Six-week-old male BALB/c mice were purchased from Southern Medical University (Guangzhou, China). The mice were housed in a specific pathogen-free (SPF) environment with a 12-h dark/light cycle. All experiments were conducted in accordance with the guidelines outlined by the committee of Southern Medical University on the use and care of animals. The protocols were approved by the Animal Subjects Committee of Nanfang Hospital. The vehicle (AOO) used to dissolve TDI consisted of a mixture of 2 volumes of acetone and 3 volumes of olive oil for dermal sensitization, and 1 volume of acetone and 4 volumes of olive oil for airway challenge. The concentrations of TDI were provided as a percentage (v/v) in AOO. TDI (toluene-2,4-diisocyanate), acetone and 1,25D₃ were obtained from Sigma-Aldrich (Shanghai, China). Enzyme-linked immunosorbent assay (ELISA) kits for IgE, interleukin (IL)-4 and interferon-γ (IFN-γ) were from Boshide Biotechnology (Wuhan, China).

All the mice were randomly divided into the following 3 groups as follows: i) the AOO group: vehicle (AOO)-sensitized/AOO-challenged and phosphate-buffered saline (PBS)-treated; ii) the TDI group: TDI-sensitized/TDI-challenged and PBS-treated; and iii) 1,25D₃ group: TDI-sensitized/TDI-challenged and 1,25D₃-treated. The construction of the model of TDI-induced asthma was carried out as previously described (8). Briefly, on days 1 and 8, the animals were dermally treated with 0.3% TDI or the vehicle on the dorsum of both ears (20 µl/ear). On days 15, 18 and 21, the mice underwent an oropharyngeal aspiration (20 µl) with 0.01% TDI or the vehicle. 1,25D₃ (100 ng/mouse) dissolved in 300 µl PBS containing 0.9% ethanol was administered to the mice by intraperitoneal injection 1 day prior to challenge with TDI for 8 consecutive days. The control mice received 300 µl PBS containing 0.9% ethanol by comparison. The methods for the measurements of airway reactivity, airway inflammation, and the expression levels of E-cadherin, ZO-1 and phosphorylated (p-) ERK1/2 in the airway mucosa of the mice were as previously described (8). The measurement of airway reactivity was carried out using methacholine. AHR to methacholine was assessed 24 h after the third challenge. Briefly, the mice were placed in a barometric plethysmographic chamber (Buxco Electronics, Troy, NY, USA). Aerosolized methacholine (Sigma-Aldrich) in increasing concentrations (0–50 mg/ml) was nebulized through an inlet of the main chamber for 3 min. Readings were taken and averaged for 3 min after each nebulization. The bronchopulmonary resistance was expressed as enhanced pause (Penh).

Hematoxylin and eosin (H&E) staining was used for the measurement of airway inflammation. Briefly, the mice were humanely euthanized with pentobarbital (100 mg/kg body weight, administered intraperitoneally) and the lungs were removed. The left lungs were infused with 4% formaldehyde. The lungs were fixed and embedded in paraffin. Sections (4-µm-thick) were cut using a Leica microtome 2030 (Leica Microsystems Nussloch GmbH, Nussloch, Germany). The slides were stained with H&E.

TDI-HSA conjugates were prepared by a modification of the method described in the study by Son et al (9) and the method used for the calculation of the amount of TDI bound to HSA was as previously described (7).

The human bronchial epithelial cell line, 16HBE, (Shanghai Fuxiang Biological Technology Co., Ltd., Shanghai, China) was used in this study due to its highly characterized intercellular adhesion properties (10). The 16HBE cells were grown in a cell culture flask in Dulbecco’s modified Eagle’s medium (DMEM) (Ginuo Biopharm Technology Co., Shanghai, China) with 10% fetal calf serum (FCS; Invitrogen, Gibco, Carlsbad, CA, USA) and placed in a humidified incubator at 37°C with 5% CO₂. When reaching 90% confluence, the cells were trypsinized and seeded into proper culture plates at a density of 10⁴–10⁵ cells/cm². When the cells had grown to complete confluence, they were pre-treated with or without 1,25D₃ (0.1 to 100 nM for 6 to 24 h) or the ERK1/2 selective inhibitor, U0126 (25 µM, for 1 h; Cell Signaling Technology, Beverly, MA, USA). Subsequently, 100 µg/ml TDI-HSA conjugate were added to the culture medium followed by incubation for 24 h. The concentration of 1,25D₃ and U0126 used in this study was in accordance with that used in previous studies (11,12). Cell viability was detected using a MTT colorimetric assay.

The method used for measuring TER was as described in the study of Sekiyama et al (13). In brief, the 16HBE cells were seeded at a density of 2×10⁵ cells/cm² onto the apical chamber of Polyester Membrane Transwell-Clear Inserts (Cat. no. 3460; Corning Inc., Corning, NY, USA) and incubated at 37°C until complete confluence was reached. Cell monolayer integrity was evaluated by measuring TER using a Millicell ERS-2 Epithelial Volt-Ohm Meter (Millipore, Billerica, MA, USA). TER (Ω × cm²) was calculated by (TER sample - TER blank) × surface area (cm²). The percentage change in TER following treatment was calculated as follows: TER(%) = (TER test/TER control) ×100, where the TER sample is the initial reading of each chamber, the TER blank is the reading of the no-cell control chamber, the TER test is the real value of each chamber, and the TER control is the reading of the no treatment control chamber.

The measurement of the FITC-Dx (70 kDa) flux was carried out using a previously described method (7). Briefly, the 16HBE cells were grown in Transwell inserts to achieve complete confluence. The cells were pre-treated with or without 1,25(OH)₂D₃, or the ERK1/2 inhibitor, U0126, and 100 µg/ml TDI-HSA conjugates were added to the culture medium followed by incubation for 24 h. At the end of the exposure period, the apical and basolateral chambers were washed twice with PBS, and FITC-Dx (0.5 mg/ml in phenol red-free DMEM) was then added to the apical chamber at a level of 0.5 ml, and 1.0 ml phenol red-free DMEM (Gibco, Carlsbad, CA, USA) was added to the basolateral chambers. The plates were then incubated at 37°C for 90 min. The samples from the apical and basolateral chambers were collected and data were read using a TECAN Infinite M200 fluorometer (Tecan, Maennedorf, Switzerland) with an excitation/emission wavelength of 495/520 nm.

The cells were grown to confluence, and then harvested and washed twice with ice-cold PBS. The cells were subsequently lysed in cell lysis buffer (KeyGen Biotech, Nanjing, China) containing protease inhibitor, calcineurin inhibitors and PMSF, and centrifuged at 12,000 x g for 15 min at 4°C. The protein products were normalized and boiled with standard SDS sample buffer, then separated by 8% (ZO-1) or 10% (E-cadherin, occludin, claudin-1/2, ERK1/2 and p-ERK1/2) sodium dodecyl sulfate-polyacrylamide (SDS) gel electrophoresis and transferred onto polyvinylidene fluoride membranes. The membranes were then blocked with 5% BSA (for p-ERK1/2 only) or skim milk at room temperature for 2 h, and incubated with anti-ZO-1 (sc-10804), anti-E-cadherin (sc-7870), anti-occludin (sc-5562), anti-claudin-1/2 (sc-28668) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-ERK1/2 and anti-p-ERK1/2 (#9101S) antibodies (Cell Signaling Technology) at 4°C overnight, and then incubated with anti-rabbit IgG (#7074; Cell Signaling Technology) at room temperature for 1 h. The immunoreactive bands were detected using an enhanced chemiluminescence ECL system (DingGuo Biotech Co., Ltd., Guangzhou, China).

In a parallel experiment, the 16HBE cells were seeded at a density of 1×10⁵ cells/cm² on a cell culture dish (35×12 mm, 15 mm glass bottom; Nest Biotechnology Co., Ltd., Shanghai, China) until confluent. The cells were then fixed with 4% para-formaldehyde at room temperature for 15 min, washed with ice-cold PBS for 15 min, incubated with 0.2% Triton X-100 in PBS for 10 min, and rinsed again with PBS. The cells were blocked with 5% skim milk for 2 h. All samples were subsequently incubated with rabbit polyclonal anti-E-cadherin and FITC-linked anti-rabbit IgG (ZF-0311; Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, China). The cell nuclei were stained with 4′,6-diamidino-2-phenylindole dihydrochloride (Sigma-Aldrich). A laser scanning confocal microscope (Olympus, Tokyo, Japan) was utilized to examine the distribution of junction proteins in the 16HBE cells. The images were processed with FV10-ASW1.7 Viewer and Photoshop.

Statistical analysis was performed using SPSS software version 13.0. Data are expressed as the means ± standard error (SE) and comparisons among groups were analyzed by one-way ANOVA accompanied by the LSD test for multiple comparisons. A value of P<0.05 was considered to indicate a statistically significant difference.

The pulmonary assessment of enhanced pause (Penh) was used to determine airway reactivity to methacholine. As shown in Fig. 1A, the Penh values were significantly increased in the TDI-treated mice compared with the controls following stimulation with methacholine (12.5, 25 and 50 mg/ml) (P<0.05); these values decreased after the administration of 1,25D₃ (methacholine, 50 mg/ml) (P<0.05). Similarly, there was a robust elevation in the serum IgE levels when the mice were sensitized and challenged with TDI; this effect was inhibited by 1,25D₃ (Fig. 1B). At the same time, the TDI-induced release of IL-4 (Th2-related) and IFN-γ (Th1-related) was also suppressed by treatment with 1,25D₃ (Fig. 1C and D).

The mice sensitized and challenged with TDI displayed marked inflammation in the peribronchial regions, as well as epithelial hyperplasia, as indicated by H&E staining of the sections (Fig. 2A). The analysis of the number of total and differential cells in the bronchoalveolar lavage (BAL) fluid revealed a significant increase in the number of total inflammatory cells, neutrophils and eosinophils following challenge with TDI (Fig. 2B). Treatment with 1,25D₃ markedly mitigated peribronchial inflammation and inflammatory cell accumulation into the airway lumen (Fig. 2A and B).

The pulmonary expression of p-ERK1/2 was determined by western blot analysis. There was a marked increase in the expression levels of activated ERK1/2 following airway challenge with TDI, which was then significantly inhibited by treatment with 1,25D₃ (n=3) (Fig. 2C).

The sections subjected to immunofluorescence staining showed a strong immunoreactivity of E-cadherin and ZO-1 at the lateral side and apicolateral border of the airway epithelial cells, while challenge with TDI resulted in much fainter staining of the two junction proteins at the epithelial cell-cell contact sites; these effects were partially reversed by treatment with 1,25D₃ (Fig. 3).

Each concentration of TDI-HSA we used had no detectable effect on cell viability (data not shown). TER was measured to determine epithelial barrier integrity. TER decreased immediately following the addition of TDI-HSA (100 µg/ml) to the culture plates and this decrease lasted for >24 h (Fig. 4A).

We then assessed the total cell lysates by western blot analysis to determine whether the epxression levels of TJ and AJ proteins were downregulated. We observed a decrease in the expression of occludin that was in parallel with the changes observed in TER. A transient abnormal expression of ZO-1 and claudin-1/2 was also detected, while the total protein expression of E-cadherin was relatively unaltered (Fig. 4B).

The TDI-HSA-treated 16HBE cells were pre-treated with 1,25D₃ at the indicated concentrations (0.1–100 nM) (Fig. 5A and B) for the indicated periods of time (0, 6, 12 and 24 h) (Fig. 5C and D). We observed that treatment with 1,25D₃ significantly reversed the decrease in TER and increased FITC-Dx permeability. Treatment with 1,25D₃ at the dose of 10 nM for 24 h achieved the most prominent protective effects.

In line with the aforementioned results using the mice with TDI-induced asthma, we observed an increase in the levels of p-ERK1/2 when the 16HBE cells were stimulated with TDI-HSA (Fig. 6A); this effect was reversed by treatment with 1,25D₃ (Fig. 6B), suggesting that the ERK1/2 pathway may be involved in this process. Thus, we compared the effects of 1,25D₃ with those of the selective ERK1/2 inhibitor, U0126, on the maintenance of airway epithelial integrity. Treatment with both 1,25D₃ and U0126 significantly increased TER (Fig. 6C), decreased FITC-Dx permeability (Fig. 6D), elevated the protein expression of occludin and claudin-2 (Fig. 6E), and inhibited the delocalization of E-cadherin (Fig. 6F).