Thirty patients with asthma and 12 age-matched control subjects were included in the study. All control subjects were healthy, nonsmoking volunteers who had no history of lung disease. All patients with asthma were recruited from respiratory outpatient clinics at our institution; they were nonsmokers and satisfied American Thoracic Society criteria for asthma. Methacholine inhalation challenge testing was performed for all study subjects as we previously described. All subjects with asthma in this study demonstrated bronchial hyperreactivity to methacholine. Exhaled nitric oxide (NO) was measured for all subjects with a chemiluminescence analyzer (CLM-500; Shimazu; Kyoto, Japan) in accordance with American Thoracic Society standards. The mean value of three expiratory NO concentrations was calculated for each subject and expressed as parts per billion. At study entry, regular medication in asthmatic patients consisted of short-acting p2-agonists on demand. No patients were receiving oral or inhaled corticosteroids. All patients with asthma were clinically stable, and none had a history of respiratory infection for at least the 4-week period preceding the study. All subjects gave their written informed consent for participation in the study, which was approved by the ethics committee of Osaka City University.
Sputum Induction and Processing
Sputum induction was performed as we previously described. The volume of sputum samples was measured, and the sample was divided into two portions. One portion was diluted with phosphate-buffered saline solution containing dithiothreitol (DTT) [a final concentration of 1 mmol/L] (WAKO Pure Chemical Industries; Osaka, Japan) was then centrifuged at 400g for 10 min, and the cell pellet was resuspended. The slides were prepared using a cytospin (Cytospin 3: Shandon; Tokyo, Japan) and stained with May-Grunwald-Giemsa stain for differential cell counts. The other portion of sputum samples, for assay of angiopoietin-1 and angiopoietin-2, was diluted with phosphate-buffered saline solution without DTT because we had preliminarily detected a detrimental effect of DTT on measurement of angiopoietin-1 and angiopoietin-2 levels in induced sputum. The supernatant was stored at – 70°C for subsequent assay of eosinophil cationic protein (ECP), VEGF, angiopoietin-1, angiopoietin-2, and albumin. VEGF, angiopoietin-1, and angiopoietin-2 concentrations were measured by quantitative sandwich enzyme immunoassays (Quantikine; R&D Systems; Minneapolis, MN). Samples were analyzed in duplicate. ECP concentration was measured using a radioimmunoassay kit (Pharmacia Diagnostics; Uppsala, Sweden). The limits of detection for VEGF, angiopoi-etin-1, and, angiopoietin-2 were 9 pg/mL, 62.5 pg/mL, and 8.29 pg/mL, respectively. Albumin concentration was measured by laser nephelometry, and then we calculated the airway vascular permeability index (the ratio of albumin concentrations in induced sputum and serum). All subjects produced an adequate specimen of sputum; a sample was considered adequate if the patient was able to expectorate at least 2 mL of sputum and if on differential cell counting the slides contained < 10% squamous cells.
A randomized study protocol was used. At the end of 2-week run-in period, all asthmatic patients were randomly assigned to receive fluticasone propionate (200 |j,g bid) or montelukast capsule (10 mg at night) for 12 weeks. Pulmonary function testing, methacholine provocation testing, determination of exhaled NO level, and sputum induction were performed on the last day of the run-in and treatment periods.
All values are presented as mean ± SD. Multiple comparisons were performed by one-way analysis of variance, followed by the Bonferroni test. The significance of correlations was evaluated by determining Spearman rank correlation coefficients. A p value < 0.05 was considered significant.