SUBJECTS

Eighteen patients with asthma were recruited from the Division of Allergy, Department of Internal Medicine, Seonam University and Chonnam University Hospital between February and August 1999. The diagnosis of asthma had been previously established in 13 patients by their symptoms of recurrent episodic wheezing, cough, and/or dyspnoea, and either by methacholine airway hyperresponsiveness in 10 patients or by an improvement of 15% after salbutamol 200 μg (when the forced expiratory volume in one second (FEV₁) was <75% predicted) in three patients. In the five remaining patients the diagnosis was confirmed by an improvement in the FEV₁(15%) following anti-asthma therapy. No subject had respiratory infection for four weeks before the study. Seonam University Hospital Research Committee approved the study and all subjects signed the informed consent forms.

STUDY DESIGN

Eosinophils and eosinophil activation markers in sputum were measured in asthmatic patients. A questionnaire on symptoms and medications was given to all patients and a spirometric test was performed. Sputum was then collected and skin prick tests and a methacholine provocation test were performed. The clinical severity of asthma was classified according to the International Consensus Report.8 Asthma exacerbations were graded by symptoms (breathlessness, talking, and alertness), signs (respiratory rate, use of accessory muscles, wheeze, pulse/min, and pulsus paradoxus), and functional assessment (peak expiratory flow rate, arterial blood gas analysis). A person blinded to the clinical characteristics of the subjects performed the sputum measurements.

Spirometric tests were performed according to the American Thoracic Society standards9 using a SensorMedics 2200 spirometer (Cardiopulmonary Care Company, California, USA). The representative values for forced vital capacity (FVC) and forced expiratory volume in one second (FEV₁) were selected according to the Intermountain Thoracic Society criteria10 and the reference values were taken from the reports by Choiet al 11 with some modifications. Methacholine challenge tests were carried out using a modification of the method described by Chai et al 12 and the results were expressed as PC₂₀ in non-cumulative units. Allergy skin prick tests were performed using 55 common allergen extracts. The wheal size was recorded at 15 minutes and scored as follows: 0 = no different from control, + = 1–2 mm larger than control, ++ = 3–5 mm larger than control, +++ = 6–8 mm larger than control, ++++ = >8 mm larger than control. The total number of pluses was added. Atopy was defined as an atopy score of >1+.

SPUTUM INDUCTION AND PROCESSING

Sputum was induced only when it could not be produced spontaneously. The sputum induction was performed as a modification of the method described by Fahy et al.13 All subjects were premedicated with two puffs of inhaled salbutamol (200 μg). Subjects inhaled 3% hypertonic saline solution aerosols generated by an ultrasonic nebuliser (NE-U03, OMRON Co, Tokyo, Japan) with a maximum output of 0.15–0.3 ml/min and a mass median aerodynamic diameter of 4.5 μm. Hypertonic saline was inhaled for 15–20 minutes according to the severity of the asthma until an adequate volume of sputum was expectorated. Subjects were asked to rinse their mouth and blow their nose to minimise 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 sample volumes and duration of sputum induction were recorded. FEV₁ was measured before, during, and after induction of sputum. Sputum induction was stopped if the FEV₁ fell by ⩾15%.

The appearance of the sputum was recorded as mucoid, mucopurulent, or purulent. Sputum was selected from saliva and processed within two hours. The method of sputum examination described by Popovet al 14 was modified.12 Sputum was treated by adding equal volumes of 0.1% dithiothreitol (Sputalysin 10%; Gibco BRL, Grandisland, New York, 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 minutes to ensure complete homogenisation. The sample was removed from the water bath periodically for further brief gentle mixing. The suspension was filtered through gauze (1 mm pore size), the filtrate was centrifuged at 1500 rpm for 10 minutes, and the supernatant was aspirated and stored in Eppendorf tubes at –70°C for later assay. The cell pellet was resuspended in 1000 μl D-PBS and the total number of non-squamous cells was counted in a modified Neubauer haemocytometer. The cell suspension was adjusted to 0.5 × 10⁵/ml and then 50 μl of cell suspension was placed in a Sakura cytocentrifuge (Model CF-127, Tokyo, Japan). Two coded cytospins were prepared at 600 rpm for five minutes. One slide was air dried and stained with Trypan blue stain (GibcoBRL, Grand Island, New York, USA) and two others were stained with Diff-Quick (Kookje Scientific Products, Tokyo, Japan). Another two slides prepared for immunocytochemical analysis were air dried for 10 minutes, fixed in 0.25% formalin for five minutes at 4°C, wrapped in pairs in foil and stored at –70°C. Cell differential counts of 400 non-squamous cells were performed on Diff-Quick stained slides by two investigators who did not know the subject's clinical history, and the results were expressed as a percentage of the total non-squamous cell count.

IMMUNOCYTOCHEMICAL ANALYSIS OF BCL-2

Immunocytochemical analysis was performed on sputum cytospin slides with Probe On Plus slide (Fisher Scientific, Pittsburgh, Pennsylvania, USA). All steps in the staining procedure were done at 50°C in a Microprobe system taking advantage of capillary gap action15 produced by two approximated Probe On Plus slides. The primary antibody was applied for 15 minutes with anti bcl-2 antibody (Dako, Copenhagen, Denmark). Antigen/antibody complexes were detected with a goat anti-mouse IgG (Sigma, St Louis, Missouri, USA) for 10 minutes followed by avidine alkaline phosphatase (Dako)16 for seven minutes after blocking endogenous alkaline phosphatase activity in Redusol (Biomeda, Foster City, California, USA) for two minutes. The chromogen reaction consisted of Fast Red TR Salt (Research Genetics, Huntsville, Alabama, USA) for 10 minutes followed by 30 seconds of haematoxylin. Bcl-2 antibodies were diluted in primary antibody diluent (Research Genetics) at the final concentration of 5 μg/ml. Positive controls for bcl-2 consisted of known positive samples of human spleen included with each staining reaction. Negative controls consisted of staining without the primary antibody. All slides were examined using standard light microscopy by two observers and scored as percentage of positive eosinophils over total eosinophils. The intensity of the staining reaction was not quantified. The results were expressed as percentage of positively stained cells over total eosinophils.

ECP AND IL-5 MEASUREMENT

The concentration of ECP in 400 μl thawed supernatant was determined by fluoroimmunoassay (UniCAP system, Pharmacia AB, Uppsala, Sweden). IL-5 was measured by quantitative sandwich enzyme immunoassay (Quantikine; R&D System Inc). Samples were analysed in duplicates. The limits of detection for the ECP and IL-5 assays were 2.0 μg/l and 3 pg/ml, respectively.

STATISTICAL ANALYSIS

All data were analysed using the SPSS version 7.5 for Windows. Data are expressed as mean (SE). Inter-group comparisons were assessed by a non-parametric method using the Mann-Whitney U test. The correlation between variables was examined by Spearman rank correlation coefficient. A p value of less than 5% was regarded as statistically significant.