Twenty five patients with asthma and ten control subjects were recruited for this study from the Division of Allergy, Department of Internal Medicine, Chonnam University Hospital between March and September, 1997 (Table 1). Control subjects, who volunteered for this study, had no history of any respiratory symptoms, and had forced expiratory volume in one second (FEV₁)>75% predicted, a ratio of FEV₁ to forced vital capacity>75% and a normal methacholine airway responsiveness [provocation concentration of methacholine producing a 20% fall of FEV₁ (PC20)>16mg; AHR]. The diagnoses of asthma were established in the patients by their symptoms of recurrent episodic wheezing, cough and/or dyspnea, accompanied either by methacholine airway hyper-responsiveness (thirteen patients) or by a significant improvement of FEV₁ (15%) following antiasthma therapy (five patients). No subject had respiratory infection for 4 weeks prior to the study. The study was approved by Chonnam University Hospital Research Committee and all subjects signed the informed consent forms.

Eosinophils and eosinophil activation markers in hypertonic saline-induced sputum were measured in asthmatic patients and compared with those in control subjects. On the first visit, a questionnaire for symptoms and medications was given and a spirometry was performed. After that, induced sputum was collected and skin prick tests and methacholine provocation tests were done. Clinical severity of asthma was classified by the method of the International Consensus Report¹⁵⁾. All measurements in sputum were obtained by a person blind to the clinical characteristics of subjects.

Spirometry was performed according to the American Thoracic Society standards¹⁶⁾ using SensorMedics 2200 spirometer (Cardiopulmonary Care Company™, Yorba Linda, California). The representative values for FVC and FEV₁ were selected according to the International Thoracic Society criteria¹⁷⁾ and the reference values were taken from the reports by Choi et al.¹⁸⁾ and by Kim et al.¹⁹⁾. Methacholine challenge tests were carried out by a modification of the method described by Chai et al.²⁰⁾ and the results were expressed as PC₂₀ in noncumulative units. Allergy skin prick tests were performed with 55 common allergen extracts. Atopy means one or more positive allergy skin prick tests.

Sputum induction was performed as a modification of the method described by Fahy et al.²¹⁾. All subjects were premedicated with inhaled salbutamol 2 puffs (200μg). Subjects inhaled 3% hypertonic saline solution aerosols generated by an ultrasonic nebulizer (NE-U03, OMRON Co., Tokyo, Japan) with maximum output of 0.15–0.3 ml/min and mass median aerodynamic diameter of 4.5μm Hypertonic saline was inhaled for 20–30 min according to the severity of asthma until an adequate volume of sputum was expectorated. Subjects were asked to rinse the mouth and blow the nose to minimize contamination with saliva and postnasal drip. They were encouraged to cough deeply and frequently during hypertonic saline inhalation. They were instructed to cough the sputum into a sterile plastic container. The volumes of samples and duration of sputum induction were recorded. FEV₁ was measured before, during and after induction of sputum. Sputum induction was stopped in subjects with a fall of the FEV₁ ≥15%.

The appearance of the sputum was recorded as mucoid, mucopurulent or purulent. Sputum was selected from saliva and processed within 2 hours. The method of sputum examination described by Popov et al.²²⁾ was modified²¹⁾. Sputum was treated by adding equal volumes of 0.1% dithiothreitol (Sputalysin 10%; Gibco BRL, USA) followed by equal volumes of Dulbecco’s phosphate buffered saline (D-PBS). The sample was then mixed gently and placed in a shaking water bath at 37°C for 15 min to ensure complete homogenization. The sample was removed from the water bath periodically for further brief gentle mixing. The suspension was filtered through a gauze (1mm pore size), the filtrate was centrifuged at 1,500 rpm for 10 min and the supernatant was aspirated and stored in Eppendorf tubes at −70°C for later assay. The cell pellet was resuspended in D-PBS, 1000μL and total nonsquamous cells were counted in a modified Neubauer hemocytometer. The cell suspension was adjusted to 0.5×10⁵/ml, and then 50 μL of cell suspension placed into cups of Sakura cytocentrifuge (Model CF-127, Tokyo, Japan), and two coded cytospins were prepared at 600 rpm at 5 min, air dried, stained by Diff-Quick® (Kookje Scientific Products, Japan) stain and stored at −70°C. Cell differentials of 400 nonsquamous cells were performed in Diff-Quick® stain slides by two investigators who did not know the subject’s history, and the results were expressed as a percentage of the total non-squamous cell count.

The concentration of ECP in 400μL in the thawed supernatant was determined using fluoroimmunoassay (Pharmacia CAP system). IL-5 was measured by quantitative sandwich enzyme immunoassay (Quantikine™; R&D Systems, Inc., MN), as described by Dickason²³⁾. Samples were analysed in duplicates. The limits of detection for ECP and IL-5 assays were 2.0 μg/L and 3 pg/ml, respectively.

All data were analyzed using the SPSS version 7.5 for Windows. Data are expressed as mean ± SEM. Comparison of variables was performed using Student’s t test and Mann-Whitney U test. One-way analysis of variance (ANOVA) was used to compare mean values among groups of subjects. When a significant difference between groups was observed, inter-group comparisons were assessed by Bonferroni’s method. Pearson’s correlations and Spearman’s correlations were used to assess relationships between variables. A p-value of <0.05 was considered significant.

Sputum induction by hypertonic saline inhalations was discontinued if the FEV₁ fell by more than 15%. The subjects experienced a salty sense in thirtyeight sputum induction trials, chest tightness in three and dyspnea in two. Mean duration of the induction was 15.6 min in asthmatics and 19.6 min in control subjects. During sputum induction, there were falls of FEV₁ ≥ 15% in two asthmatics which required additional inhalations of salbutamol aerosols. The maximal fall in FEV₁ from baseline during the hypertonic saline inhalation was significantly greater in the asthmatics than in the control subjects (5.2 ± 0.6% vs 1.5 ± 0.9%, p<0.01).

Thirty eight sputum samples from asthmatics and 10 sputum samples from control subjects were analyzed. The sputum had less than 30% of squamous epithelial cells and greater than 85% of cell viability. Differential counts in the induced sputum are shown in Figure 1. The proportion of eosinophils and neutrophils was significantly higher in asthmatics than in control subjects (eosinophils: 25.6 ± 4.6% vs 1.7 ± 0.2%, p<0.01; neutrophils: 20.3 ± 2.4% vs 9.4 ± 1.8%, p<0.01), whereas the proportion of macrophage was lower (42.8 ± 5.3% vs 84.0 ± 1.6%, p<0.01). No significant differences were noted between asthmatics and control subjects in either total cell count (7.4 ± 2.2 × 10⁵/ml vs 5.3 ± 0.9 × 10⁵/ml) or the absolute or relative numbers of lymphocytes and epithelial cells.

The level of ECP in the sputum was significantly increased in asthmatics compared with that in control subjects (1117.8±213.9μg/L vs 154.6 ± 47.4μg/L, p<0.01, Figure 2). IL-5 was detected more frequently in asthmatics than in control subjects [11/25 (44%) vs 1/10 (10%), p<0.05].

The proportions of eosinophils and ECP level in the sputum were significantly higher in moderate to severe asthma (n=18) than in mild asthma (n=7) (p<0.01, respectively, Figure 3). IL-5 was significantly increased in moderate to severe asthmatics compared to mild asthmatics (34.2 ± 8.5pg/ml vs 0pg/ml).

The proportion of eosinophils in induced sputum was inversely correlated with FEV₁ and FEV₁/FVC (Table 2). The eosinophil activation markers (ECP and IL-5) had also an inverse relationship with FEV₁ and FEV₁/FVC. Eosinophil activation markers did not correlate atopy score, methacholine-AHR.

The proportions of sputum eosinophils were positively correlated with the ECP level and the IL-5 level (Table 2). The level of ECP was positively correlated with the level of IL-5.