Nasal polyps were obtained from eight patients who were undergoing endoscopic sinus surgery. Patients were excluded if they had an allergy or they had received systemic or topical steroids or antibiotics during the four weeks preceding the study. This study was approved by the Institutional Review Board of Daegu Catholic University Medical Center and each patient signed a consent form that outlined the objectives of the research and experiments. The specimens were placed in Ham's F-12 medium supplemented with 100 international units penicillin, 100 µg/mL streptomycin and 2 µg/mL amphotericin B, and they were transported to the laboratory. The nasal polyp epithelial cells were isolated using protease digestion methods. The polyps were rinsed with Ham's F-12 medium supplemented with antibiotics and they were incubated with Dispase (Roche, Mannheim, Germany) in Ham's F-12 medium for 16 hr at 4℃. After incubation, the nasal epithelial cells were isolated by gentle agitation. The cell suspensions were filtered through a No. 60 mesh cell dissociation sieve and then centrifuged at 1,000 rpm for 5 min at room temperature. The cell pellet was then resuspended in Ham's F-12 medium supplemented with antibiotics, 150 µg/mL glutamine, 5 µg/mL transferrin, 25 ng/mL, epithelial growth factor, 15 µg/mL endothelial cell growth supplement, 5 IU/mL insulin, 200 pM triiodothyronin, 100 nM hydrocortisone and 15% fetal calf serum (FCS). The cell suspensions (10⁶ cells/mL) were plated on to six-well culture plates and these were placed in a 5% CO₂ humidified incubator at 37℃. The culture medium was changed after 24 hr and then every two days thereafter. When the epithelial cell cultures reached confluence, Ham's F-12 medium was changed to RPMI-1640 medium supplemented with 10% FCS and 50 µg/mL gentamycin. The nasal epithelial cells were activated with endotoxin removed DP and DF at 10, 25, and 50 µg/mL, respectively, (Greer Lab, Lenoir, NC, USA). After 48 hr of incubation, the supernatants and epithelial cells were harvested and stored at -70℃ until they were assayed. Cytokine production (IL-8, GM-CSF) was quantified using commercially available ELISA kits (R&D System, Minneapolis, MN, USA).

The confluent nasal epithelial cells were incubated with the DP and DF for 48 hr, and then they were treated with 1% paraformumaldehyde in phosphate buffer solution for 1 hr at room temperature to inhibit the synthesis of cytokines that maintain the intercellular interaction. The nasal epithelial cells were co-cultured with PBMCs for 72 hr. PBMCs were isolated from healthy volunteers after they gave their informed consent, which was approved by the Institutional Review Board of Daegu Catholic University Medical Center. The PBMCs were isolated by density gradient centrifugation on Histopaque (Sigma, St. Louis, MO, USA). The interphase cells were harvested, washed and resuspended at 1×10⁶ cells/ml in RPMI-1640 medium (Gibco, Rockville, MD, USA) and they were supplemented with 10% FBS. After 72 hr, the supernatants were harvested to measure the tumor necrosis factor (TNF)-α, IL-5 and interferon (INF)-γ using commercially available ELISA kits (R&D system).

After stimulated with the HDMs, the cell pellets were placed in cryo-tubes and 1 mL of Trizole reagent was added. RNA was extracted according to the manufacture's instructions (Roche). The RNA's purity and concentration were measured using a spectrophotometer (Beckman, Mountain View, CA, USA). One microliter of RNA was used for the reverse transcription PCR amplifications, which were performed in a PerkinElmer (Norwalk, CT, USA) thermal cycler. The primers used in this study were as follows: ICAM-1 (sense, AGG TCC ACC ACT GAC ACG TT, antisense CCA ATA TGA TTC CAC CCA, 348 bp) and β-actin (sense, CTG GCC GGG ACC TGA CT, antisense, GCA GCC GTG GCC ATC TC, 268 bp). The amplification reaction consisted of an initial denaturation step at 94℃ for 2 min, followed by three-step cycling: denaturation at 94℃ for 30 sec, annealing at a temperature optimized for each primer pair (ICAM-1: 58℃ and β-actin: 58℃) for 30 sec, and extension at 72℃ for 2 min, all for 32 cycles. The amplification products were electrophoresed on 2% agarose gels and they were visualized by ethidium bromide staining. The band densities were measured using the multi Gauge ver. 2.02 (Fujifilm, Tokyo, Japan). The band intensities were expressed as a percentage of the density of β-actin.

Immunocytochemical staining was performed with the monoclonal anti-ICAM-1 antibody (Sigma). The cytospin preparations of the nasal epithelial cells were obtained with a sample application of 1×10⁵ cells per slide. The cytospin slides were air-dried and treated with 3% H₂O₂. Non-specific binding was blocked by 10% goat serum. Incubation with the diluted specific anti-ICAM-1 antibody was performed at 37℃ for 1 hr, and then this was again done overnight at 4℃ in a moist chamber. After incubation with the corresponding diluted secondary antibody, the antigen-antibody complex was visualized using a commercial AEC kit (DAKO Co., Carpentaria, CA, USA) in accordance with the manufacturer's instructions. The immunostained cell samples were counted and three visual fields on every slide were evaluated (Fig. 1). The results were expressed as the percentages of positive cells. Immunolabelling was carried out in a single series to minimize the batch-to-batch variation.

Epithelial ICAM-1 was neutralized with the monoclonal antibody anti-CD54 (mAb 1379; Chemicon, Hampshire, UK). An IgG2a isotype antibody was used as a control. ICAM-1 and isotype control antibodies were added 1 hr prior to co-culture with the PBMCs. The cultured supernatants were collected after 72 hr to measure the cytokines.

All the experiments were performed at least five times and they revealed comparable results. The results are presented as the mean±SD. Statistical significance was determined using Student's t test and the Kruskal-Wallis Test (SPSS ver. 14.0; SPSS Inc., Chicago, IL, USA). A P-value of 0.05 or less was considered significant.

In the presence of HDM extracts, the nasal polyp epithelial cells produced larger amounts of IL-8 and GM-CSF compared to the non-stimulated epithelial cells. Maximal cytokine production was found at 50 µg/mL with DF and DP. The DF showed a time dependent increase in chemical mediator production up until 48 hr, but the cytokine productions by DP were slightly decreased after 24 hr (Fig. 2).

The activated epithelial cells were fixed with paraformumaldehyde to inhibit the cytokine secretion from the nasal epithelial cells. When the activated nasal epithelial cells were co-cultured with PBMCs, the PBMCs produced larger amounts of IL-5 and TNF-α, but not INF-γ. Compared with the co-culture of non-activated epithelial cells and PBMCs, the enhancement of TNF-α occurred at 50 and 25 µg/mL of DF, and at 25 µg/mL of DP, and the enhancement of IL-5 occurred at 50 and 25 µg/mL of DF (Fig. 3).

After stimulation with HDMs for 48 hr, relatively high values for the ICAM-1 mRNA were detected by stimulation with DP. Next, we evaluated the ICAM-1 expression on the cell membrane by performing immunocytochemical staining. After stimulation with HDMs, the epithelial cell membrane ICAM-1 expression was increased by both DP and DF (Fig. 4).

To evaluate the role of ICAM-1 in the interaction between epithelial cells and PBMCs, the epithelial cells were pretreated with CD54 monoclonal antibody to block the binding of leukocyte function associated antigen-1 to ICAM-1. After 72 hr of coculture, the production of IL-5, INF-γ and TNF-α was not inhibited by ICAM-1 antibody pretreatment.