Sixty-four consecutive patients with suspected asthma, who had all undergone both a combined HS challenge and sputum induction procedure, and a MCh challenge at Chonnam National University Hospital, participated in this study. The diagnosis of asthma was based on a clinical history of episodic wheezing, breathlessness, chest tightness or cough, and an improvement in forced expiratory volume in one second (FEV₁) of ≥12% and at least 200 mL after anti-asthma treatment for two weeks. The remaining patients were considered as non-asthma, although some of them may have subclinical or mild asthma. The Institutional Review Board of Chonnam National University Hospital approved the study. All patients were informed of the experimental procedures and provided written informed consent.

On the first study day, baseline spirometry, allergy skin-prick tests and routine laboratory tests were performed after history taking and physical examination. Airway responsiveness to HS was measured, and then two puffs of salbutamol were inhaled and sputum induction using HS was continued. On the next day, at the same time of day as the HS-AHR, a MCh-AHR test was performed. The subjects were instructed in the use of a metered-dose inhaler through a valved holding chamber, and they were asked to complete a PEF diary. Anti-asthma medications, including fluticasone (1,000 μg/day) and prednisolone (30 mg/day for one week), were prescribed and a clinical evaluation was conducted at least two weeks later.

Allergy skin-prick tests were carried out using 55 common allergen extracts with histamine and saline solution as positive and negative controls, respectively. After 15 minutes, a wheal more than 3 mm larger than that seen in the negative control was considered a positive prick test result, and subjects with at least one positive test result were considered atopic.

Airway responsiveness to hypertonic saline solution was assessed using the method of Iredale et al.⁸⁾ with 4.5% NaCl aerosol delivered from an ultrasonic nebulizer (Ultra-neb 2000, DeVilbiss, PA), through a Hans Rudolph 2700 two-way nonrebreathing valve box with a rubber mouthpiece and nose clips. The subjects inhaled 4.5% NaCl for doubling time periods (0.5, 1.0, 2.0, 4.0 and 8.0 minutes). FEV₁ was measured in triplicate before the test (baseline), and in duplicate (30 and 90 sec) after each inhalation period with a spirometer (Spiro Analyzer ST-250, Fukuda Sangyo, Tokyo, Japan). The next challenge period followed within three minutes of the end of the previous one. If the FEV₁ fell by less than 10%, the exposure time was doubled. If the fall in FEV₁ was between 10 and 15%, the exposure time was kept the same. If the fall in FEV₁ exceeded 15%, or after the maximal dose of solution had been delivered to the inspiratory port of the valve, the challenge was stopped. A dose-response curve was constructed by plotting the % change in FEV₁ against the cumulative dose of aerosol delivered, expressed in mL on a log scale for each inhalation period. The provocative dose of HS causing a 15% fall in FEV₁ (PD₁₅) was calculated by linear interpolation of the log-dose-response curve. The degree of airway responsiveness to HS was categorized as severe AHR if PD₁₅ was <2 mL, mild to moderate AHR if PD₁₅ was ≥2 but ≤20 mL, and normal if PD15 was >20 mL. Patients not responding to the highest dose of HS were assigned a value twice the highest dose applied. Another index of airway responsiveness, the dose response ratio (DRR), was calculated by dividing the maximal % fall in FEV₁ by the dose of saline required to produce it⁹⁾.

The MCh challenge test followed a standardized tidal breathing method¹⁰⁾. MCh in isotonic saline was aerosolized at room temperature in a DeVilbiss 646 nebulizer (DeVilbiss Co., Somerset, PA; output 0.13 mL/min). Dilution increments were 0.075, 0.15, 0.31, 0.62, 1.25, 2.5, 5.0, 10 and 25 mg/mL. The aerosols were inhaled by tidal breathing over 2 min at 5-min intervals, through the mouth with the nose clipped. The challenge test was discontinued if FEV₁ dropped 20% or more from the baseline or if the maximal concentration of MCh was administered. The provocation concentration of MCh resulting in a 20% fall in FEV₁ (PC₂₀) was calculated by linear interpolation of the log-dose-response curve. The degree of airway responsiveness to MCh was categorized as moderate to severe AHR if PC₂₀ was < 1 mg/mL, mild AHR if PC₂₀ was ≥1 but ≤4 mg/mL, borderline AHR if PC₂₀ was >4 but ≤16 mg/mL, and normal if PC₂₀ was >16 mg/mL. Patients not responding to 25 mg/mL of MCh or HS were assigned a PC₂₀ of 50 mg/mL. MCh-DRR was calculated by dividing the maximal % fall in FEV₁ by the dose of MCh required to produce it.

The subjects were encouraged to expectorate into a sterile container after each dose of HS solution. Immediately after the HS challenge, the patients inhaled 200 μg of salbutamol and additional 4.5% saline until sputum was induced. Sputum was processed as previously described¹¹⁾. Briefly, opaque sputum portions were selected and digested with 0.1% dithiothreitol (Sigma, MO), and filtered through 53-μm nylon gauze (Spectrum Laboratories Inc., CA). Total cell counts were determined and cytospin (CF-120, Japan) slides were prepared. Differential cell counts were expressed as the percentage of non-squamous cells in a sample of 300 cells on each of two slides stained with Diff-Quik. The interferon (IFN)-γ, interleukin (IL)-4 and IL-5 levels in sputum were measured using commercially available ELISA kits (IFN-γ: Endogen, USA; IL-4 & IL-5: Quantikine HS, R&D Systems, MN).

Variables were summarized as the mean±SEM. All calculations of PD₁₅ or PC₂₀ were performed after log transformation. Data were compared using an unpaired Student’s t-test. Associations between variables were examined with the Spearman rank correlation coefficient. A value of p<0.05 was considered statistically significant.