AMP is also more sensitive to changes in airway inflammation, since it improves to a greater extent after therapy with corticosteroids than does PC20 methacholine (8, 9). However, it is still unknown whether this greater improvement in PC20 AMP is related to reduction in airway inflammation. The aim of our study was to investigate whether steroid-induced improvement in PC20 AMP is more closely associated with the concomitant reduction in airway inflammation than is improvement in PC20 methacholine. To investigate this, we tapered the dose of inhaled corticosteroids in 120 patients with mild to moderately severe asthma (10). Corticosteroids were subsequently started, with assessment of the relationship between the corticosteroid-induced improvement in both PC20 methacholine and PC20 AMP and the concomitant increase in the level of FEV1 % predicted and reduction in airway inflammation. The last of these was assessed from the volume of exhaled air and the number of inflammatory cells and concentration of eosinophil cationic protein (ECP) obtained by sputum induction.
Patients aged 18 to 65 yr with a diagnosis of asthma were included if they met the following criteria: PC20 methacholine ⩽ 8 mg/ml, at least one positive skin prick test among tests for 17 common aeroallergens, reversibility to β2-agonist ⩾ 9% of the predicted FEV1, and ability to expectorate sputum after inhaltion of hypertonic saline.
Doses of inhaled corticosteroids were tapered as described in a previous study (10). Patients were asked to visit the hospital on two consecutive days either when they had discontinued their inhaled corticosteroids completely for 3 wk, or before this, if they experienced subjective symptoms of a pending asthma exacerbation for which they felt that treatment with corticosteroids was desirable. Patients were then randomized to receive treatment for 2 wk with prednisone (30 mg/d), fluticasone (2,000 μg/d), or fluticasone (500 μg/d). Before and after 2 wk of treatment with corticosteroids, measurements were made of PC20 methacholine and PC20 AMP as well as sputum induction, and nitric oxide (NO) in exhaled air, all as described previously. The study protocol was approved by the local medical ethics committee, and all participants gave their written informed consent.
Exhaled NO was measured with the tidal breathing method, using a chemiluminescence analyzer (CLD 700 AL; ECO Physics, Duernten, Switzerland) as described previously (10).
FEV1 was measured with a calibrated water-sealed spirometer according to standardized guidelines (10, 11). Provocation tests were performed with a 2-min tidal breathing method adapted from Cockcroft and coworkers (12, 13). After an initial nebulized saline challenge, subjects inhaled doubling concentrations, ranging from 0.04 to 320 mg/ml, of AMP, and 0.038 to 19.2 mg/ml of methacholine-bromide, at 5-min intervals.
Sputum was induced by inhalation of hypertonic saline aerosols as previously described (10). Fifteen minutes after salbutamol (200 μg) inhalation, hypertonic saline (3%, 4%, and 5%) was nebulized for each concentration over a period of 7 min. Whole sputum samples were processed according to the method of Fahy and colleagues with some modifications (10, 14). Eosinophil cationic protein (ECP) was measured with the ImmunoCAP technique (Pharmacia, (Uppsala, Sweden) according to instructions provided by the manufacturer.
All calculations of PC20 were made with the base-2 logarithm (log2), since this reflects doubling concentrations and normalizes the distribution. For purposes of analysis, patients responding to saline were assigned a PC20 value that was half of the lowest concentration applied (15). Patients not responding to the highest concentration of methacholine or AMP were assigned a value of twice the highest concentration applied. The normality of distributions was assessed with the Kolmogorov–Smirnov test. If this test resulted in a value of p < 0.05, normalization by logarithmic transformation was attempted. Correlations between variables were calculated with Pearson's rank correlation test in cases of normal distribution or with Spearman's rank correlation test otherwise. To determine independent prognostic factors for PC20 methacholine and PC20 AMP, we used multivariate regression analysis in a stepwise algorithm (SPSS PC+ 10.0; SPSS Inc., Chicago, IL), with treatment group, use of corticosteroids (yes/no), and number of days for which corticosteroids were stopped at randomization being entered as covariates.
Between September 1995 and July 1997, 120 patients were enrolled in the study (Table 1). After the start of treatment, two of the 120 patients were lost from the study (one because of pregnancy and one because of loss of study medication). In this study, it was mandatory that patients completely discontinued their inhaled corticosteroids for at least 3 wk. During the steroid tapering period, 16 patients returned to the hospital earlier than scheduled because of symptoms of a pending asthma exacerbation. Of these 16 patients, six patients were still using inhaled corticosteroids at the start of the treatment period (three patients using 400 μg/d budesonide or beclomethasone, two patients 250 μg/d fluticasone, and one patient using 800 μg/d budesonide); the remaining 10 patients had discontinued their inhaled corticosteroids for a median period of 12 d (range: 2 to 21 d). It was not possible to perform hyperresponsiveness testing in all patients, owing to asthma symptoms and low values of FEV1. Thus, we were able to measure the PC20 methacholine and the PC20 AMP both before and after treatment with corticosteroids in 111 (94%) and 108 (92%) patients, respectively. Before treatment with corticosteroids, all patients were, by design, responsive to methacholine (PC20 ⩽ 8 mg/ml), and of 114 patients challenged with AMP, 102 (89%) were responsive (PC20 ⩽ 320 mg/ml). After treatment with corticosteroids, 97 of the 111 (87%) patients were responsive to methacholine, whereas 78 of the 108 (72%) patients were responsive to AMP.
| Before Therapy | After Therapy | Change (Δ) | p Value | |||||
|---|---|---|---|---|---|---|---|---|
| Age, yr | 27 (22–38) | |||||||
| Sex, M/F | 41/79 | |||||||
| Smoking, % | ||||||||
| Current | 26 | |||||||
| Nonsmoker | 74 | |||||||
| FEV1, %pred | 81 (67–92) | 85 (73–97) | 5.4 (−0.8–12.3) | < 0.01 | ||||
| PC20 methacholine, mg/ml | 0.54 (0.02–7.9)‡ | 1.5 (0.02–39.1)‡ | 1.5 (0.6–2.5)† | < 0.01 | ||||
| PC20 AMP, mg/ml | 4 (0.02–640)‡ | 42.8 (0.02–640)‡ | 3.1(0.4–5.6)† | < 0.01 | ||||
| Exhaled NO, ppb§ | 14 (11–20) | 11 (7–15) | −3.6 (−7.4–0.4) | < 0.01 | ||||
| Sputum ECP, ng/ml | 76 (33–250) | 35 (17–101) | −27.8 (−143–0.7) | < 0.01 | ||||
| Sputum | ||||||||
| Squamous cells, % | 7 (3–16) | 8 (4–19) | ||||||
| Total cell count, 103/ml | 519 (230–699) | 479 (328–810) | 84 (−142–272) | = 0.10 | ||||
| Macrophages, 103/ml | 198 (109–369) | 275 (142–423) | 47 (−41–190) | < 0.01 | ||||
| Lymphocytes, 103/ml | 9 (4–17) | 6 (3–12) | − 1 (−8–4) | = 0.06 | ||||
| Neutrophils, 103/ml | 138 (64–298) | 165 (80–295) | 16 (−85–119) | = 0.17 | ||||
| Eosinophils, 103/ml | 22 (7–60) | 3 (0–12) | −11 (−42–−1) | < 0.01 | ||||
| Bronchial epithelial cells, 103/ml | 10 (4–26) | 13 (5–29) | 2 (−10–14) | = 0.36 |
Both the PC20 methacholine and the PC20 AMP improved significantly with steroid therapy, by factors of 1.5 and 3.1 doubling concentrations, respectively (Table 1). Furthermore, the FEV1 % pred increased significantly after therapy with corticosteroids, whereas the number of eosinophils and macrophages in sputum, the concentration of ECP in sputum, and the concentration of NO in exhaled air all decreased significantly after therapy with corticosteroids.
A significant positive correlation was found between the change in PC20 methacholine and PC20 AMP with the change in the level of FEV1 % pred (r = 0.31, p < 0.01, and r = 0.36, p < 0.01, respectively) (Table 2; Figure 1). There was a significant negative correlation between the change in PC20 methacholine and PC20 AMP on one hand and the change in the number of sputum eosinophils on the other hand, the correlation being stronger for the change in PC20 AMP (r = −0.28, p < 0.01, and r = −0.43, p < 0.01, respectively) (Figure 2). Furthermore, there were significant negative correlations between changes in PC20 AMP and the change in the concentration of ECP in sputum and NO in exhaled air, and significant positive correlations between changes in PC20 methacholine and the change in the number of sputum lymphocytes and macrophages. Other correlations were not significant.
| ΔPC20 Methacholine† | ΔPC20 AMP† | |||||||
|---|---|---|---|---|---|---|---|---|
| ΔFEV1 %predicted | r = 0.31 | p = 0.001 | r = 0.36 | p = 0.0001 | ||||
| Sputum differential | ||||||||
| ΔEosinophils†, 103/ml | r = −0.28 | p = 0.004 | r = − 0.43 | p = 0.000005 | ||||
| ΔLymphocytes†, 103/ml | r = 0.27 | p = 0.052 | r = 0.15 | p = 0.132 | ||||
| ΔMacrophages†, 103/ml | r = 0.25 | p = 0.01 | r = 0.01 | p = 0.901 | ||||
| ΔNeutrophils†, 103/ml | r = 0.14 | p = 0.15 | r = − 0.08 | p = 0.441 | ||||
| ΔBronchial epithelial cells†, 103/ml | r = 0.05 | p = 0.59 | r = − 0.14 | p = 0.17 | ||||
| ΔSputum ECP†, ng/ml | r = − 0.07 | p = 0.483 | r = − 0.24 | p = 0.015 | ||||
| ΔNO exhaled breath, ppb | r = − 0.18 | p = 0.065 | r = − 0.38 | p = 0.0001 | ||||
Fig. 1. Relationship of improvement in PC20 methacholine and in PC20 AMP (expressed in doubling concentrations [DC]) to the increase in FEV1 % predicted.
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Fig. 2. Relationship of improvement in PC20 methacholine and in PC20 AMP (expressed in doubling concentrations, [DC]) to the decrease in the number of sputum eosinophils. The value for the number of sputum eosinophils is log transformed to normalize the distribution.
[More] [Minimize]In a multivariate linear stepwise regression model, changes in both PC20 methacholine and in PC20 AMP were independently negatively correlated with the change in the number of eosinophils and positively correlated with the change in the number of lymphocytes in sputum (Table 3). In addition, the change in FEV1 % pred was independently and positively correlated with the change in PC20 methacholine but not with the change in PC20 AMP. Both the change in the concentration of NO in exhaled air and the change in the number of bronchial epithelial cells in sputum were independently and negatively correlated with the change in PC20 AMP. The total explained variance was larger for changes in PC20 AMP than for changes in PC20 methacholine (total explained variance = 36% and 22%, respectively). In the model, three additional covariates were entered, but remained non-significant: treatment group, the use of inhaled corticosteroids at randomization (yes/no), and the number of days without inhaled corticosteroids before randomization.
| ΔPC20 methacholine | ΔPC20 AMP | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| β* | Explained Variance | β* | Explained Variance | |||||||
| ΔSputum lymphocytes, 103/ml | 0.31 | ΔSputum eosinophils, 103/ml | − 0.39 | |||||||
| ΔSputum eosinophils, 103/ml | −0.26 | ΔSputum lymphocytes, 103/ml | 0.34 | |||||||
| ΔFEV1 %pred | 0.18 | ΔNO exhaled air, ppb | − 0.29 | |||||||
| ΔSputum bronchial epithelial cells, 103/ml | − 0.19 | |||||||||
| 22% | 36% |
This study demonstrates that corticosteroid-induced improvement in PC20 AMP is more closely associated with the concomitant reduction in airway inflammation than is improvement in PC20 methacholine. First, improvement in PC20 AMP was solely associated with reduction in airway inflammation in the multivariate regression analysis (i.e., as indicated by the change in the number of sputum eosinophils, lymphocytes, and epithelial cells and the concentration of NO in exhaled air), but was not associated with the increase in FEV1 % predicted. In contrast, improvement in PC20 methacholine was associated with both reduction in airway inflammation (i.e., the change in the number of sputum lymphocytes and eosinophils) and the increase in FEV1 % predicted. Second, the total explained variance of the model estimating the improvement in BHR with corticosteroid administration was much greater for AMP than for methacholine (36% versus 22%). The results of this analysis were independent of the treatment group (prednisone at 30 mg/d, fluticasone at 2,000 μg/d, or fluticasone at 500 μg/d).
Improvement in PC20 methacholine was associated with a reduction in airway inflammation in both monovariate and multivariate regression analyses. It has now been generally accepted that airway inflammation contributes to the presence and severity of BHR. However, the direct association between airway inflammation and BHR has been the subject of much controversy, with almost as many negative as positive reports in the literature (16-20). Therefore, other factors are likely to be involved in the development and maintenance of BHR (21). A possible explanation for this can be as follows: BHR consists of a variable component and a fixed component. The variable component is largely caused by airway inflammation and has been illustrated to a great extent in allergen exposure tests (18, 22). Activation of different inflammatory cells induces vascular leakage and edema, with a concomitant increase in airway wall thickness (23). In addition, airway smooth-muscle cells become activated, and together, this contributes to an increase in bronchial responsiveness after inhalation of a bronchoconstrictor stimulus (23). The fixed component of BHR is caused by structural changes in the airway wall, which have been consistently found in asthmatic airways and are generally referred to as “airway remodeling.” Airway remodeling causes thickening of the airway wall due to the deposition of fibrous proteins, an increase in airway smooth-muscle mass due to hypertrophy and hyperplasia, and hypertrophy of mucus-secreting glands (24, 25). Furthermore, the contractile force of airway smooth muscle increases as a result of hypertrophy and hyperplasia (26). It has indeed been demonstrated that a greater degree of reticular basement membrane thickening, a component of airway remodeling consistently found in asthmatic airways, is associated with more severe BHR and a lower FEV1 (27-29). It is difficult to dissect the variable from the fixed component of BHR in cross-sectional studies, in which only one measurement is available. Longitudinal studies can dissect the variable component from the fixed component, since by definition it is the variable component that changes over time.
In accord with this, Ichinose and colleagues recently showed that the variable component of PC20 methacholine (i.e., its change after therapy with corticosteroids) is associated with a reduction in airway inflammation, whereas no association between PC20 methacholine and airway inflammation could be found at baseline (30). The findings of Ichinose and colleagues are compatible with ours. We extended the observation of Ichinose and colleagues in two ways. First, we measured bronchial responsiveness to both a direct stimulus (methacholine) and an indirect stimulus (AMP). In monovariate regression analysis, improvement in PC20 AMP was associated with a reduction in eosinophils, as was PC20 methacholine. Additionally, improvement in PC20 AMP was associated with a decrease in the concentration of ECP in sputum. Second, we performed a multivariate regression analysis.
Interestingly, multivariate regression analysis revealed that steroid-induced improvement in PC20 AMP, acting indirectly via the release of mediators from immunologically primed mast cells, is more closely associated with reduction in airway inflammation than is improvement in PC20 methacholine. The most likely explanation for this is that a generally decreased inflammatory process results in a decreased production of cytokines and other mediators that stimulate mast cell chemotaxis, maturation, or activation directly or indirectly. Alternatively, the number or activation state of eosinophils may be reduced by a decreased production of inflammatory mediators by mast cells. Another possible explanation may be that corticosteroids exert an effect on the adenosine receptors or the postreceptor mechanisms of mast cells, although such an effect has not so far been reported.
In concordance with others, we found that PC20 AMP improved to a greater extent after treatment with corticosteroids than did PC20 methacholine (3.1 versus 1.5 doubling concentrations). This was probably due to a rapid (within 2 wk) reduction in cellular activity (8, 9). It has been shown that PC20 methacholine continues to improve for at least 1 yr with steroid therapy (15). Whether this is also the case for PC20 AMP is unknown, but the bronchoconstrictor response to another indirect stimulus, exercise, has been shown to reach a plateau after 2 mo (31). In this context it is interesting to speculate that the improvement in PC20 methacholine will “catch up” with the improvement in PC20 AMP after a longer period.
Improvements in both PC20 methacholine and PC20 AMP were associated with the change in the number of sputum lymphocytes. In a recent study by Lemière and coworkers, it was shown that the interobserver repeatability is low for sputum lymphocytes and bronchial epithelial cells (32). Thus, it is the more remarkable that we were able to find significant differences, since greater variability of a test result will decrease the statistical power for detecting changes. The positive association between improvement in both PC20 methacholine and PC20 AMP and the change in the number of lymphocytes suggests that lymphocytes have a protective effect against BHR. This was an unexpected finding, since it has been shown that lymphocytes contribute to the inflammatory process in asthma (33, 34). However, the exact meaning of lymphocytes in sputum is not yet entirely clear. In our study, we did not measure activation markers and subsets of lymphocytes. These measurements should be able to more precisely help define the exact role of these inflammatory cells in sputum in acute and chronic asthma in future studies.
We found that improvement in PC20 AMP but not in PC20 methacholine was associated with a decrease in the number of bronchial epithelial cells in sputum. Epithelial cell shedding is an important feature of asthma (35). It has been shown that epithelial cell clumps (creola bodies) are present in increased numbers in the sputum of asthma patients. Furthermore, partial epithelial denudation of the basement membrane is frequently observed in mucosal biopsy specimens derived from asthmatic airways (36). The decrease in the number of epithelial cells may reflect the begining of restoration of the barrier function of the airways, leading to a decrease in BHR. However, in our study, the decrease in the level of bronchial responsiveness to AMP, but not to methacholine, was associated with a decrease in the number of bronchial epithelial cells. It therefore seems more likely that the decrease in the number of epithelial cells in sputum after therapy with corticosteroids reflects an overall decrease in the cellular activation state in asthma, which is associated with the improvement in PC20 AMP but not in PC20 methacholine.
A further finding was that the improvement in PC20 AMP but not PC20 methacholine was associated with a decrease in the concentration of NO in exhaled air in both monovariate and multivariate regression analysis. It has been suggested in a number of studies that measurement of NO in exhaled air may give information about the degree of airway inflammation in asthmatic patients. The concentration of NO in exhaled air is increased in patients with asthma (37). In addition, the concentration of NO decreases after therapy with corticosteroids and increases after allergen exposure (38, 39). Moreover, it has been shown that proinflammatory cytokines increase the expression of inducible NO synthase in cultured human airway epithelial cells. Thus, our finding that improvement in PC20 AMP is more closely associated with a decrease in the concentration of NO in exhaled air is compatible with the thesis of improvement in PC20 AMP being more closely associated with reduction in airway inflammation.
Sputum induction has been shown to be a reliable and reproducible method to assess the extent of airway inflammation (40). In general, the percentages of inflammatory cells in sputum are analyzed. We have chosen not to present the percentages of inflammatory cells in sputum, since a decrease in the percentage of one cell type is inevitably associated with an increase in another cell type. Therefore this mode of expression will influence the results of steroid-induced changes in sputum cell differential counts. To avoid this adverse effect, we have used absolute cell counts in sputum. We have also analyzed the percentages of inflammatory cells in sputum. That analysis similarly shows that improvement in PC20 methacholine was associated with both an increase in FEV1 % predicted and a reduction in airway inflammation (i.e., percentage of sputum eosinophils and lymphocytes), whereas improvement in PC20 AMP was more closely related to a reduction in inflammation and not to a reduction in FEV1 % predicted. The improvement in PC20 AMP was independently associated with the decrease in the concentration of NO in exhaled air, and with changes in the percentages of lymphocytes, macrophages, bronchial epithelial cells, and eosinophils in sputum. The current analysis of absolute cell counts shows that the change in percentages of sputum macrophages was not a valid association, but was merely due to concomitant changes in the percentages of sputum eosinophils and lymphocytes.
In conclusion, this study shows that improvement in PC20 AMP with steroid treatment is more closely associated with reduction in airway inflammation than is improvement in PC20 methacholine. This finding suggests that PC20 AMP is a more powerful tool to monitor active inflammation in the airway wall.
The authors thank Pharmacia Uppsala for generously providing the kits for ECP determination, and B. Dijkhuizen and J. Zonderland for their laboratory assistance.
Supported by a grant from the University of Groningen, the University Hospital of Groningen, and GlaxoSmithKline.
| 1. | Finney MJ, Karlsson JA, Persson CGEffects of bronchoconstrictors and bronchodilators on a novel human small airway preparation. Br J Pharmacol8519852936 |
| 2. | Phillips GD, Ng WH, Church MK, Holgate STThe response of plasma histamine to bronchoprovocation with methacholine, adenosine 5′-monophosphate, and allergen in atopic nonasthmatic subjects. Am Rev Respir Dis1411990913 |
| 3. | Polosa R, Ng WH, Crimi N, Vancheri C, Holgate ST, Church MK, Mistretta ARelease of mast-cell-derived mediators after endobronchial adenosine challenge in asthma. Am J Respir Crit Care Med1511995624629 |
| 4. | Phillips GD, Rafferty P, Beasley R, Holgate STEffect of oral terfenadine on the bronchoconstrictor response to inhaled histamine and adenosine 5′-monophosphate in non-atopic asthma. Thorax421987939945 |
| 5. | Pauwels RA, Van der Straeten MEAn animal model for adenosine-induced bronchoconstriction. Am Rev Respir Dis1361987374378 |
| 6. | Crimi N, Palermo F, Oliveri R, Polosa R, Settinieri I, Mistretta AProtective effects of inhaled ipratropium bromide on bronchoconstriction induced by adenosine and methacholine in asthma. Eur Respir J51992560565 |
| 7. | Van den Berge M, Meijer RJ, Kerstjens HAM, De Reus DM, Koeter GH, Kauffman HF, Postma DSPC20 AMP is more closely associated with airway inflammation in asthma than PC20 methacholine. Am J Respir Crit Care Med163200115461550 |
| 8. | Weersink EJ, Douma RR, Postma DS, Koeter GHFluticasone propionate, salmeterol xinafoate, and their combination in the treatment of nocturnal asthma. Am J Respir Crit Care Med155199712411246 |
| 9. | O'Connor BJ, Ridge SM, Barnes PJ, Fuller RWGreater effect of inhaled budesonide on adenosine 5′-monophosphate-induced than on sodium-metabisulfite-induced bronchoconstriction in asthma. Am Rev Respir Dis1461992560564 |
| 10. | Meijer RJ, Kerstjens HAM, Arends LR, Kauffman HF, Koeter GH, Postma DSEffects of inhaled fluticasone and oral prednisolone on clinical and inflammatory parameters in patients with asthma. Thorax541999894899 |
| 11. | Quanjer PH, Tammeling GJ, Cotes JE, Pedersen OF, Peslin R, Yernault JCLung volumes and forced ventilatory flows. Report of the Working Party on Standardization of Lung Function Tests, European Community for Steel and Coal. Official Statement of the European Respiratory Society. Eur Respir J Suppl161993540 |
| 12. | Cockcroft DW, Killian DN, Mellon JJ, Hargreave FEBronchial reactivity to inhaled histamine: a method and clinical survey. Clin Allergy71977235243 |
| 13. | Oosterhoff Y, Jansen MA, Postma DS, Koeter GHAirway responsiveness to adenosine 5′-monophosphate in smokers and nonsmokers with atopic asthma. J Allergy Clin Immunol921993773776 |
| 14. | Fahy JV, Liu J, Wong H, Boushey HACellular and biochemical analysis of induced sputum from asthmatic and from healthy subjects. Am Rev Respir Dis147199311261131 |
| 15. | Kerstjens HAM, Brand PL, Hughes MD, Robinson NJ, Postma DS, Sluiter HJ, Bleecker ER, Dekhuijzen PN, de Jong PM, Mengelers HJA comparison of bronchodilator therapy with or without inhaled corticosteroid therapy for obstructive airways disease. Dutch Chronic Non-Specific Lung Disease Study Group. N Engl J Med327199214131419 |
| 16. | Sont JK, Han J, van Krieken JM, Evertse CE, Hooijer R, Willems LN, Sterk PJRelationship between the inflammatory infiltrate in bronchial biopsy specimens and clinical severity of asthma in patients treated with inhaled steroids. Thorax511996496502 |
| 17. | Lim S, Jatakanon A, John M, Gilbey T, O'Connor BJ, Chung KF, Barnes PJEffect of inhaled budesonide on lung function and airway inflammation: assessment by various inflammatory markers in mild asthma. Am J Respir Crit Care Med.15919992230 |
| 18. | Aalbers R, De Monchy JG, Kauffman HF, Smith M, Hoekstra Y, Vrugt B, Timens WDynamics of eosinophil infiltration in the bronchial mucosa before and after the late asthmatic reaction. Eur Respir J61993840847 |
| 19. | Alvarez MJ, Olaguibel JM, Garcia BE, Rodriquez A, Tabar AI, Urbiola EAirway inflammation in asthma and perennial allergic rhinitis: relationship with nonspecific bronchial responsiveness and maximal airway narrowing. Allergy552000355362 |
| 20. | Bradley BL, Azzawi M, Jacobson M, Assoufi B, Collins JV, Irani AM, Schwartz LB, Durham SR, Jeffery PK, Kay ABEosinophils, T-lymphocytes, mast cells, neutrophils, and macrophages in bronchial biopsy specimens from atopic subjects with asthma: comparison with biopsy specimens from atopic subjects without asthma and normal control subjects and relationship to bronchial hyperresponsiveness. J Allergy Clin Immunol881991661674 |
| 21. | Crimi E, Spanevello A, Neri M, Ind PW, Rossi GA, Brusasco VDissociation between airway inflammation and airway hyperresponsiveness in allergic asthma. Am J Respir Crit Care Med157199849 |
| 22. | Gauvreau GM, Lee JM, Watson RM, Irani AM, Schwartz LB, O'Byrne PMIncreased numbers of both airway basophils and mast cells in sputum after allergen inhalation challenge of atopic asthmatics. Am J Respir Crit Care Med161200014731478 |
| 23. | Busse WWInflammation in asthma: the cornerstone of the disease and target of therapy. J Allergy Clin Immunol1021998S17S22 |
| 24. | Busse W, Elias J, Sheppard D, Banks-Schlegel SAirway remodeling and repair. Am J Respir Crit Care Med160199910351042 |
| 25. | Fish JE, Peters SPAirway remodeling and persistent airway obstruction in asthma. J Allergy Clin Immunol1041999509516 |
| 26. | Holgate ST, Davies DE, Lackie PM, Wilson SJ, Puddicombe SM, Lordan JLEpithelial-mesenchymal interactions in the pathogenesis of asthma. J Allergy Clin Immunol1052000193204 |
| 27. | Hoshino M, Nakamura Y, Sim J, Shimojo J, Isogai SBronchial subepithelial fibrosis and expression of matrix metalloproteinase-9 in asthmatic airway inflammation. J Allergy Clin Immunol1021998783788 |
| 28. | Chetta A, Foresi A, Del Donno M, Consigli GF, Bertorelli G, Pesci A, Barbee RA, Olivieri DBronchial responsiveness to distilled water and methacholine and its relationship to inflammation and remodeling of the airways in asthma. Am J Respir Crit Care Med1531996910917 |
| 29. | Bousquet J, Lacoste JY, Chanez P, Vic P, Godard P, Michel FBBronchial elastic fibers in normal subjects and asthmatic patients. Am J Respir Crit Care Med153199616481654 |
| 30. | Ichinose M, Takahashi T, Sugiura H, Endoh N, Miura M, Mashito Y, Shirato KBaseline airway hyperresponsiveness and its reversible component: role of airway inflammation and airway calibre. Eur Respir J152000248253 |
| 31. | Waalkens HJ, Essen-Zandvliet EE, Gerritsen J, Duiverman EJ, Kerrebijn KF, Knol KThe effect of an inhaled corticosteroid (budesonide) on exercise-induced asthma in children. Dutch CNSLD Study Group. Eur Respir J61993652656 |
| 32. | Lemièire C, Taha R, Chaboilez S, Hamid QComparison of cellular composition of induced sputum analysed by Wright staining and immunocytochemistry. J Allergy Clin Immunol1072001S36 |
| 33. | Gratziou C, Carroll M, Walls A, Howarth PH, Holgate STEarly changes in T lymphocytes recovered by bronchoalveolar lavage after local allergen challenge of asthmatic airways. Am Rev Respir Dis145199212591264 |
| 34. | Kidney JC, Wong AG, Efthimiadis A, Morris MM, Sears MR, Dolovich J, Hargreave FEElevated B cells in sputum of asthmatics: close correlation with eosinophils. Am J Respir Crit Care Med1531996540544 |
| 35. | Beasley R, Roche WR, Roberts JA, Holgate STCellular events in the bronchi in mild asthma and after bronchial provocation. Am Rev Respir Dis1391989806817 |
| 36. | Jeffery PKBronchial biopsies and airway inflammation. Eur Respir J9199615831587 |
| 37. | Alving K, Weitzberg E, Lundberg JMIncreased amount of nitric oxide in exhaled air of asthmatics. Eur Respir J6199313681370 |
| 38. | Kharitonov SA, Yates DH, Barnes PJInhaled glucocorticoids decrease nitric oxide in exhaled air of asthmatic patients. Am J Respir Crit Care Med1531996454457 |
| 39. | Baraldi E, Carra S, Dario C, Azzolin N, Ongaro R, Marcer G, Zacchello FEffect of natural grass pollen exposure on exhaled nitric oxide in asthmatic children. Am J Respir Crit Care Med1591999262266 |
| 40. | in't Veen JCCM, de Gouw HW, Smits HH, Sont JK, Hiemstra PS, Sterk PJ, Bel EH. Repeatability of cellular and soluble markers of inflammation in induced sputum from patients with asthma. Eur Respir J 1996;9:2441–2447. |
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