The original stock of male ICR mice (20–30 g) was purchased from the Dae-Han Biolink Co. Ltd. (Daejeon, Korea). The animals were maintained in the Laboratory Animal Research Center (LARC) of Gyeongsang National University. The care and treatment of the mice were in accordance with the guidelines established by the Public Health Service Policy on the Human Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee.

Piceatannol (trans-3,4,3′,5′-tetrahydoxystilbene) (PIC) (Fig. 1) was purchased from Sigma-Aldrich and dissolved in dimethyl sulfoxide (DMSO), so that the final PIC concentrations in culture media were 1, 10, 50 and 100 μM. Compound 48/80, anti-dinitrophenyl (DNP) IgE, DNP-human serum albumin (HSA), pyrrolidine dithiocarbamate (PDTC), phorbol 12-myristate 13-acetate (PMA), the calcium ionophore A23187 (calcimycin) and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The easy-BLUE™ Total RNA Extraction kit was purchased from Intron Biotechnology. Iscove’s modified Dulbecco’s medium (IMDM) and Dulbecco’s modified Eagle’s medium (DMEM) were obtained from Gibco BRL (Grand Island, NY, USA). Anti-human TNF-α and IL-8 antibodies, and biotinylated anti-human TNF-α and IL-8 antibodies were obtained from BD Pharmingen (San Diego, CA, USA); antibodies to ERK, p-ERK, JNK, p-JNK, p38 and p-p38 were obtained from Cell Signaling (Beverly, MA, USA).

As an in vivo model for the immediate-type allergic reaction, compound 48/80-induced systemic reaction was carried out as previously described (24). Briefly, the mice (n=10/group) received an intraperitoneal injection of 8 mg/kg body weight (BW) of the mast cell degranulator, compound 48/80. PIC was dissolved in saline and administered intraperitoneally at doses of 10–1000 mg/kg BW 1 h before the compound 48/80 injection. In the time-dependent experiment, PIC (1000 mg/kg) was administered 5, 10, 15 and 20 min after compound 48/80 injection (n=10/group). Mortality was monitored for 1 h after induction of anaphylactic shock.

PCA reaction was carried out as previously described (25). Briefly, mice were injected intradermally with 0.5 μg of anti-DNP IgE. After 48 h, each mouse (n=10/group) received an injection of 1 mg of DNP-HSA in PBS containing 4% Evans blue (1:1) via the tail vein. PIC (10–1000 mg/kg BW) was intraperitoneally administered 1 h before the challenge. Thirty minutes after the challenge, the mice were sacrificed and the dorsal skin (diameter, 1 cm) was removed in order to measure the pigment area. The amount of dye was determined colorimetrically after extraction with 1 ml of 1 M KOH and 9 ml of a mixture of acetone and phosphoric acid (5:13). The intensity of the absorbent was measured at 620 nm in a spectrophotometer (Biochrom Libra S22 UV; Biochrom, Holliston, MA, USA).

The human mast cell line HMC-1 (a kind gift from Dr H.M. Kim of Kyeonghee University, Seoul, Korea) was grown in IMDM, supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin and 10% heat-inactivated fetal bovine serum (FBS), in a humidified incubator at 37°C in 5% CO₂. The adherent rat basophilic leukemia RBL-2H3 1 cell line (Korean Cell Line Bank no. 22256) was grown as a monolayer culture in complete DMEM (containing 10% heat-inactivated fetal bovine serum, 4 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin). For experiments, the cells were detached by trypsinization, washed once with complete DMEM, once with serum-free DMEM, and then were resuspended in serum-free DMEM.

HMC-1 and RBL-2H3 cells were treated with PIC (1, 10, 50 and 100 μM) for 30 min. The cells were then stimulated by 50 nM of PMA plus 1 μM of A23187 and incubated at 37°C for the indicated time periods.

Cell viability was determined by an MTT assay. Briefly, cells were pretreated with various concentrations of PIC (1, 10, 50 and 100 μM) for 30 min and then incubated for 12 h in the absence or presence of PMA plus A23187. After a 12-h incubation, 50 μl of MTT solution was added to the culture (total volume, 500 μl). After an 8-h incubation, the absorbance was measured at 540 nm with a microplate reader (PRS Scientific LLC, Frederick, MD, USA). The absorbance correlates with cell viability, and the number of cells (% of control) was calculated using the following formula: Cell number (% of control) = (absorbance of treated cells - absorbance of blank well)/(absorbance of control cells - absorbance of blank well) × 100. The control was not treated, and the blank control consisted of PMA- plus A23187-stimulated HMC-1 cells without PIC treatment.

Preparation of serum and determination of histamine contents were performed as previously described (26). Trypsinized RBL-2H3 cells were resupended in serum-free DMEM (1×10⁶ cells/ml) in a 24-well cell culture plate and stabilized for 1 h in an incubator. The cells were pretreated with various concentrations of PIC (1, 10, 50 and 100 μM) for 30 min and then incubated for 8 h with PMA plus A23187. After treatments, the cell suspensions were centrifuged at 13,000 × g for 5 min and the pellets were discarded. The concentrations of released histamine were determined using a histamine enzyme immunoassay kit (Oxford Biomedical Research, Oxford, MI, USA), and 50-μl samples of supernatants were tested following the manufacturer’s instructions. Fifty microliter samples or the standard, plus the enzyme conjugate were added to the 96-well plate of the kit, which was coated with monoclonal antibody, followed by incubation for 45 min at room temperature. The plate was then washed 3 times, and after the addition of 150 μl of tetramethylbenzidine (TMB) substrate, it was incubated for 15–20 min. The reaction was stopped using 50 μl of 1 N HCl, and the absorbance was read at 450 nm. All subsequent steps took place at room temperature, and all standards and samples were assayed in triplicate.

Mast cell degranulation was determined by measuring the release of a granule marker, β-hexosaminidase. RBL-2H3 cells were seeded into 24-well plates (1×10⁶ cells/well) and incubated overnight. Cells were washed 3 times with Siraganian buffer (125 mM NaCl, 5 mM KCl, 1.5 mM CaCl₂, 1.5 mM MgCl₂, 20 mM HEPES, pH 7.3). The cells were then stimulated with 400 ng/ml dinitrophenol-bovine serum albumin (DNP-BSA) (Sigma-Aldrich) suspended in 500 μl Siraganian buffer containing 0.1% BSA, and incubated at 37°C for 1 h. Following incubation, 50 μl samples of the cell culture supernatant were each incubated with 200 μl of 1 mM p-nitrophenyl N-acetyl-β-D-glucosamine (Sigma) in 0.05 M citrate buffer (pH 4.5) for 3 h at 37°C. The enzyme reaction was terminated by the addition of 500 μl of 0.05 M sodium carbonate buffer (pH 10.0), and the absorbance of each reaction was determined at 405 nm.

The effects of various treatments on β-hexosaminidase release were described as the percentage of the IgE-only control absorbance value: % of inhibition = (Treated - Blank - Spontaneous)/(Control - Blank - Spontaneous); Control, normal allergen-IgE response evoked with test material not added; Treated, normal allergen-IgE response evoked with test material added; Blank, only test material and substrate added to ELISA well; Spontaneous, allergen-IgE response was not evoked with test material not added.

The secretion of TNF-α and IL-8 was measured by an enzyme-linked immunosorbent assay (ELISA). HMC-1 cells were stimulated with PMA (50 nM) and A23187 (1 μM) for 8 h with or without PIC. The ELISA was performed by coating 96-well plates with 6.25 ng/well of monoclonal antibodies with specificity for TNF-α or IL-8. Before use and between subsequent steps in the assay, the coated plates were washed twice with washing buffer (PBS containing 0.05% Tween-20). For the standard curve, recombinant TNF-α and IL-8 were added to serum which was previously determined to be negative for endogenous TNF-α and IL-8. After exposure to the cell supernatant from the experiment, the assay plates were exposed sequentially to biotinylated 2,2′ azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) substrate. The absorbances at 405 nm were determined within 10 min after addition of the substrate. All subsequent steps took place at room temperature, and all standards and samples were assayed in triplicate.

HMC-1 cells (1×10⁶/ml) were pretreated with PIC for 30 min before PMA (50 nM) plus A23187 (1 μM) stimulation for 2 h. Total RNA was isolated according to the manufacturer’s specifications using an easy-BLUE RNA extraction kit. The concentration of total RNA was determined by spectrophotometry. Each sample was reverse transcribed to cDNA for 60 min at 45°C using a cDNA synthesis kit (Amersham Biosciences, Piscataway, NJ, USA). The primers and PCR conditions are shown in Table I. Products were electrophoresed on a 1.5% agarose gel and visualized by staining with ethidium bromide. The gels were photographed using a Kodak DC 290 digital camera (Eastman Kodak, Rochester, NY, USA) and digitized using UN-SCAN-IT software (Silk Scientific, Orem, UT, USA). Relative band intensity was analyzed by using ImageJ image analyzing software.

HMC-1 cells were washed twice with PBS and resuspended for protein extraction with reagents from an Intron Pro-Prep Protein Extraction kit. Processed samples were subjected to 8% SDS-PAGE and transferred to a PVDF membrane. The membrane was then washed with 5% skim milk in PBST (0.05% Tween-20 in PBS) for 1 h at room temperature and then incubated overnight with anti-MAPK antibodies. After the blot was washed 3 times with PBST, it was incubated with the secondary antibody for 1 h. The antibody-specific proteins were then visualized using an enhanced chemiluminescence detection kit (Amersham Biosciences), and the blot was exposed to X-ray film.

Statistical significances were compared between each treated group and analyzed by the Student’s t-test. Data from the experiments are presented as means ± SEM. The numbers of independent experiments assessed are provided in the figure legends.

To determine the effect of PIC on allergic reaction, an in vivo model of a systemic reaction was used. Compound 48/80 (8 mg/kg) was used as a model of induction for a systemic fatal allergic reaction. After the intraperitoneal injection of compound 48/80, the mice were monitored for 1 h, after which the mortality rate was determined. As shown in Table II, injection of compound 48/80 into mice induced fatal shock in 100% of the animals. When PIC was intraperitoneally administered at concentrations ranging from 10 to 1000 mg/kg BW 1 h prior to exposure to compound 48/80, the mortality rate was dose-dependently reduced. In addition, the mortality of mice administered PIC (1000 mg/kg) following compound 48/80 injection increased in a time-dependent manner (Table III). The effect of PIC on the compound 48/80-induced serum histamine release was investigated. PIC was administered at concentrations ranging from 10 to 1000 mg/kg 1 h prior to the compound 48/80 injection. Injection of compound 48/80 caused a marked increase in serum histamine release, and this induction was inhibited by PIC treatment in a dose-dependent manner (Fig. 2A). To confirm the anti-allergic effects of PIC, we used an IgE-mediated passive cutaneous anaphylaxis (PCA) model. PCA is one of the most important in vivo models of local allergic reaction. A local extravasation was induced by a local injection of IgE followed by an antigenic challenge. Intraperitoneal injection of PIC dose-dependently inhibited the PCA reaction (Fig. 2B).

To evaluate the cytotoxic effects of PIC, an MTT viability assay in HMC-1 and RBL-2H3 cells was performed. Fig. 3 shows the viability of cells incubated 24 h after stimulation in the absence or presence of PIC (1, 10, 50 and 100 μM). Stimulation and treatment with PIC did not appreciably affect cell viability. Kang et al (27). reported on the cytotoxic effect of resveratrol, the precursor of PIC, on HMC-1 cells using the MTT assay. Resveratrol was not cytotoxic against HMC-1 and RBL-2H3 cells up to 100 μM.

The release of chemical mediators such as histamine plays an important role in allergic inflammation (27). Therefore, we examined the effects of PIC on histamine release. We evaluated the ability of PIC to inhibit PMA and A23187-induced histamine release from RBL-2H3 cells (Fig. 4A). Histamine (~188 ng/ml) was detectable in unstimulated cells; however, RBL-2H3 cells released ~275 ng/ml of histamine when stimulated with PMA plus A23187. PIC inhibited PMA and A23187-induced histamine release in a dose-dependent manner as follows: 22.14% inhibition at 1 μM, 30.18% inhibition at 10 μM, 51.92% inhibition at 50 μM and 61.61% inhibition at 100 μM (Fig. 4B).

The rat basophilic leukemia RBL-2H3 cell line, a tumor analog of mast cells, exhibits phenotypic characteristics of mucosal mast cells. After stimulation with antigens, cells release β-hexosaminidase, which is a marker of mast cell degranulation. Thus, RBL-2H3 cells are considered a good model for the comprehensive study of events induced by multivalent allergens in mast cells (27–29). In our study, PIC strongly suppressed PMA plus A23187-induced degranulation in a dose-dependent manner in the RBL-2H3 cells (Fig. 5).

TNF-α and IL-8 are the most important pro-inflammatory cytokines. Therefore, we tested the effect of PIC on the gene expression of TNF-α and IL-8 induced by PMA plus A23187 in HMC-1 cells. Gene expression of pro-inflammatory cytokines was then analyzed using RT-PCR. HMC-1 cells were treated with PMA plus A23187 in the presence or absence of PIC (1, 10, 50 and 100 μM) for 2 h and then harvested for transcriptional analysis via RT-PCR. Enhanced TNF-α and IL-8 mRNA expression induced by PMA plus A23187 was inhibited by pretreatment of HMC-1 cells with PIC. Pretreatment with PIC at concentrations of 1, 10, 50 and 100 μM inhibited PMA and A23187-induced gene expression of TNF-α and IL-8. Gene expression of TNF-α and IL-8 was decreased in a dose-dependent manner by pretreatment with PIC (Fig. 6).

To confirm the effect of PIC on the gene expression of pro-inflammatory cytokines, culture supernatants were assayed for TNF-α and IL-8 levels by ELISA methods. HMC-1 cells were pretreated with PIC (1, 10, 50 and 100 μM) before stimulation with PMA (50 nM) and A23187 (1 μM) for 8 h, and culture supernatants were analyzed using ELISA. As shown in Fig. 7A and B, the concentrations of TNF-α and IL-8 were considerably increased after HMC-1 cells were stimulated with PMA plus A23187. PIC pretreatment strongly inhibited these increases in a dose-dependent manner.

MAPK pathways play a crucial role in the regulation of pro-inflammatory molecules affecting cellular responses. In order to elucidate the mechanisms underlying the effects of PIC, we examined the possible effects of PIC on the activation of MAPKs (Fig. 8). The stimulation of HMC-1 cells with PMA plus A23187 resulted in increased phosphorylation of all 3 types of MAPKs (p38, JNK, and ERK) 30 min post-treatment. Pretreatment with PIC (1, 10, 50 and 100 μM) attenuated PMA and A23187-induced p38 and JNK MAPK activation but did not significantly affect the phosphorylation of ERK.