Because imperatorin (IPT), the furanocoumarins exhibits anti-inflammatory activity, we reasoned that IPT might modulate the allergic rhinitis (AR). The aim of this study was to analyze the regulation of AR by IPT. Here, we show the effect and mechanism of IPT in an ovalbumin (OVA)-induced AR model. The number of rubs after the OVA challenge in the OVA-sensitized mice was significantly higher than that in the OVA-unsensitized mice. The increased number of rubs was inhibited by the oral administration of IPT. The increased levels of IgE and histamine in the OVA-sensitized mice were reduced by IPT administration. The levels of interferon-γ were enhanced, whereas the levels of interleukin (IL)-4 were reduced on the spleen tissue of the IPT-administered AR mice. Protein levels of IL-1β, macrophage inflammatory protein-2, intercellular adhesion molecule-1, and cyclooxygenase-2 were reduced by IPT administration in the nasal mucosa of the OVA-sensitized mice. In the IPT-administered mice, the number of eosinophils and mast cells infiltration increased by OVA-sensitization were also decreased. In addition, IPT inhibited caspase-1 activity in the same nasal mucosa tissue. In activated human mast cells, the receptor-interacting protein 2 (RIP2), IκB kinase (IKK)-β, nuclear factor-κB (NF-κB)/RelA, and caspase-1 activation were increased, but increased RIP2, IKK-β, NF-κB/RelA, and caspase-1 activation were inhibited by the treatment of IPT. In addition, IPT inhibited caspase-1 activity and IL-1β production in IgE-stimulated bone marrow-derived mast cells. We can conclude that IPT exerts significant effects by regulating of caspase-1 activation in AR animal and in vitro models.
Allergic rhinitis (AR) is a global health problem that causes major illness and disability and common manifestation of allergic diseases, affecting approximately 500 million people worldwide (Bousquet et al., 2008). Many of the symptoms of patients with AR, including sneezing, itching, and respiratory obstruction, cause a lot of pain. However, the symptoms of AR do not end here. If prolonged, AR can cause problems in the nasal voice box and can cause very severe eye and ear symptoms. These symptoms are attributed to the release of histamine and other active substances by mast cells, which stimulate the dilation of blood vessels, irritate nerve endings, and increase the secretion of tears (Whitcup, 2006).
Since the discovery by Coffman and colleagues of two distinct types of T helper (Th) cells in mice (Mosmann et al., 1986), mutual regulation between Th1 cells and Th2 cells has been considered important for homeostatic maintenance of the immune system in the whole body. Dysregulated Th1 and Th2 responses lead to excessive Th1-cell or Th2-cell activation, resulting in the development of autoimmune diseases associated with the accumulation of Th1 cells or in an induction of allergic diseases because of the accumulation of Th2 cells, respectively (Bach, 2002). In response to exposure to allergens, patients with AR present an inflammatory IgE-mediated response characterized by a Th2-immunologic pattern with mast cells and eosinophil activation and the release of inflammatory mediators interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (Johansson et al., 2001). Leukotrienes and prostanoids produced by the 5-lipoxygenase and cyclooxygenase (COX)-2 pathways have potent proinflammatory and vascular actions that implicate them in the allergic and inflammatory reactions (Montuschi et al., 2007). Eosinophils are innate effector cells that are important in immune responses against helminth parasitic infections and contribute to the pathology associated with allergic inflammatory conditions. Mast cells contribute to the induction and/or maintenance of eosinophilic inflammation by a variety of mechanisms, including IgE-dependent and IgE-independent processes (Pawankar et al., 2007). The recruitment of these mast cells to inflammatory sites occurs in response to chemotactic and activation signals (Bournazou et al., 2010). Minimal persistent inflammation is a physiopathological phenomenon referring to the presence of an inflammatory cell infiltrate (eosinophils and neutrophils) associated with the expression of intercellular adhesion molecule-1 (ICAM-1) in the epithelial cells of the mucosa exposed to the allergen in the absence of clinical symptoms. ICAM-1 is still only expressed in the mucosal epithelial cells of allergic patients. ICAM-1 was considered a marker of allergic inflammation (Montoro et al., 2007). Macrophage-inflammatory protein 2 (MIP-2) is a potent chemoattractant for immune cells (Gupta et al., 1996).
Caspase-1 is a member of the cysteine-aspartic acid protease (caspase) family (Stutz et al., 2009). Caspase-1 is characterized by its ability to activate the inactive precursors of IL-1β and IL-18 that are involved in inflammation. Caspase-1 contains an N-terminal caspase recruitment domain (CARD). This CARD promotes the proteolytic activation of the recruited caspase-1 in inflammation (Stutz et al., 2009). Caspase-1 is activated within inflammasome, a large cytosolic protein complex that is induced by a growing number of endogenous, microbial, chemical or environmental stimuli (Stutz et al., 2009). Specific adaptor molecules of the receptor-interacting protein-2 (RIP2, CARD-containing kinase) regulate the activation of caspase-1 through the CARD–CARD interaction (Kobayashi et al., 2002). RIP2 then recruits the IκB kinase (IKK) complex through the direct interaction of its intermediate domain with IKK-β, leading to the activation of the nuclear factor (NF)-κB (Inohara et al., 2000).
Imperatorin (IPT) (Fig. 1A) is one of the furanocoumarins. Furanocoumarins are depicted with miscellaneous biological functions, including vasorelaxation of corpus cavernosum (Chen et al., 2000), increased cell differentiation in osteoblasts (Kuo et al., 2005), anticonvulsant (Luszczki et al., 2009), antidiabetic (Liang et al., 2009), vascular vasodilation (He et al., 2007), and reduction in liver steatosis (Ogawa et al., 2007). Lin et al. (2010) demonstrated that glutamate release was facilitated by IPT in rat hippocampal nerve terminals. In addition, Adebajo et al., 2009 reported that the IPT had the main antitrichomonal activity.
This present study was designed to investigate the possibility of applying this IPT for the regulation of AR. Furthermore, we aimed to validate a possible mechanism in the ovalbumin (OVA)-induced AR models and activated mast cells.
Materials and Methods
IPT, dexamethasone (DEX), OVA, phorbol 12-myristate 13-acetate, calcium ionophore (A23187), O-phthaldialdehyde, avidin peroxidase, 2′-azino-bis(3-ethylbenzithiazoline-6-sulfonic acid) tablets substrate, bicinchoninic acid (BCA), antidinitrophenyl (DNP) IgE, DNP-human serum albumin (HSA), and other reagents were purchased from Sigma-Aldrich (St. Louis, MO). Fetal bovine serum (FBS), Iscove's modified Dulbecco's medium, and streptomycin were purchased from Invitrogen (Carlsbad, CA). Anti-mouse IgE/IL-1β [mature form detection antibody (Ab)]/IL-4/IFN-γ Ab, biotinylated anti-mouse IgE/IL-1β/IL-4/IFN-γ Ab, recombinant mouse IgE/IL-1β/IL-4/IFN-γ, anti-human IL-1β Ab, biotinylated anti-human IL-1β Ab, and recombinant human IL-1β were purchased from BD Biosciences Pharmingen (San Diego, CA). Ab for IKK-β, RIP2, caspase-1, COX-2, NF-κB/RelA, IκBα, histone, and actin were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The mouse OVA-specific IgE kit was purchased from DS Pharma Biomedical Co. Ltd. (Osaka, Japan) The caspase-1 assay kit was supplied by R&D Systems, Inc. (Minneapolis, MN).
OVA-Induced AR Animal Model.
We maintained 6-week-old female BALB/c (Charles River Laboratories, Inc., Wilmington, MA) mice under pathogen-free conditions. Mouse care and experimental procedures were performed under the approval from the animal care committee of Kyung Hee University [Approval no. KHUASP (SE)-10-016]. The mice were sensitized on days 1, 5, and 14 by intraperitoneal injection of 100 μg of OVA emulsified and 20 mg of aluminum hydroxide (Sigma-Aldrich) in a 100 μl of phosphate-buffered saline (PBS) and challenged intranasally with 2 μl of 1.5 mg of OVA or PBS. The mice were challenged intranasally with PBS in a similar manner for the negative control. IPT (0.1 and 1 mg/kg), DEX (5 mg/ml), or a control vehicle (distilled water.) was administrated orally for 10 days before the intranasal OVA challenge (Fig. 1B). Bain et al. (2011) reported that OVA-primed mice displayed a significant increase in sneezing behavior when challenged intranasally with OVA. In our study, although the frequency of sneezing was increased by the OVA challenge, there were no appreciable differences in the appearance of the mice between the OVA-unsensitized and OVA-sensitized groups (data not shown). Nasal symptoms were only evaluated by counting the number of nasal rubs that occurred in the 10 min after OVA intranasal provocation at the 10-day mark after the challenge. We measured OVA-specific IgE as the relevant endpoint for AR. The number of mice in each group was 5.
Culture of Human Mast Cell Line-1 Cells.
The human mast cell line (HMC-1) was generously provided by Eichi Morri (Osaka University, Osaka, Japan). HMC-1 cells were grown in Iscove's modified Dulbecco's medium supplemented with 100 units/ml penicillin, 100 mg/ml streptomycin, and 10% heat-inactivated FBS at 37°C 5% CO2 and 95% humidity. HMC-1 cells (3 × 105 cells/ml) were treated with IPT (1 and 10 μg/ml) for 1 h before stimulation with phorbol 12-myristate 13-acetate and A23187 (PMACI) was incubated for 2 or 8 h.
The histamine was measured from HMC-1 cells and serum according to the manufacturer's specifications using the histamine assay kit supplied by Oxford Biomedical Research (Oxford, MI).
Enzyme-Linked Immunosorbent Assay.
HMC-1 cells (3 × 105) were treated with IPT (1 and 10 μg/ml) for 1 h before stimulation with PMACI incubated for 8 h. Cytokines of serum, mucosa, and spleen tissue and supernatant were measured by an enzyme-linked immunosorbent assay (ELISA). The ELISA was performed by coating 96-well plates with 1 μg/well capture Ab. Before the subsequent steps in the assay, the coated plates were washed twice with 1× PBS containing 0.05% Tween 20. All reagents and coated wells used in this assay were incubated for 2 h at room temperature. The standard curve was generated from the known concentrations of cytokine, as provided by the manufacturer. After exposure to the medium, the assay plates were exposed sequentially to each of the biotin-conjugated secondary antibodies and an avidin peroxidase and 2′-azino-bis(3-ethylbenzithiazoline-6-sulfonic acid) tablet substrate solution containing 30% H2O2. The plates were read at 405 nm. Appropriate specificity controls were included, and all samples were run in duplicate. The OVA-specific IgE was measured from the serum according to the manufacturer's specifications using an OVA-specific IgE kit. Cytokine levels in the spleen and nasal mucosa were divided according to the total protein. The protein was estimated using the BCA method with the BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA). This method combines the reduction of cupric ions to cuprous ions by the protein in an alkaline medium and then the subsequent reaction of the cuprous ions with two molecules of BCA to give an intense purple color read at 560 nm.
Reverse Transcription-Polymerase Chain Reaction.
HMC-1 cells (3 × 106) were treated with IPT (1 and 10 μg/ml) for 1 h before stimulation with PMACI incubated for 6 h. The total RNA was isolated from the cells and nasal mucosa according to the manufacturer's specification using an easy-BLUE RNA extraction kit (iNtRON Biotechnology, Kyunggi-do, Korea). The concentration of total RNA in the final elutes was determined by spectrophotometry. Total RNA (2.5 μg) was heated at 65°C for 10 min and then chilled on ice. Each sample was reverse-transcribed to cDNA for 90 min at 37°C using a cDNA synthesis kit (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK). The polymerase chain reaction (PCR) was performed with the following primers for the mouse IL-1β (5′-AGG CCA CAG GTA TTT TGT CG-3′; 5′-GCC CAT CCT CTG TGA CTC AT-3′), mouse GAPDH (5′-TTC ACC ACC ATG GAG AAG GC-3′; 5′-GGC ATG GAC TGT GGT CAT GA-3′), human IL-1β (5′-GGG GTA CCT TAG GAA GAC ACA AAT TG-3′; 5′-CCG GAT CCA TGG CAC CTG TAC GAT CA-3′), and human GAPDH (5′-CCT GCT TCA CCA CCT TCT TG-3′; 5′-CAA AAG GGT CAT CAT CTC TG-3′). GAPDH was used to verify whether equal amounts of RNA were used for reverse transcription (RT) and PCR amplification from different experimental conditions. Saturation curves for PCR were obtained from various experimental conditions (RNA concentrations, annealing temperatures, and PCR cycle numbers). We determined the optimal amplification conditions (annealing temperature and PCR cycle number) of primers for the PCR. The annealing temperature was 50°C for mouse and human IL-1β and 60°C for mouse and human GAPDH, respectively. Products were electrophoresed on a 1.5% agarose gel and visualized by staining with ethidium bromide.
Western Blot Analysis.
HMC-1 cells (3 × 106) were treated with IPT (1 and 10 μg/ml) for 1 h before stimulation with PMACI incubated for 2 h. Western blot analysis was used for nasal mucosa tissue extracts, and cell extracts were prepared by a detergent lysis procedure. Samples were heated at 95°C for 5 min and briefly cooled on ice. After the centrifugation at 15,000g for 5 min, 50-μg aliquots were resolved by 10% SDS-polyacrylamide gel electrophoresis. The resolved proteins were electrotransferred overnight to nitrocellulose membranes in 25 mM Tris, pH 8.5, 200 mM glycerin, and 20% methanol at 25 V. Blots were blocked for at least 2 h with 1× PBS containing 0.05% Tween 20 containing 5% nonfat dry milk and then incubated with primary antibodies for 1 h at room temperature. Blots were developed by peroxidase-conjugated secondary antibodies, and proteins were visualized by enhanced chemiluminescence procedures (GE Healthcare) according to the manufacturer's instructions.
Tissue samples were immediately fixed with 10% formaldehyde and embedded in paraffin. The sections of the nasal mucosa sample were 4 μm thick. Each section was stained with hematoxylin and eosin (for eosinophils), Alcian blue and safranin O (for mast cells), CD4 (for T cells; Abbiotec, San Diego, CA), F4/80 (for macrophages; eBioscience, San Diego, CA), or immunohistochemical stain (for IL-1β) before dewaxing and dehydration. The numbers of eosinophils, mast cells, T cells, macrophages, and IL-1β on both sides of the septal mucosa were counted. Sections were coded and randomly analyzed by two blinded observers.
To evaluate the effect of IPT on neutrophil infiltration, the activity of tissue myeloperoxidase (MPO) was assessed. A biopsy was placed in 0.75 ml of 80 mM PBS, pH 5.4, containing 0.5% hexadecyltrimethylammonium bromide and homogenized (45 s at 0°C) in a motor-driven homogenizer. The homogenate was decanted into a microcentrifuge (Microfuge; Eppendorf, Westbury, NY) tube, and the vessel was washed with a second 0.75-ml aliquot of hexadecyltrimethylammonium bromide in a buffer. The wash was added to the tube, and the 1.5-ml sample was centrifuged at 12,000g at 4°C for 15 min. Samples of the resulting supernatant were added to 96-well microliter plates in triplicate at a volume of 30 μl. For the MPO assay, 200 μl of a mixture containing 100 μl of 80 mM PBS, pH 5.4, 85 μl of 0.22 M PBS, pH 5.4, and 15 μl of 0.017% hydrogen peroxide were added to the wells. The reaction was started by the addition of 20 μl of 10 mM O-dianisidine dihydrochloride in 80 mM PBS, pH 5.4. The plates were incubated at 37°C for 3 min and then placed on ice. The reaction was stopped by the addition of 30 μl of 1.46 M sodium acetate, pH 3.0. Enzyme activity was determined colorimetrically using a plate reader set to measure absorbance at 460 nm and is expressed as optical density milligram per tissue.
HMC-1 cells (3 × 106) were treated with IPT (1 and 10 μg/ml) for 1 h before stimulation with PMACI incubated for 2 h. The caspase-1 assay used nasal mucosa tissue and cell extracts. Caspase-1 activity was measured according to the manufacturer's specifications using a caspase assay kit (R&D Systems, Inc.). Equal amounts of the total protein were quantified by a BCA protein quantification kit (Sigma-Aldrich) in each lysate. Catalytic activity of caspase-1 from the cell lysate was measured by the proteolytic cleavage of WEHD-pNA or YVAD-pNA (BioVision, Inc., San Francisco, CA) for 4 h or various times at 37°C. The plates were read at 405 nm.
Transient Transfection and Luciferase Assay.
For the transfection, we seeded the HMC-1 cells (1 × 107) in a 100-mm culture dish. We then used Lipofectamine 2000 (Invitrogen) to transiently transfect reporter gene constructs into HMC-1 cells. HMC-1 cells (3 × 106) were treated with IPT (1 and 10 μg/ml) for 1 h before stimulation with PMACI incubated for 24 h. We mixed 20 μl of cell extract and 100 μl of the luciferase assay reagent at room temperature. To measure the luciferase activity, we used a luminometer (1420 luminescence counter; PerkinElmer Life and Analytical Sciences) in accordance with the manufacturer's protocol. The transfection experiments were performed in at least three different experiments, all with similar results. The relative luciferase activity was defined as the ratio of firefly luciferase activity to Renilla luciferase activity.
An IKK-β kinetic assay was performed according to the manufacturer's specifications using an IKK-β activity assay kit (EMD Chemical, Gibbstown, NJ).
Preparation of Mouse Bone Marrow-Derived Mast Cells.
Bone marrow-derived mast cells (BMMCs) were generated from the femoral bone marrow cells of mice as described previously. Cells were incubated in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 100 μM 2-mercaptoethanol, 10 mM sodium pyruvate, 10 μM minimal essential medium nonessential amino acid solution (Invitrogen), 100 U/ml murine IL-3 (R&D Systems, Inc.), and 0.5 U/ml murine stem cell factor (R&D Systems, Inc.) at 37°C in a humidified atmosphere in the presence of 5% CO2. After 4 weeks of being cultured, more than 96% of the cells were identifiable as mast cells as determined by toluidine blue staining. BMMCs were treated with anti-DNP IgE (10 μg/ml) and HSA (50 ng/ml).
The experiments shown are a summary of the data from at least three experiments, and statistical analyses were performed using an SPSS statistical software (SPSS 11.5; SPSS Inc., Chicago, IL). Treatment effects were analyzed by one-way analysis of variance, offered by Tukey's multiple range tests, and P < 0.05 was used to indicate significance.
Effects of IPT on Clinical Symptoms and Histamine, IgE, IL-4, IFN-γ, and IL-1β Levels in the AR Model.
To investigate the inhibitory effects of IPT in the AR model, we sensitized mice on days 1, 5, and 14 by intraperitoneal injections of 100 μg of OVA emulsified in 20 mg of aluminum hydroxide and the challenged mice with 1.5 mg of OVA. DEX (5 mg/kg) used as a positive control. The numbers of nasal and ear rubs after the OVA challenge in the OVA-sensitized mice were significantly higher than those in the OVA-unsensitized mice. Increased rub scores were inhibited by treatment with IPT (Fig. 2A). The spleen weights after the OVA challenge in the OVA-sensitized mice were significantly higher than those in the OVA-unsensitized mice. Increased spleen weights were reduced by IPT administration (Fig. 2B). Histamine levels in the serum were reduced by IPT (Fig. 2C). Levels of IgE in the AR mice were significantly higher than those in the serum, spleen, and nasal mucosa tissues of the OVA-unsensitized mice (Fig. 2, D and E). Total IgE and OVA-specific IgE levels increased by the OVA in the serum were reduced by IPT. To identify the Th1/Th2 immune reaction in IPT-administered mice, we measured IL-4 and IFN-γ levels in the spleen. As shown in Fig. 2, F and G, the levels of IL-4 in the AR mice significantly increased compared with those in the normal mice. IL-4 levels significantly decreased in the IPT-administered AR mice. However, IFN-γ levels increased by OVA were not changed in the IPT-administered AR mice. The protein levels of IL-1β in the serum and spleen tissue were increased in the AR mice compared with those levels in the control mice (Fig. 2H). However, protein levels of IL-1β were significantly reduced by IPT administration. IPT did not affect various allergic factors by itself.
Effects of IPT on IL-1β, MIP-2, ICAM-1, and COX-2 Levels in the Nasal Mucosa Tissue of the AR Model.
To evaluate the regulatory effect of IPT on IL-1β expression, we measured the protein and mRNA levels of IL-1β in the AR model. The protein and mRNA levels of IL-1β in the nasal mucosa tissue were increased in the AR mice compared with those levels in the control mice (Fig. 3, A and B). However, protein and mRNA levels of IL-1β were reduced by IPT administration. Levels of MIP-2, ICAM-1, and COX-2 in the AR mice were significantly higher than those in the nasal mucosa tissues of the OVA-unsensitized mice (Fig. 3, C–F). Increased levels of MIP-2, ICAM-1, and COX-2 were inhibited by IPT (Fig. 3, C–F).
Effects of IPT on Inflammation of Nasal Mucosa Tissue.
The respective numbers of inflammatory cells (eosinophil, mast cell, neutrophils, macrophages, and T cells) in the nasal mucosa in the AR mice were significantly higher than those in the control mice. In the IPT-administered mice, the increase in eosinophil, mast cells, macrophages, and T-cell infiltration (MPO activation) by OVA sensitization was decreased. However, IPT did not affect neutrophil infiltration (Fig. 4B). Immunohistochemical analysis of the nasal mucosa sections in the AR mice revealed that IL-1β is highly expressed, whereas in the IPT-administered mice, it is decreased (Fig. 4A).
Effects of IPT on Caspase-1 Activation in the Nasal Mucosa Tissues.
Caspase-1 plays a key role in inflammatory responses by cleaving pro-IL-1β into secreted proinflammatory cytokines. To investigate the effect of IPT on caspase-1 activation, a caspase-1 assay was performed with the nasal mucosa tissues. As shown in Fig. 5A, IPT (1 mg/kg) and DEX (5 mg/kg) inhibited OVA-induced caspase-1 activation. IPT also reduced the expression of caspase-1 in the nasal mucosa tissue (Fig. 5, B and C).
Effects of IPT on PMACI-Induced IL-1β Expression and Histamine Release in HMC-1 Cells.
Mast cells play a major role in the inflammatory reaction of AR (Pawankar et al., 2007). Because IL-1β is a major cytokine released from mast cells after allergic responses, we examined the effect of IPT on the expression of IL-1β in human mast cells. The protein and mRNA levels of IL-1β were significantly inhibited by IPT (Fig. 6, A and B). In addition, the inhibitory effects of IPT on PMACI-induced histamine release from HMC-1 cells are shown in Fig. 6C. IPT did not affect IL-1β and histamine by itself. We examined cell viability using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay and found that IPT had no effect on cell viability (Fig. 6D).
Effects of IPT on PMACI-Induced NF-κB Activation in HMC-1 Cells.
To assess the regulatory mechanism of IPT on allergic inflammation in the in vitro model, we examined the effect of IPT on PMACI-induced NF-κB activation, which is known to be important for cytokine expression in HMC-1 cells. Because the suppression of NF-κB is linked with anti-inflammation, we postulated that IPT mediates its effects at least partly through the suppression of NF-κB activation. The pretreatment with IPT (1 and 10 μg/ml) inhibited the PMACI-induced NF-κB/RelA levels in the nuclear extract (Fig. 7A). As a marker of NF-κB activation, the degradation of IκB-α in cell lysates was detected. We showed that IPT (1 and 10 μg/ml) inhibited the PMACI-induced IκB-α degradation (Fig. 7A). Histone and actin expression levels were not changed by any treatment in the nuclear and cytoplasmic extracts. Next, we examined whether IPT could modulate the luciferase expression specifically via the NF-κB. The NF-κB luciferase reporter gene constructs (pNF-κB-LUC, plasmid containing NF-κB binding site; Stratagene, La Jolla, CA) were transiently transfected into HMC-1 cells, which was treated by IPT and then stimulated by PMACI. As shown in Fig. 7B, PMACI increased reporter gene activity. However, the increased activity was decreased by the treatment of IPT (P < 0.05). IPT did not affect NF-κB activation by itself.
Effects of IPT on PMACI-Induced RIP2/IKK-β/Caspase-1 Activation in HMC-1 Cells.
RIP2 is a CARD-containing kinase that interacts with caspase-1 and plays an important role in NF-κB activation. Apoptosis-associated speck-like protein containing a CARD is a pyrin- and CARD-containing molecule, important in the induction of apoptosis and caspase-1 activation (Sarkar et al., 2006). Expressions of RIP2 and IKK-β were inhibited by IPT in HMC-1 cells (Fig. 8A). In the IKK-β assay, IKK-β activity was also inhibited by IPT (Fig. 8B). To evaluate whether IPT regulated the caspase-1 activation, we performed a Western blot analysis and caspase-1 assay. As shown in Fig. 8, C and D, caspase-1 activity increased by PMACI was inhibited by IPT. A caspase-1 kinetic assay was used to evaluate the binding affinity of IPT for the caspase-1 catalytic domain. Furthermore, recombinant caspase-1 (BioVision, Inc.) was also used to confirm the effect of IPT in the kinetic assay. As shown in Fig. 8E, caspase-1 activity was increased after a treatment with YVAD-pNA (caspase-1 substrate), but the IPT or caspase-1 inhibitor inhibited the binding of YVAD-pNA with the caspase-1-catalytic domain. It is noteworthy that IPT (10 μg/ml) exhibited a more potent binding affinity than did the caspase-1 inhibitor. In addition, caspase-1 activity and IL-1β production increased by anti-DNP IgE and HSA were inhibited by IPT in BMMCs (Fig. 8, F and G). IPT did not affect the expression of RIP2, activation of IKK-β and caspase-1, and production of IL-1β by itself.
In this study, IPT reduced the allergic inflammatory reaction in the AR model. AR is characterized by a two-phase allergic reaction. In the early-phase inflammatory response, allergen-IgE-dependent activation of mast cells and basophils results in the production of pharmacologically active mediators, such as histamine, prostaglandins, leukotrienes, and cytokines that produce sneezing, rhinorrhea, and itching (Jeong et al., 2009). The late-phase of AR shows accumulations of mast cells, eosinophils, and basophils in the epithelium and an accumulation of eosinophils in the deeper lamina propria (Fuentes-Beltrán et al., 2009). Recruitment of inflammatory cells, including eosinophils, mast cells, basophils, and T cells, results in the further release of histamine and leukotrienes, as well as in the release of other compounds, including proinflammatory cytokines, COX-2, and chemokines, which sustain the allergic response and promote the late phase response (Fuentes-Beltrán et al., 2009; Fukui et al., 2009). In previous studies, polyphenolic phytochemicals including rosmarinic acid have been shown to inhibit the IgE response (Makino et al., 2001) and inflammation characterized by polymorphonuclear leukocyte (eosinophils and neutrophils) infiltration (Osakabe et al., 2002). Glucocorticosteroid (GC) is the most effective drug for AR. GC inhibits the function of infiltrating inflammatory cells and their recruitment into the nasal mucosa. GC inhibits the maturation, cytokine production, COX-2 expression, FcεRI expression, and mediator release of mast cells (Smith et al., 2002; Takano et al., 2004). We observed that IPT inhibited the IgE production, inflammatory cytokine production, chemokine production, and COX-2 expression in the mice AR model. DEX also reduced the allergic and inflammatory reaction. Therefore, our results suggest that the effect of IPT is similar to the mechanism of rosmarinic acid or GC. We can also deduce that IPT has an antiallergic effect.
Inflammasomes are multiprotein cytoplasmic complexes that mediate the activation of inflammatory caspase-1 (Stutz et al., 2009). Inflammasome regulates the activation and secretion of caspase-1-regulated IL-1β and IL-18. Caspase-1(−/−) mice show the decreased production of IL-6 after stimulation with lipopolysaccharide (Martinon and Tschopp, 2005). Grzegorczyk et al. (2002) have reported a significant increase in caspase-1 level in serum from allergic asthmatic patients compared with a control group. Caspase-1 activity was increased in patients with the Netherton syndrome (Hosomi et al., 2008). In this study, we observed that caspase-1 was activated in the AR mice. IPT inhibited caspase-1 activity and IL-1β production. Therefore, we postulate that the inhibitory effect of IPT on inflammatory cytokine production might be derived from the regulation of caspase-1 activation.
Mast cells arise from pluripotential stem cells and mature in the tissue. They have the ability to generate immune reactions after exposure to a variety of receptor-mediated signals initiated by both innate and acquired immune-response mechanisms. Activated mast cells release a broad spectrum of mediators, including cytokines such as IL-1β (Druce, 1998; Johansson et al., 2001). Suppression of NF-κB activation has been linked to the inhibition of inflammatory cytokine (IL-1β, IL-6, and tumor necrosis factor-α) expression (Inohara et al., 2000). Activation by RIP2 induces caspase-1 oligomerization and promotes caspase-1 activation, with the latter inducing cytokine stimulation (Inohara et al., 2000). RIP2 and IKK complexes may play an important role for NF-κB activation (Inohara et al., 2000). In this study, IPT inhibited the IL-1β expression and production. We also confirmed that IPT suppressed the RIP2/IKK-β/caspase-1 activation in mast cells. This result suggested that the inhibitory effect of IPT on the allergic inflammatory reaction might be derived through the regulation of RIP2/IKK-β/caspase-1 activation.
IPT has not been elucidated for the effect and mechanism on inflammatory reactions. For the first time, we observed that IPT can regulate the reduction of inflammatory cytokine expression and that the inhibition of caspase-1 activation, which causes the symptoms of AR, nasal itching, and mucous membrane inflammation of the eye, was found to alleviate symptoms. Therefore, we see the potential that the drugs of IPT have on inhibitory action on AR.
Participated in research design: Kim and Jeong.
Conducted experiments: Oh.
Contributed new reagents or analytic tools: Jeong and Oh.
Wrote or contributed to the writing of the manuscript: Kim, Jeong, and Oh.
This work was supported by the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology [Grant 2009-0090401].
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
- allergic rhinitis
- receptor-interacting protein 2
- HMC-1 cell
- human mast cell line
- IκB kinase
- nuclear factor-κB
- intercellular adhesion molecule-1
- macrophage-inflammatory protein 2
- caspase recruitment domain
- reverse transcription
- polymerase chain reaction
- phosphate-buffered saline
- calcium ionophore
- phorbol 12-myristate 13-acetate and A23187
- Tyr-Val-Ala-Asp, p-nitroanilide
- bicinchoninic acid
- T helper
- human serum albumin
- fetal bovine serum
- enzyme-linked immunosorbent assay
- bone marrow-derived mast cell
- phosphate-buffered saline
- Received May 19, 2011.
- Accepted July 1, 2011.
- Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics