You are here

  • Home
  • Archive
  • Volume 54, Issue 5
  • Effect of inhaled steroids on airway hyperresponsiveness, sputum eosinophils, and exhaled nitric oxide levels in patients with asthma

Article Text

Article menu

Download PDFPDF

Original article
Effect of inhaled steroids on airway hyperresponsiveness, sputum eosinophils, and exhaled nitric oxide levels in patients with asthma
Free
  1. Elizabeth L J van Rensena,
  2. Karin C M Straathofa,
  3. Maud A Veselic-Charvatb,
  4. Aeilko H Zwindermanc,
  5. Elisabeth H Bela,
  6. Peter J Sterka
  1. aDepartment of Pulmonology, bDepartment of Cytology, cDepartment of Medical Statistics, dLeiden University Medical Center, Leiden, The Netherlands
  1. Professor P J Sterk, Lung Function Laboratory, C2-P Department of Pulmonology, Leiden University Medical Center, P O Box 9600, NL-2300 RC Leiden, The Netherlands.

Abstract

BACKGROUND Airway hyperresponsiveness, induced sputum eosinophils, and exhaled nitric oxide (NO) levels have all been proposed as non-invasive markers for monitoring airway inflammation in patients with asthma. The aim of this study was to compare the changes in each of these markers following treatment with inhaled glucocorticosteroids in a single study.

METHODS In a randomised, double blind, placebo controlled, parallel study 25 patients with mild asthma (19–34 years, forced expiratory volume in one second (FEV1) >75% predicted, concentration of histamine provoking a fall in FEV1 of 20% or more (PC20) <4 mg/ml) inhaled fluticasone propionate (500 μg twice daily) for four weeks. PC20 to histamine, sputum eosinophil numbers, and exhaled NO levels were determined at weeks 0, 2, and 4, and two weeks after completing treatment. Sputum was induced by inhalation of hypertonic (4.5%) saline and eosinophil counts were expressed as percentage non-squamous cells. Exhaled NO levels (ppb) were measured by chemiluminescence.

RESULTS In the steroid treated group there was a significant increase in PC20, decrease in sputum eosinophils, and decrease in exhaled NO levels compared with baseline at weeks 2 and 4 of treatment. Subsequently, each of these variables showed significant worsening during the two week washout period compared with week 4. These changes were significantly different from those in the placebo group, except for the changes in sputum eosinophils and exhaled NO levels during the washout period. There were no significant correlations between the changes in the three markers in either group at any time.

CONCLUSIONS Treatment of asthmatic subjects with inhaled steroids for four weeks leads to improvements in airway hyperresponsiveness to histamine, eosinophil counts in induced sputum, and exhaled nitric oxide levels. The results suggest that these markers may provide different information when monitoring anti-inflammatory treatment in asthma.

  • airway hyperresponsiveness
  • asthma
  • fluticasone propionate
  • inhaled corticosteroids
  • nitric oxide
  • induced sputum

https://doi.org/10.1136/thx.54.5.403

Statistics from Altmetric.com

    Request Permissions

    If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.

    Asthma is an inflammatory disease of the airways associated with airway hyperresponsiveness to various bronchoconstrictor stimuli such as histamine.1 The accompanying inflammation is characterised by the presence of inflammatory cells such as T lymphocytes, neutrophils and eosinophils and their cytokines in the airway mucosa, as demonstrated in bronchial biopsy specimens.2 ,3 The current treatment of asthmatic patients is based on the belief that reducing airway inflammation is essential, and that control of such inflammation can be indirectly assessed by optimising symptoms and lung function.1 ,4 However, monitoring airway inflammation more closely by measurement of non-invasive and sensitive markers of inflammation, such as airway hyperresponsiveness,5 sputum eosinophils,6 or exhaled NO levels,7 may provide additional information for assessing asthma control.

    Inhaled glucocorticosteroids are currently the most effective treatment for asthma, not only reducing symptoms and airway hyperresponsiveness8 but also leading to an improvement in airway inflammation.9 However, recent evidence has suggested that such treatment often provides only partial suppression of airway inflammation, as shown by persisting eosinophilic inflammation in the bronchial (sub)mucosa after long term inhaled steroid treatment in some patients.5

    Among the non-invasive techniques, hypertonic saline induced sputum has been shown to be a reliable method for measuring eosinophilic airways inflammation.6 ,10 ,11 The number of eosinophils in sputum is associated with asthma severity10 and decreases following treatment with inhaled steroids.12 In addition, nitric oxide levels in exhaled air have also been proposed as a marker for disease severity in asthma.7 ,13 Indeed, inhaled glucocorticosteroids decrease the levels of exhaled NO in patients with asthma14 in a dose dependent way.15

    Although the effects of inhaled steroids on sputum eosinophils and exhaled NO have been well established, comparative analysis is required before any of these markers can be recommended in the monitoring of asthma treatment. In the present study we investigated treatment induced changes in airway hyperresponsiveness, sputum eosinophils, and exhaled NO levels in asthma. To that end we performed histamine challenge, induced sputum, and exhaled NO measurements before, during, and after four weeks of treatment with fluticasone propionate or placebo in steroid naive patients with asthma.

    Methods

    SUBJECTS

    Twenty five non-smoking atopic patients (16 men, age range 19–34 years) with mild persistent asthma1 volunteered to participate in the study (table 1). Symptoms of episodic chest tightness and wheezing were treated by on-demand usage of inhaled salbutamol alone, which was discontinued at least eight hours before the measurements. Two weeks before the study all subjects were free from symptoms of respiratory tract infection. Atopy was indicated by a positive skin prick test (>3 mm weal) to one or more of 10 common airborne allergen extracts (Vivodiagnost, ALK, The Netherlands). The forced expiratory volume in one second (FEV1) was more than 75% of the predicted value16 and all subjects were hyperresponsive to inhaled histamine (provocative concentration causing a fall in FEV1 of 20% or more (PC20) of <4 mg/ml).17 The study was approved by the medical ethics committee of the Leiden University Medical Center and written informed consent was obtained from all volunteers.

    View this table:
    Table 1

    Characteristics of the subjects

    DESIGN OF STUDY

    The study was of a randomised, double blind, placebo controlled, parallel design. During screening the selection criteria were checked for all subjects. Before entering the treatment period baseline values of PC20 histamine and percentage eosinophils in induced sputum were determined. These two measurements were carried out on two separate days with a 2–4 day interval between them. Prior to histamine challenge and sputum induction, baseline values of FEV1 and exhaled NO were recorded. This sequence of measurements was used at all time points during the study.

    Immediately after the second baseline visit the subjects were treated with inhaled fluticasone propionate (500 μg twice daily) or placebo for a period of four weeks. The measurement of PC20histamine, sputum eosinophils, FEV1, and exhaled NO were repeated during the treatment period (at weeks 2 and 4) and during the washout period at two weeks after the treatment period.

    HISTAMINE CHALLENGE

    Histamine challenges were performed according to a standardised methodology.17 Histamine-di-phosphate (Sigma Chemicals, St Louis, MO, USA) in phosphate buffered saline (PBS) was stored at 4°C and administered at room temperature. Doubling concentrations between 0.06 and 16 mg/ml were used. The aerosols were generated by a DeVilbiss 646 nebuliser (output 0.13 ml/min), connected to an inspiratory and expiratory valve box with an expiratory aerosol filter (Pall Ultipor BB50T). Each dose was inhaled through the mouth by tidal breathing for two minutes at five minute intervals with the nose clipped.17

    The airway responses to the inhaled aerosols were measured using FEV1, recorded by a dry rolling seal spirometer (Morgan Spiroflow, Morgan UK) and monitored on-line by a personal computer with a special software program. Before each test FEV1 was measured in triplicate for calculation of mean baseline levels.17 The airway response was recorded at 30 and 90 seconds after each dose. After each inhalation the lowest technically satisfactory FEV1 value was applied in the analysis to calculate the percentage fall in FEV1 from baseline. The test was discontinued if FEV1 decreased by 20% or more. The PC20 was calculated by log-linear interpolation of the final two data points.

    SPUTUM INDUCTION

    Sputum was induced and processed by the so called full sample method18 according to a protocol that has been validated in our laboratory.6 Hypertonic saline aerosols (NaCl 4.5%) were generated at room temperature by a DeVilbiss Ultraneb 2000 ultrasonic nebuliser with a calibrated particle size (MMAD 4.5 μm) at maximal output (2.5 ml/min). The aerosols were administered to the subjects through a 100 cm long tube with an internal diameter of 22 mm and inhaled via the mouth through a two way valve (No. 2700; Hans-Rudolph, Kansas City, MO, USA) with the nose clipped. Before inhalation of the aerosols baseline FEV1 was recorded and, for safety reasons, 400 μg salbutamol was administered through a metered dose inhaler (Volumatic). The subjects then inhaled hypertonic saline aerosols during 2 × 5 minute and 1 × 10 minute intervals. After each inhalation, or as soon as the subjects started coughing, they were asked to blow their nose, to rinse their mouth and throat with water, and to expectorate sputum into a clean plastic container by coughing. After testing, FEV1 was measured and salbutamol was administered if needed.

    SPUTUM PROCESSING AND CELL DIFFERENTIAL COUNTS

    The volume of the induced sputum samples was determined and mixed with an equal volume of 0.1% sputolysin (dithiotreitol, Calbiochem, USA).6 To ensure complete homogenisation the samples were placed in a shaking water bath at 37°C for 15 minutes and then gently mixed. The homogenised sputum was centrifuged (350g) for 10 minutes at room temperature. The cell pellet was resuspended in PBS to a final volume of 2–5 ml, then filtered through a gauze (pore size approximately 1 mm) to remove clumps. Total cell counts were performed in a haemacytometer (Tamson, Zoetermeer, The Netherlands). The sample was then diluted with PBS to a final concentration of ±0.3 × 106 cells/ml which was used for preparation of the cytocentrifuge slides (1500 rpm, three minutes, 50 μl/slide; Shandon 3, Life Sciences International, Veldhoven, The Netherlands).

    Differential counts of eosinophils, neutrophils, lymphocytes, macrophages, epithelial and squamous cells were performed on Diff-Quik stained cytospins by a qualified cytopathologist. To correct for the variable salivary contamination, differential leucocyte and cylindrical epithelial cell counts were expressed as a percentage of 250 nucleated cells, excluding squamous cells. For each sample differential cell counts were performed twice by the same observer and the mean values were used in the analysis. A sputum sample was considered adequate when the percentage squamous cells was less than 80%. The reproducibility of the sputum cell counts obtained by this method has been shown to be satisfactory.6 To ensure a blind analysis of the sputum samples all cytocentrifuge slides were coded before analysis by an investigator who was not involved in the counting.

    EXHALED NO

    Exhaled NO levels were measured by a chemiluminescence analyser (Sievers NOA 270B) according to a standardised procedure7which has previously been used by our laboratory.19 The subjects were connected to a closed system to avoid contamination of the measurements with ambient NO. Pressurised air with a low NO concentration (<1 ppb) was administered through a 150 litre reservoir connected to the inspiratory side of a Hans-Rudolph three way valve. The subjects performed a slow vital capacity manoeuvre with a constant expiratory flow of 10 l/min against an expiratory resistance of 3–4 cm H2O. The expiratory NO concentration was sampled continuously from the centre of the mouthpiece at a flow rate of 440 ml/min and the average concentration (in parts per billion; ppb) was determined for a period of 10 seconds.7 Baseline values of exhaled NO were obtained from the mean values of the two NO measurements recorded before histamine challenge and sputum induction because their reproducibility was good (intraclass correlation coefficient, R i >0.92).

    ANALYSIS OF DATA

    PC20 was log transformed before statistical analysis and expressed as geometric mean (SD) doubling doses. Based on their close to normal distribution, the percentage of eosinophils in sputum and the levels of exhaled NO were expressed as mean (SD). To test for differences between and within the treatment groups in general, multivariate analysis of variance (MANOVA) was applied for FEV1 and log PC20, whilst the Kruskal-Wallis test was used for sputum eosinophils and exhaled NO. The changes in PC20 (expressed in doubling doses), sputum eosinophils, and exhaled NO levels within each treatment group were analysed using the Student’s paired t test whilst changes in PC20, sputum eosinophils, and exhaled NO levels between both groups were tested using the Student’s unpairedt test, providing the 95% confidence intervals (95% CI). Finally, Pearson correlation analysis was used to examine the relationship between the changes in PC20, sputum eosinophils, and exhaled levels of NO. The results were considered significant if the p value was <0.05. All statistical analyses were performed using the SPSS program.

    Results

    Three of the subjects dropped out during the washout period between weeks 4 and 6 because of a history of respiratory tract infection (nos 5 and 6) or because they were taking an antihistamine (no. 11). Three subjects (nos 9, 18 and 23) did not produce adequate sputum at baseline, whilst subjects 21 and 7 were not able to produce sputum at week 2 and week 4, respectively. These time points were handled as missing data.

    LUNG FUNCTION AND HISTAMINE CHALLENGE

    At baseline there were no significant differences in FEV1 and PC20 between the groups (p>0.19; table 1). During the study there were no significant changes in FEV1 in the two groups (p>0.96, MANOVA). In the placebo group there were no significant changes in PC20 (p = 0.92, MANOVA) while in the steroid treated group PC20 increased significantly at week 4 compared with baseline values (mean change 2.01 (95% CI 0.683 to 2.090); p = 0.001; fig 1). After a two week washout period PC20 decreased again compared with week 4 by –1.75 (–1.831 to –0.582) doubling doses (p = 0.002; table 2, fig 1). These changes were significantly different from the changes in the placebo group (p<0.003; table 3).

    Figure 1
    Figure 1

    Airway hyperresponsiveness to histamine (PC20) at baseline, at weeks 2 and 4 of treatment, and after two weeks of washout in the steroid treated (closed bars) and placebo groups (open bars) shown as geometric mean doubling doses; *significant difference between the two groups.

    View this table:
    Table 2

    Airway hyperresponsiveness, sputum eosinophils and exhaled nitric oxide (NO) levels during and after steroid and placebo treatment

    View this table:
    Table 3

    Changes in airway hyperresponsiveness, sputum eosinophil counts, and exhaled NO levels between steroid and placebo treatment groups

    SPUTUM EOSINOPHILS

    The mean (SD) percentage of squamous cells in this study was 33.4 (17.6)%. Baseline sputum eosinophils were not significantly different in the two groups (p = 0.31; table 1). There were no significant changes in sputum eosinophils within the placebo group (p = 0.85, MANOVA), but in the steroid treated group a significant decrease in sputum eosinophils was observed compared with baseline values (mean change at week 4 –2.46 (95% CI –4.260 to –0.660)%; p = 0.01) with a subsequent worsening in the washout period compared with week 4 (mean change 6.13 (95% CI 0.804 to 11.459)%; p = 0.03; table 2, fig 2). The changes in sputum eosinophils were not significantly different between the two groups when baseline values were compared with week 4, or week 4 values were compared with those in the washout period (table3).

    Figure 2
    Figure 2

    Mean eosinophil counts in induced sputum at baseline, at weeks 2 and 4 of treatment, and after two weeks of washout in the steroid treated (closed bars) and placebo groups (open bars); *significant difference between the two groups.

    EXHALED NO

    At baseline exhaled NO levels were not significantly different in the two groups (p = 0.55; table 1). During the study there were no significant changes in exhaled NO levels in the placebo group (p = 0.54, MANOVA; table 2) but in the steroid treated group the levels of exhaled NO decreased significantly at week 4 compared with baseline values with a mean change of –4.88 (95% CI –6.862 to –2.892) ppb (p < 0.001), with a subsequent increase during the washout period compared with week 4 of 3.65 (95% CI 0.882 to 6.423) ppb (p = 0.016; table 2, fig 3). These changes in exhaled NO levels were significantly different from the changes in the placebo group between baseline and week 4 (p = 0.005; table 3).

    Figure 3
    Figure 3

    Mean levels of exhaled nitric oxide (NO) at baseline, at weeks 2 and 4 of treatment, and after two weeks of washout in the steroid treated (closed bars) and placebo groups (open bars); *significant difference between the two groups.

    Figure 4
    Figure 4

    Relationship between the change in sputum eosinophils and the change in PC20 histamine at week 4 compared with baseline (• steroid group; ○ placebo group).

    Figure 5
    Figure 5

    Relationship between the change in exhaled NO levels and the change in PC20 histamine at week 4 compared with baseline (• steroid group; ○ placebo group).

    Figure 6
    Figure 6

    Relationship between the change in sputum eosinophil numbers and the change in exhaled NO levels at week 4 compared with baseline (• steroid group; ○ placebo group).

    RELATIONSHIPS BETWEEN CHANGES IN PARAMETERS

    Within the steroid group there were no significant correlations between the changes in PC20, sputum eosinophils, and exhaled NO at any time point (Pearson’s r<0.56, p >0.15; figs 4-6).

    Discussion

    The results of this study indicate that four weeks of treatment with inhaled steroids leads to improvements in airway hyperresponsiveness, sputum eosinophil numbers, and levels of exhaled NO in patients with mild atopic asthma. In addition, it appears that the improvements in these markers are lost two weeks after cessation of treatment. This suggests that each of these markers may be useful for monitoring patients with asthma, even though there might be small differences between the markers in their earliest response to anti-inflammatory treatment.

    To our knowledge this is the first study to compare the treatment induced changes in airway hyperresponsiveness to histamine, eosinophil counts in induced sputum, and exhaled NO levels in a group of asthmatic patients. Our study confirms and extends the results of others who have shown the beneficial effect of glucocorticosteroids on each of these markers separately. Like Kraan et al, we found an improvement of two doubling doses in airway hyperresponsiveness after four weeks of treatment with inhaled steroids.20 Furthermore, our findings are in agreement with those of Keatings et al 12and Kharitonov et al 14 who demonstrated a decrease in sputum eosinophils and exhaled NO levels, respectively, after treatment with inhaled steroids.

    Although cross sectional relationships between airway hyperresponsiveness, sputum eosinophils, and exhaled NO levels in patients with asthma have been reported previously,10 ,21only limited data are available on the comparison of within subject changes in these markers during treatment follow up. Our results are in agreement with those of Baraldi et al who also failed to find a correlation between steroid induced changes in PD20 and sputum eosinophils.22 The absence of such relationships may reflect the partially distinct pathophysiological backgrounds of these markers and might indicate the possible independent complementary clinical information during anti-inflammatory therapy.

    We do not believe that our data were influenced by measurement errors since we used validated and reproducible methods.6 ,7 ,17 ,19 All subjects in the study were carefully selected as non-smokers with stable atopic asthma who had not used inhaled steroids for at least one month prior to the study. We chose a relatively high dose of inhaled steroid to ensure an optimal anti-inflammatory effect. To avoid carryover effects the histamine challenge for determination of PC20 and the sputum induction were separated by 2–4 days. Furthermore, exhaled NO levels on these two days appeared to be highly reproducible. Our inability to show a significant improvement in lung function following steroid treatment may be due to the normal baseline levels of FEV1in our study (77–111% of the predicted value).

    How can the present findings be interpreted? Firstly, corticosteroids are likely to decrease the percentage of eosinophils in the sputum by reducing the release and subsequent effects of cytokines such as interleukin 5 (IL-5) and granulocyte-macrophage colony-stimulating factor (GM-CSF) on eosinophil infiltration and survival.23-25 Secondly, the steroid induced reduction in exhaled NO levels can be explained by the inhibition of inducible NO synthase (iNOS) expression directly and/or indirectly by reduction in the levels of stimulatory cytokines, for instance in epithelial cells.26 Finally, the improvement in the physiological marker PC20 is likely to be due to effects of steroids on the presence and activity of multiple (infiltrative and resident) cells.5 ,8 ,9 ,27 Hence, it may not be surprising that the steroid induced changes in the three markers were not significantly correlated with each other. It would appear that early improvement of eosinophils in sputum in response to steroid treatment is somewhat out of phase with the other two markers. However, we believe that this has few implications, given the consistency in the changes between the markers after four weeks of treatment.

    What are the clinical implications of the present findings? Treatment according to the current guidelines is based on minimising symptoms and optimising lung function.1 However, frequently this fails to provide complete suppression of airway inflammation.5It has been postulated that persistent airway inflammation in asthma leads to airway remodelling and an irreversible loss of lung function.28 ,29 This may require the use of more direct markers for monitoring airway inflammation.10 ,30 Indeed, a recent study by Sont et al showed that the adjustment of long term inhaled steroid treatment, additionally guided by the level of airway hyperresponsiveness, leads to a significantly better clinical, as well as histological, outcome than treatment guided by symptoms and lung function alone.31 Based on the present data, it is now necessary to determine in long term prospective trials whether monitoring sputum eosinophils and/or exhaled NO levels can provide similar benefits in the management of asthma.

    Acknowledgments

    The authors thank all the volunteers who participated in this study and Glaxo Wellcome, The Netherlands, for supporting the study with a grant for material expenses.

    References

    1. ↵
      1. National Institutes of Health, National Heart, Lung, and Blood Institute
      (January 1995) Global initiative for asthma. Publication 95-3659, .
    2. ↵
      1. Djukanovic R,
      2. Roche WR,
      3. Wilson JW,
      4. et al.
      (1990) Mucosal inflammation in asthma. Am Rev Respir Dis 142:434–457, .
      OpenUrlCrossRefPubMedWeb of Science
    3. ↵
      1. Ying S,
      2. Humbert M,
      3. Barkans J,
      4. et al.
      (1997) Expression of IL-4 and IL-5 mRNA and protein product by CD4+ and CD8+ T cells, eosinophils, and mast cells in bronchial biopsies obtained from atopic and nonatopic (intrinsic) asthmatics. J Immunol 158:3539–3544, .
      OpenUrlAbstract
    4. ↵
      1. British Thoracic Society
      (1997) The British guidelines on asthma management: 1995 review and position statement. Thorax 52 (Suppl 1):S1–S21, .
      OpenUrlFREE Full Text
    5. ↵
      1. Sont JK,
      2. Van Krieken JHJM,
      3. Evertse CE,
      4. et al.
      (1996) Relationship between the inflammatory infiltrate in bronchial biopsy specimens and clinical severity of asthma in patients treated with inhaled steroids. Thorax 51:496–502, .
      OpenUrlAbstract/FREE Full Text
    6. ↵
      1. In’t Veen JCCM,
      2. De Gouw HWFM,
      3. Smits HH,
      4. et al.
      (1996) Repeatability of cellular and soluble markers of inflammation in induced sputum from patients with asthma. Eur Respir J 9:2441–2447, .
      OpenUrlAbstract
    7. ↵
      1. Kharitonov SA,
      2. Alving K,
      3. Barnes PJ
      (1997) Exhaled and nasal NO measurements: recommendations. Eur Respir J 10:1683–1693, .
      OpenUrlCrossRefPubMedWeb of Science
    8. ↵
      1. Barnes PJ
      (1995) Inhaled glucocorticoids for asthma. N Engl J Med 332:868–875, .
      OpenUrlCrossRefPubMedWeb of Science
    9. ↵
      1. Trigg CJ,
      2. Manolitsas ND,
      3. Wang J,
      4. et al.
      (1994) Placebo-controlled immunopathologic study of four months of inhaled corticosteroids in asthma. Am J Respir Crit Care Med 150:17–22, .
      OpenUrlCrossRefPubMedWeb of Science
    10. ↵
      1. Pizzichini E,
      2. Pizzichini MMM,
      3. Efthimiadis A,
      4. et al.
      (1996) Indices of airway inflammation in induced sputum: reproducibility and validity of cell and fluid-phase measurements. Am J Respir Crit Care Med 154:308–317, .
      OpenUrlCrossRefPubMedWeb of Science
    11. ↵
      1. Grootendorst DC,
      2. Sont JK,
      3. Willems LNA,
      4. et al.
      (1997) Comparison of inflammatory cell counts in asthma: induced sputum vs bronchoalveolar lavage and bronchial biopsies. Clin Exp Allergy 27:769–779, .
      OpenUrlCrossRefPubMedWeb of Science
    12. ↵
      1. Keatings VM,
      2. Jatakanon A,
      3. Worsdell M,
      4. et al.
      (1997) Effects of inhaled and oral glucocorticoids on inflammatory indices in asthma and COPD. Am J Respir Crit Care Med 155:542–548, .
      OpenUrlCrossRefPubMedWeb of Science
    13. ↵
      1. Massaro AF,
      2. Gaston B,
      3. Kita D,
      4. et al.
      (1995) Expired nitric oxide levels during treatment of acute asthma. Am J Respir Crit Care Med 152:800–803, .
      OpenUrlCrossRefPubMedWeb of Science
    14. ↵
      1. Kharitonov SA,
      2. Yates DH,
      3. Barnes PJ
      (1996) Inhaled glucocorticoids decrease nitric oxide in exhaled air of asthmatic patients. Am J Respir Crit Care Med 153:454–457, .
      OpenUrlCrossRefPubMedWeb of Science
    15. ↵
      1. Kharitonov SA,
      2. Yates DH,
      3. Chung KF,
      4. et al.
      (1996) Changes in the dose of inhaled steroid affect exhaled nitric oxide levels in asthmatic patients. Eur Respir J 9:196–201, .
      OpenUrlAbstract
    16. ↵
      1. Quanjer Ph H,
      2. Tammeling GJ,
      3. Cotes JE,
      4. et al.
      (1993) Lung volumes and forced ventilatory flows. Eur Respir J 6:5–40, .
      OpenUrlFREE Full Text
    17. ↵
      1. Sterk PJ,
      2. Fabbri LM,
      3. Quanjer Ph H,
      4. et al.
      (1993) Airway responsiveness: standardized challenge testing with pharmacological, physical and sensitizing stimuli in adults. Eur Respir J 6:53–83, .
      OpenUrlAbstract/FREE Full Text
    18. ↵
      1. Fahy J,
      2. Lui J,
      3. Wong H,
      4. et al.
      (1993) Cellular and biochemical analysis of induced sputum from asthmatic and from healthy subjects. Am Rev Respir Dis 147:1126–1131, .
      OpenUrlCrossRefPubMedWeb of Science
    19. ↵
      1. De Gouw HWFM,
      2. Grünberg K,
      3. Schot R,
      4. et al.
      (1998) The relationship between exhaled nitric oxide and airway hyperresponsiveness following experimental rhinovirus infection in asthmatic subjects. Eur Respir J 11:126–132, .
      OpenUrlAbstract/FREE Full Text
    20. ↵
      1. Kraan J,
      2. Koëter GH,
      3. Van der Mark Th W,
      4. et al.
      (1985) Changes in bronchial hyperreactivity induced by 4 weeks of treatment with antiasthmatic drugs in patients with allergic asthma: a comparison between budesonide and terbutaline. J Allergy Clin Immunol 76:628–636, .
      OpenUrlCrossRefPubMedWeb of Science
    21. ↵
      1. Jatakanon A,
      2. Lim S,
      3. Chung KF,
      4. et al.
      (1997) Correlation between exhaled nitric oxide, sputum eosinophils, and methacholine responsiveness. Am J Respir Crit Care Med 155:A819, .
      OpenUrl
    22. ↵
      1. Baraldi E,
      2. Maestrelli P,
      3. Semenzato R,
      4. et al.
      (1997) Comparison of exhaled NO values, eosinophil counts in the induced sputum and eosinophil cationic protein (ECP) in asthmatic children. Eur Respir J 10:158s, .
      OpenUrl
    23. ↵
      1. Wallen N,
      2. Kita H,
      3. Weiler D,
      4. et al.
      (1991) Glucocorticoids inhibit cytokine-mediated eosinophil survival. J Immunol 147:3490–3495, .
      OpenUrlAbstract/FREE Full Text
    24. ↵
      1. Hallswoth MP,
      2. Litchfield TM,
      3. Lee TH
      (1992) Glucocorticoids inhibit granulocyte-macrophage colony-stimulating factor-1 and interleukin-5 enhanced in vitro survival of human eosinophils. Immunology 75:382–385, .
      OpenUrlPubMedWeb of Science
    25. ↵
      1. Corrigan CJ,
      2. Hamid Q,
      3. North J,
      4. et al.
      (1995) Peripheral blood CD4 but not CD 8 T-lymphocytes in patients with exacerbations of asthma transcribe and translate messenger RNA encoding cytokines which prolong eosinophil survival in the context of a Th2-type pattern: effect of glucocorticoid therapy. Am J Respir Cell Mol Biol 12:567–578, .
      OpenUrlCrossRefPubMedWeb of Science
    26. ↵
      1. Moncada S,
      2. Palmer RMJ,
      3. Higgs EA
      (1991) Nitric oxide: pathophysiology and pharmacology. Pharmacol Rev 42:109–142, .
    27. ↵
      1. Haley KJ,
      2. Drazen JM
      (1998) Inflammation and airway function in asthma. What you see is not necessarily what you get. Am J Respir Crit Care Med 157:1–3, .
      OpenUrlPubMedWeb of Science
    28. ↵
      1. Lange P,
      2. Parner J,
      3. Vestbo J,
      4. et al.
      (1998) A 15-years follow-up of ventilatory function in adults with asthma. N Engl J Med 339:1194–1200, .
      OpenUrlCrossRefPubMedWeb of Science
    29. ↵
      1. Haahtela T,
      2. Järvinen M,
      3. Kava T,
      4. et al.
      (1994) Effects of reducing or discontinuing inhaled budesonide in patients with mild asthma. N Eng J Med 331:700–705, .
      OpenUrlCrossRefPubMedWeb of Science
    30. ↵
      1. Barnes PJ,
      2. Kharitonov SA
      (1996) Exhaled nitric oxide: a new lung function test. Thorax 51:233–237, .
      OpenUrlFREE Full Text
    31. ↵
      1. Sont JK,
      2. Willems LNA,
      3. Bel EH,
      4. et al.
      (1999) The clinical control and histopathological outcome of asthma using airway hyperresponsiveness as an additional guide of long-term treatment. Am J Respir Crit Care Med, (in press).