Lung specimens were obtained from 15 subjects undergoing resection for a small peripheral carcinoma (< 3 cm in diameter). They were all lifelong nonsmokers. None had a central tumor or obstructive pneumonia. Pulmonary function tests of eight patients showed normal spirometric results and normal lung volume (control group) (Table 1). Seven patients had bronchial asthma (asthma group); the diagnosis of asthma was supported by recurrent episodes of airflow limitation. The airflow limitation was improved in FEV_1.0 of greater than 15% after inhaled salbutamol (200 μg) in the asthmatics. Inflammation in large airways was histologically confirmed together with airway wall remodeling. Four asthma subjects were atopic and the remaining were nonatopic. Therapeutic periods in the asthmatics were more than 5 yr. All were treated with an inhaled β₂-agonist only when needed, and with a stable daily dose of inhaled steroid for greater than 6 mo.

Lung specimens without lung cancer were fixed with 10% formalin, and embedded in paraffin; at least 5 blocks were made in each case. The sections of 5 μm thickness were deparaffinized with xylene and stained with hematoxylin–eosin and with periodic acid–Schiff/alcian blue; sections were also immunostained for CC10 and different inflammatory cell markers by using the avidin/biotin peroxidase complex method. The monoclonal antibodies used were anti-CD3 (pan T lymphocyte; Novacastra Laboratories, Newcastle, UK), EG2 (activated eosinophils, secreting eosinophil cationic protein [ECP]; Kabi Pharmacia, Uppsala, Sweden), antitryptase (mast cells; Dako, Glostrup, Denmark), and TY-5 (CC10/UP-1) (13). In order to retrieve antigen, sections were incubated with 0.1% trypsin and 0.1% CaCl₂ 2H₂O in 50 mM Tris buffer at pH 7.4 at 37° C for 120 min in case of immunostaining of tryptase, and sections were pretreated with autoclave in 0.01 M sodium citrate buffer (pH 6.0) at 120° C for 20 min in case of immunostaining of CD3. The sections were soaked in absolute methanol containing 0.3% hydrogen peroxide, for 30 min at room temperature, to eliminate endogenous peroxidase activity. The sections were incubated with 1.5% nonimmunized goat serum for 30 min at room temperature, then incubated with each monoclonal antibody for 60 min at room temperature, and washed three times with phosphate-buffered saline (PBS) for 30 min. Thereafter, the sections were incubated with biotinylated goat anti-mouse Ig serum for 60 min. The biotinylated goat anti-mouse Ig serum was preapplied human-Ig- coupled Sepharose-4B to remove nonspecific binding to human tissue. After being washed with PBS, the sections were reacted with avidin/ biotin peroxidase complex (Vector, Burlingame, CA). The enzyme reaction was developed as described previously (12). Nuclei were lightly counterstained with hematoxylin. Normal mouse Ig was used as a negative control. No significant reaction occurred in this case.

We measured inner perimeter (Pi) defined by the luminal border of the airways and the area of small airways according to the method described by Carroll and coworkers (14) with modification. Briefly, using a light microscope, we took microphotographs of small airways that were cut in transverse section, and analyzed measurements of airways on a minicomputer using images of microphotographs. Airways with Pi < 6 mm were assessed as small airways. We evaluated immunoreactive cells (T cells, activated eosinophils, and mast cells) and CC10-positive epithelial cells in the small airways using serial sections. CC10-positive cells in the small airways were expressed as percentages of CC10-positive epithelial cells to total epithelial cells. In case goblet cell metaplasia greater than 5% in total epithelial cells assessed by sections staining with periodic acid–Schiff/alcian blue was found in the small airways, they were excluded in this analysis. Immunoreactive cells were expressed as numbers per square millimeter of the small airway wall.

Data were expressed as means ± SD. The Mann-Whitney U test was used to compare paired sets of data and the Pearson's least squares linear regression analysis was used to determine correlations. The level of critical significance was assigned at a p value less than 0.05.