The study population consisted of 16 patients with IPF, ten with idiopathic NSIP, and eight normal controls. All subjects were given a chest radiograph, HRCT, pulmonary function test, and blood gas analysis (Table 1). The diagnoses of IPF and NSIP were based on the international consensus statement (21). Two of the NSIP patients had inflammation and fibrotic type pneumonia (group II) and eight had interstitial fibrotic type pneumonia (group III). BAL and open -lung or video-guided thoracoscopic biopsies were performed on all IIP patients. BAL was performed at least 3 days before lung biopsy and without any immunosuppressive therapies. In normal subjects, BAL was performed on the right middle lobe. Exclusion criteria consisted of other known causes of interstitial lung disease, such as drug reactions, environmental exposures, and collagen vascular diseases. The normal controls were all healthy volunteers, free of symptoms, not taking any medications, and who had normal chest radiographs. For immunohistochemical staining, the healthy tissues of biopsies of solitary pulmonary nodules were used.

BAL was performed as previously described (17), using a flexible fiberoptic bronchoscope (Olympus B2-10; Olympus Optical Co., Tokyo, Japan). Briefly, all study subjects were pretreated with atropine (0.5 mg) immediately before the BAL procedure. Bronchial trees were instilled four times with 50 mL of normal saline, and fluid was removed each time by gentle suction with negative pressure below 50 mmHg. The total volume of recovered fluids was measured, and the supernatant was separated from the cell pellets by centrifugation at 500×g for 5 min. The supernatants were frozen and stored at -80℃ for later use. A differential count of 500 cells was performed on slides prepared by cytocentrifugation and Diff-Quik staining (Scientific Products, Gibbstowne, NJ, U.S.A.). No complications were experienced during the BAL procedures.

The IL-13, IL-4, and IFN-γ levels were measured using quantitative sandwich enzyme immunoassays with ELISA kits (BD Bioscience, San Diego, CA, U.S.A.), according to the manufacturer's recommendations. The detection limits were 2.0 pg/mL, 3.5 pg/mL, and 1.0 pg/mL for IL-4, IL-13, and IFN-γ, respectively, and values below these thresholds were assigned a value of 0 pg/mL. For validation of assay, we performed spiking experiment using 10 samples and the recovery rates of IL-4, IL-13, and IFN-γ were 86.2±0.1%, 93.5±0.6%, 89.3±0.8%, respectively. The inter-assay coefficient of variability for all assays was less than 10%, and the intraassay coefficient of variability was less than 10% across the range of concentrations.

A VectaStatin goat ABC Elite kit (Vector Laboratories, Burlingame, CA, U.S.A.) was used for immunostaining. Tissue sections (5 µm) were deparaffinized, endogenous peroxidase was blocked with 1.4% H₂O₂ in MeOH for 30 min, and nonspecific binding was blocked with 1.5% normal rabbit serum for 30 min. The sections were incubated with goat polyclonal antihuman IL-13 antibody (1:100, Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.) and goat polyclonal antihuman IL-13Rα1 and IL-13Rα2 antibody (1:200, R&D, Minneapolis, MN, U.S.A.) at 4℃ for 16 hr. After washing with TBS, the sections were sequentially incubated with biotinylated rabbit anti-goat immunoglobulin G (H+L) (1:200, Vector Laboratories, Burlingame, CA, U.S.A.) and avidin-biotin peroxidase complex (1:50, ABC kit, Vector Laboratories) for 30 min. The color reaction was developed with 3,3'-diaminobenzidine tetrachloride (Zymed Laboratories, South San Francisco, CA, U.S.A.), and Harris hematoxylin stain was applied as a counter stain. The intensity of immunostaining was graded from absent (-) to strongly positive (+++) for the following tissues: alveolar epithelium, bronchial epithelium, smooth muscle, alveolar macrophage, endothelial cells, and interstitium. Grading was as follows: (-), no staining; (±), very faintly brown; (+), faintly brown; (++), moderately brown; and (+++), dark brown. Histological sections were assessed independently by two observers who were blind to the clinical information of the study subjects.

The SPSS/PC+ program (Chicago, IL, U.S.A.) was used for statistical analyses. Differences between groups were compared with the nonparametric Kruskal-Wallis test for continuous data. Significant relationships between groups were further analyzed using the Mann-Whitney U test. Spearman's rank correlation was used to analyze the relationships of IL-4, IL-13, and IFN-γ concentrations to the lung functions. Differences were considered significant at P<0.05. The data are expressed as mean±SEM.

The clinical and BAL profiles are summarized in Table 1. Age, smoking status, and sex ratio were comparable among the three groups. The NSIP and IPF patients had a significant reduction in forced vital capacity (FVC), total lung capacity (TLC), and diffusing capacity of carbon monoxide (DLCO) compared with controls (P<0.05). Both the cell number and percentage of neutrophils in 1 mL of BAL fluid were significantly higher in patients with NSIP and in those with IPF than in control subjects (P<0.01 and P<0.05, respectively). There were no difference in the percentages of lymphocytes and macrophages between NSIP and IPF patients, but IPF patients had a significantly higher percentage of eosinophils than controls (P<0.01).

IL-13 activity was detected in the BAL fluid of all patients and in seven of the eight control subjects. The IL-13 levels were higher in IPF patients than in NSIP patients (249.0±83.1 vs. 137.7±37.9 pg/mL, P=0.032) and controls (249.0±83.1 vs. 49.0±13.1 pg/mL, P=0.012). The IL-13 levels were also higher in NSIP patients than in controls (P=0.041; Fig. 1A). IL-4 activity was detected in 12 of 16 IPF patients, eight of ten NSIP patients, and three of eight controls. The mean IL-4 level was significantly higher in the IPF and NSIP groups than in the control group (16.3±4.5 and 12.3±3.35 pg/mL vs. 4.67±2.28 pg/mL, P=0.008 and P=0.034, respectively). The IL-4 level was not significantly different between the IPF and NSIP groups (P>0.05; Fig. 1B). IFN-γ activity was detected in 14 of 16 IPF patients, nine of ten NSIP patients, and seven of eight controls. Both patient groups had low INF-γ levels compared with the control group (7.72±3.02 and 3.82±1.59 pg/mL vs. 11.32±3.64 pg/mL; Fig. 1C), but only the difference between NSIP patients and controls was significant (P=0.047). The IL-13 level was inversely correlated with DLCO (% pred) (n=26, r=-0.58, P=0.014) and FVC (% pred) (n=26, r=-0.47, P=0.043) in NSIP and IPF patients (Fig. 2). Neither the IL-4 nor INF-γ level correlated with the physiological parameters (data not shown).

Immunohistochemical staining of IL-13, IL-13Rα1, and IL-13Rα2 was performed in the lung tissues of controls (n=4), IPF patients (n=6), and NSIP patients showing fibrosis (n=6). To avoid the smoking effect, never smokers were selected from the study subjects. Control subjects had undergone a lobectomy to remove localized solitary pulmonary nodules (two lung cancer and two tuberculoma). Both IL-13 and IL-13Rα1 were strongly expressed in the bronchial epithelial cells, alveolar macrophages, endothelial cells, and smooth muscle of all patients (Fig. 3), although semiquantitative evaluation showed that their intensities were much stronger in IPF patients than in NSIP patients (Table 2). IL-13Rα2 showed the same pattern of expression as IL-13Rα1 but weaker staining in both patient groups (Table 2, Fig. 3). Neither IL-13Rα1 nor IL-13Rα2 expression was detected in the four control lung tissues.