Normal airway epithelial cells were cultured by a modification of the explant method of Lechner and coworkers (23). Nasal tissue was obtained from 10 nonallergic patients who had undergone surgery to relieve nasal obstruction. Bronchial tissue was obtained from 10 patients who had undergone lobectomy for lung cancer. Tissues were cut into 0.5 × 0.5 cm pieces and explanted to six-well culture plates. Two milliliters of LHC-1 medium (MCDB 151 with the Ca²⁺ concentration increased to 0.1 mM and supplemented with 5 μg/ml insulin, 5 ng/ml epidermal growth factor, 10 μg/ml transferrin, 5 × 10⁻⁴ mM each of phosphoethanolamine and ethanolamine, 5 × 10⁻⁴ mM hydrocortisone, and 50 μg/ml each of gentamicin and kanamicin) (Sigma Chemical Co., St. Louis, MO) with 10% fetal bovine serum (FBS) (GIBCO, Grand Island, NY) was then added to the plates, which were then incubated in a humidified 5% CO₂ incubator at 36.5°C.

After 7 to 11 d, outgrowths of epithelial cells were cultured in serum-free LHC-1 medium. After 7 d, cells were dissociated with trypsin (Böehringer-Mannheim, Mannheim, Germany) and inoculated into six-well culture plates coated with collagen (Böttger GmbH, Berlin, Germany). The identity of cultured cells was confirmed on the basis of morphologic characteristics and results of immunocytochemical staining with antibodies against cytokeratin, fibroblasts, and macrophages (Dako Japan, Kyoto, Japan) (24). The morphologic characteristics and positive staining for cytokeratin indicated that more than 96% of cells were epithelial cells. However, less than 3% of cells reacted with antibodies against fibroblasts and macrophages.

Influenza virus A/Sisen/2/92 (a kind gift from Dr. M. Toda, Showa University, Tokyo, Japan) was grown in Madin– Darby canine kidney (MDCK) cells (American Type Culture Collection, Rockville, MD) in minimal essential medium containing 2% FBS at 33.5°C under 5% CO₂. The infected culture fluid was subjected to centrifugation to produce a clear fluid, which was stored in aliquots at −80°C.

Epithelial cells were cultured to near confluence in six-well tissue culture plates. Influenza virus A (H3N2) was added to the plates at a multiplicity of infection of 0.2 (this viral titer was selected because severe cell damage was not observed and epithelial cells could produce cytokines with this viral titer) and allowed to be adsorbed by the cells for 1 h with gentle rocking of the plates every 10 min. After 1 h, virus was removed and 2 ml of serum-free LHC-1 medium was added. Infected cells were incubated at 36.5°C under 5% CO₂ in humidified air. For analysis of viral RNA, infected cells were harvested 1, 8, 24, 48, and 72 h after infection. For analysis of RANTES mRNA, nasal and bronchial epithelial cells, which were either infected or uninfected, were collected 24 h after infection or when media were exchanged. Culture media were collected 8, 24, 48, and 72 h after infection for enzyme-linked immunosorbent assay (ELISA).

Nasal and bronchial epithelial cells were cultured to near confluence in 24-well tissue culture plates. To analyze whether inflammatory cytokines could stimulate RANTES production, cytokines were added to the plates and the culture media were collected 24 h after stimulation to assay RANTES concentration. Added cytokines were as follows: 10 ng/ml, IL-1β; 100 U/ml, TNF-α; 100 U/ml, interferon γ (IFN-γ); and cytokine mix (a mixture of these three cytokines).

Total RNA was extracted with guanidium thiocyanate from samples of about 5 × 10⁵ cells (25). First-strand complementary DNAs (cDNAs) were synthesized by means of incubation of 1-μg RNA templates in the presence of Moloney murine leukemia virus reverse transcriptase (Pharmacia, Tokyo, Japan), random primers, and reaction buffer at 37°C for 1 h. After cDNAs were denatured at 94°C for 5 min, the polymerase chain reaction (PCR) was performed with 5 pmol each of the 5′ and 3′ primers, 5 μl of cDNA, 0.6 U of Taq polymerase (Böehringer-Mannheim), and 2.5 μl of PCR reaction buffer (100 mmol/liter, Tris-HCl; 15 mmol/liter, MgCl₂; 500 mmol/liter, KCl; Böehringer-Mannheim), distilled water was added until the reaction volume was 25 μl. The PCR primers were synthesized according to the sequence of hemagglutinin gene type 3 (H3, positions 337–1063) of influenza virus A (26). The sequences of the primers were 5′-TTGTTGAACGCAGCAAAGCT-3′ and 5′-TCTAGTTTGTTTCTCTGGTA-3′. The amplification reaction was performed for 25 cycles with denaturation at 94°C for 45 s, annealing at 61°C for 45 s, and extension at 72°C for 1 min (Perkin-Elmer Cetus, Norwalk, CT) (27). After incubation at 72°C for 10 min, PCR-amplified products were analyzed with 1.8% agarose gel electrophoresis and ethidium bromide staining followed by visualization with an ultraviolet transilluminator.

Total RNA was extracted from 5 × 10⁵ airway epithelial cells. The RT-PCR was performed using the method described previously. The PCR primers were as follows (1):

RANTES: 5′-ATGAAGGTCTCCGCGGCACGCCT-3′

5′-CTAGCTCATCTCCAAAGAGTTG-3′

β-actin: 5′-GTGGGGCGCCCCAGGCACCA-3′

5′-CTCCTTAATGTCACGCACGATTTC-3′

The amplification reaction was performed for 30 cycles with denaturation at 94°C for 45 s, annealing at 61°C for 45 s, and extension at 72°C for 1 min.

After incubation at 72°C for 10 min, PCR-amplified products were analyzed by 1.8% agarose gel electrophoresis. The gel was soaked in a denaturing solution containing 1.5 M sodium chloride and 0.5 M sodium hydroxide for 45 min with gentle shaking, then soaked in a neutralizing solution containing 1.5 M sodium chloride and 1 M Tris-EDTA, pH 7.4, for 45 min. The PCR products were transferred onto a nitrocellulose filter in 10× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) for 10 h with capillary action. The filter was dried at 80°C for 1 h. Prehybridization was carried out at 65°C for 2 h in a solution containing 6× SSC, 0.5% sodium dodecyl sulfate (SDS), 5× Denhardt's solution, and 100 μg/ml salmon sperm DNA. Hybridization was carried out at 65°C for 10 h in a solution containing 5× SSPE (1× SSPE is 0.18 M NaCl, 10 mM NaPO₄, and 1 mM EDTA [pH 7.7]), 0.2% SDS, 5× Denhardt's solution, 100 μg/ml salmon sperm DNA, and the probes for RANTES and β-actin, which encoded the internal region of the PCR products (R&D Systems, Minneapolis, MN) and were end labeled with ³²P by means of polynucleotide kinase. The filter was washed twice with 2× SSC and 0.1% SDS at room temperature for 30 min and then washed at 65°C for 2 h with gentle shaking in 0.1× SSC and 0.1% SDS. The filter was dried and used to expose autoradiography film at −80°C (28).

Concentrations of RANTES in the collected culture medium were determined with a commercially available system for ELISA (Amersham Life Science, Amersham, UK). The standards and samples were added to the 96 wells of a microtiter plate coated with anti-RANTES antibody. After incubation at room temperature for 2 h, each well was washed with wash buffer four times. Then, polyclonal antibody against RANTES, which was conjugated to horseradish peroxidase, was added to the well. After incubation at room temperature for 2 h, each well was washed with wash buffer four times. Substrate solution (stabilized hydrogen peroxide and tetramethylbenzidine) was added to each well, after which the plate was incubated at room temperature for 20 min. Sulfuric acid was then added to each well, and the absorbance was measured at 450 nm.

A standard curve was generated by plotting the logarithm of the optical density against the logarithm of the concentration of the standard; the concentration of each sample was then calculated. The minimal detectable concentration was 2.5 pg/ml.

To determine whether inflammatory cytokines mediated the RANTES production, concentrations of IL-1β, TNF-α, and IFN-γ in the culture medium collected 24 h after infection were also analyzed using commercially available ELISA kits (IL-1β and TNF-α [R&D Systems]; IFN-γ [Genzyme, Cambridge, MA]) with the same method as described previously. The minimal detectable concentration was as follows: IL-1β, 1 pg/ml; TNF-α, 4.4 pg/ml; and IFN-γ, 3 pg/ml.

We also examined a blocking assay to analyze the intervention of IL-1β and TNF-α in RANTES production induced by influenza virus. Anti-IL-1β antibodies (40 μg/ml; R&D Systems) and anti-TNF-α antibodies (40 μg/ml; R&D Systems) were added at the time of inoculation of influenza virus. Twenty-four hours after, the medium of the cells was collected and the concentrations of RANTES in the medium were analyzed by ELISA.

The peripheral blood of healthy volunteers was heparinized and overlaid onto Mono-Poly solution (Dai Nippon Pharmaceutical Co., Ltd., Osaka, Japan) (29) and then centrifuged at 400 × g at room temperature for 20 min. The polymorphonuclear leukocyte-rich fraction was aspirated and washed twice with phosphate-buffered saline (centrifugation at 400 × g for 10 min). Eosinophils were purified by negative selection with immunomagnetic microbeads coated with antibody against CD16 (30). Fifty microliters of MACS CD16 microbeads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) per 5 × 10⁷ cells was added to the cell pellets, after which the cells were incubated for 30 min at 6°C. The cells were suspended in 1 ml of phosphate-buffered saline, and the cell suspension was applied to the column in a MACS separator. The CD16-negative cells were collected and the eosinophil purity was determined by microscopic examination of Giemsa-stained cytospin preparations.

Eosinophil chemotaxis was assayed by means of a modification of Junger's method (31) using a 96-well chamber (Neuro Probe, Inc., MD). Eosinophils were suspended at 2.5 × 10⁵ cells/ml in the LHC medium. Each supernatant of cultured epithelial cells (uninfected cells and cells infected with influenza virus A) with or without anti-RANTES neutralizing antibodies (50 μg/ml, R&D Systems), a volume of 410 μl, was added to each well of a flat-bottom 96-well microtiter plate (Neuro Probe, Inc.). The microtiter plate was placed into the bottom tray of the chamber and covered with a polycarbonate filter (5-μm pore size, Neuro Probe, Inc.). Eosinophil suspension (200 μl) was then added to the upper wells after the chamber was tightened and allowed to migrate for 60 min at 37°C in 5% CO₂ atmosphere. The upper wells were then emptied by suction, and the microtiter plate with the attached filter was removed. The upper surface of the filter was wiped with a tissue to remove residual cell suspension, and the cells in the microtiter plate were sedimented by centrifugation at 250 × g for 15 min. The filter was detached and sedimented cells were counted by phase-contrast microscopy. The numbers of cells were counted in five random fields (one field = 1 mm²) of each duplicate well. LHC medium only was used as a negative control, and platelet-activating factor (PAF) (10⁻⁷ M; Sigma) or recombinant RANTES (1, 10, and 100 ng/ml; R&D Systems) was used as a positive control for eosinophil chemotaxis.

Analysis of variance was used to evaluate the statistical significance of the data in the analysis of cytokines concentration in the culture medium and eosinophil chemotaxis. Differences with P values less than 0.05 were considered significant.