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Toxicology in Vitro xxx (2006) xxx窶度xx www.elsevier.com/locate/toxinvit

Anti-inXammatory eVect of Poncirus trifoliata fruit through inhibition of NF- B activation in mast cells Tae-Yong Shin a, Jay Min Oh b, Byung-Ju Choi c, Won-Hwan Park d, Cheorl-Ho Kim e, Chang-Duk Jun f, Sang-Hyun Kim g,ﾂ､ a

College of Pharmacy, Woosuk University, Jeonbuk 565-701, Republic of Korea b Wonkwang University Medical School, Jeonbuk 570-749, Republic of Korea c College of Dentistry, Kyungpook National University, Daegu 702-412, Republic of Korea d Cardiovascular Medical Research Center and Dongguk University College of Oriental Medicine, Kyung-Ju 780-714, Republic of Korea e Department of Biological Science, SungKyunKwan University, Suwon 440-746, Republic of Korea f Department of Life Science, Gwangju Institute of Science and Technology, Gwangju 500-712, Republic of Korea g Kyungpook National University Medical School, Daegu 700-422, Republic of Korea Received 25 October 2005; accepted 10 February 2006

Abstract Mast cell-mediated allergic inXammation is involved in many diseases such as asthma, sinusitis, and rheumatoid arthritis. Mast cells induce synthesis and production of pro-inXammatory cytokines including tumor necrosis factor (TNF)- and interleukin (IL)-6 with immune regulatory properties. We investigated the eVect of the fruits of Poncirus trifoliata (L.) Raf (Rutaceae) (FPT) on expression of pro-inXammatory cytokines by activated human mast cell line, HMC-1. FPT dose dependently decreased the gene expression and production of TNF- and IL-6 on phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187-stimulated HMC-1 cells. In addition, FPT attenuated PMA and A23187-induced activation of NF- B indicated by inhibition of degradation of I B , nuclear translocation of NF- B, NF- B/DNA binding, and NF- B-dependent gene reporter assay. Our in vitro studies provide evidence that FPT might contribute to the treatment of mast cell-derived allergic inXammatory diseases. ﾂｩ 2006 Elsevier Ltd. All rights reserved. Keywords: Fruits of Poncirus trifoliata; Mast cell; Allergic inXammation; TNF- ; IL-6; NF- B

1. Introduction Mast cells, which are constituents of virtually all organs and tissue, are important mediators of inXammatory responses such as allergic inXammation and hypersensitivity. In response to the antigen cross-linking of immunoglobulin E (IgE) receptor or direct activation, mast cells start releasing mediators, such as histamine, inXammatory cytokines, and products of arachidonic acid metabolism

* Corresponding author. Address: Department of Pharmacology, School of Medicine, Kyungpook National University, Daegu, 700-422, Republic of Korea. Tel.: +82 53 420 4838; fax: +82 53 423 4838. E-mail address: shkim72@knu.ac.kr (S.-H. Kim).

(Beaven and Metzger, 1993; Church and Levi-SchaVer, 1997; Metcalfe et al., 1981). Several inXammatory and chemotactic cytokines, such as tumor necrosis factor (TNF)- , interleukin (IL)-6, IL-8, IL-4, IL-13 and the transforming growth factor- are produced from activated mast cells (Bradding et al., 1993; Burd et al., 1989; Lin et al., 2000; Plaut et al., 1989). Pro-inXammatory cytokines, including TNF- and IL-6 support the well-recognized role of mast cells in allergic inXammation and hypersensitivity. TNF- induces tissue damage and is considered a major initiator of inXammation. IL-6 is a pleiotropic inXammatory cytokine produced by mast cells (Mican et al., 1992; Walsh et al., 1991). The transcriptional NF- B is important as a mediator of cellular responses to extracellular signals. NF- B plays

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a pivotal role in inXammation by virtue of its ability to induce transcription of an array of inXammatory genes, especially to the regulation of pro-inXammatory molecules such as TNF- and IL-6 (Azzolina et al., 2003). The fruits of Poncirus trifoliata (L.) Raf (Rutaceae) (FPT) are widely used in oriental medicine as a remedy for allergic inXammation (Cai, 1995; Chun and Sankawa, 1989). This crude drug shows an beneWcial eVect on the type I hypersensitivity, IgE production and IgE-mediated local anaphylaxis (Kim et al., 1999; Lee et al., 1996, 1997). In the present study we aimed to investigate the anti-inXammatory eVect of FPT on phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187-induced expression of pro-inXammatory cytokines and activation of NF- B on human mast cells. 2. Materials and methods 2.1. Reagents and cell culture PMA, calcium ionophore A23187, and pyrrolidine dithiocarbamate (PDTC) were purchased from the Sigma Chemical Co. (St Louis, MO). Anti-human TNF- and IL6 antibody, rTNF- , and rIL-6 were purchased from R&D Systems Inc. (Minneapolis, MN). Anti-human I B and NF- B (p65) antibodies were procured from Santa Cruz Biotech (Santa Cruz, CA). The human mast cell line (HMC-1) was grown in Iscove’s media (Life Technologies, Grand Island, NY) supplemented with 10% FBS and 2 mM glutamine at 37 °C in 5% CO2. 2.2. Preparation of FPT FPT was purchased from Bohwa Dang (Jeonbuk, Korea). A voucher specimen (number WSP-04-10) was deposited at the Herbarium of the College of Pharmacy, Woosuk University. FPT was grinded (1000 rpm, 30 s) at room temperature using Micro Hammer-Cutter Mill (Culatti Co., Zurich, Swiss). The particle size was 0.5–2 mm after grinding. The sample (80 g) was extracted with puriWed water (500 ml) at 70 °C for 5 h in water bath. The yield of dried extract from starting crude materials was about 8.3%. The extract was Wltered and lyophilized. The dried extract was dissolved in PBS before use.

2.4. RNA extraction and mRNA detection The total cellular RNA was isolated from the cells (1 £ 106/well in 24-well plate) after stimulation of PMA and A23187 with or without FPT using a TRI reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer’s protocol. The Wrst strand complimentary DNA (cDNA) was synthesized using the Superscript II reverse transcriptase enzyme (Life Technologies, Grand Island, NY). A reverse-transcriptase polymerase chain reaction (RT-PCR) was used to analyze the expression of mRNA for TNF- , IL-6, and -actin (internal control). The conditions for the reverse transcription and PCR steps were similar to those previously described (Kim and Sharma, 2004). The primer sets were chosen by the Primer 3 program (Whithead Institute, Cambridge, MA). The cycle number was optimized in order to ensure product accumulation in the exponential range. The ampliWed products were separated by electrophoresis on 2% agarose gel containing ethidium bromide, documented using a Kodak DC 290 digital camera, and digitized with UNSCAN-IT software (Silk ScientiWc, Orem, UT). The band intensity was normalized to that of -actin in the same sample. 2.5. Assay of TNF- and IL-6 secretion TNF- and IL-6 secretion were measured by modiWcation of an enzyme-linked immunosorbent assay (ELISA). HMC-1 cells were sensitized with PMA (20 nM) and A23187 (1 M) for 8 h with or without FPT. The ELISA was performed by coating 96-well plates with 6.25 ng/well of monoclonal antibody with speciWcity for TNF- and IL6 respectively. Before use and between subsequent steps in the assay, the coated plates were washed twice with PBS containing 0.05% Tween-20 and twice with PBS alone. For the standard curve, rTNF- and rIL-6 were added to the serum which was previously determined to be negative to endogenous TNF- and IL-6. After exposure to the medium, the assay plates were exposed sequentially to biotinylated anti-human TNF- or IL-6, and 2,2⬘-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) tablet substrates. Optical density was read within 10 min of the addition of the substrate with a 405 nm Wlter.

2.3. Cytotoxicity assay 2.6. Nuclear protein extraction The 3(4,5-dimethyl thiazolyl-2)2,5-diphenyl tetrazolium bromide (MTT, Sigma) cytotoxicity assay was used to measure the cytotoxic response to FPT, as previously described (Kim et al., 2005). HMC-1 cells were seeded at 1 £ 105 cells/ well in 96-well microplates (Falcon, Becton–Dickinson, Franklin Lakes, NJ). After 24 h of incubation with FPT, 20 l of MTT (5 mg/ml) was added per well and incubated for 4 h. The formazan crystals dissolved in isopropyl alcohol with 0.04 N HCl and absorbance was read at 570 nm using a PowerWavex Microplate spectrophotometer (BioTek Instrument, Inc., Winooski, VT).

Preparation of nuclear extract was basically as described elsewhere. BrieXy, after cell activation for the times indicated, cells were washed in 1 ml of ice-cold PBS, centrifuged at 1200 rpm for 5 min, resuspended in 400 l of ice-cold hypotonic buVer (10 mM HEPES/KOH, 2 mM MgCl2, 0.1 mM EDTA, 10 mM KCl, 1 mM DTT, 0.5 mM PMSF, pH 7.9), left on ice for 10 min, vortexed, and centrifuged at 15,000g for 30 s. Pelleted nuclei were gently resuspended in 50 l of ice-cold saline buVer (50 mM HEPES/KOH, 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 10% glycerol, 1 mM

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DTT, 0.5 mM PMSF, pH 7.9), left on ice for 20 min, vortexed, and centrifuged at 15,000g for 5 min at 4 °C. Aliquots of the supernatant that contained nuclear proteins were frozen in liquid nitrogen and stored at ¡70 °C. 2.7. Western blot analyses Samples were electrophoresed using 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as described elsewhere (Kim and Sharma, 2004), and then transferred to a nitrocellulose membrane. The I B and p65 NF- B were assayed using anti-I B and -NF- B (p65) antibodies (Santa Cruz Biotech). Immunodetection was done using an enhanced chemiluminescence detection kit (Amersham Pharmacia, Piscataway, NJ). 2.8. Electrophoretic mobility shift assays (EMSA) Nuclear protein (10 g) was incubated for 20 min at room temperature with 20 g of bovine serum albumin, 2 g of poly (dI-dC) from Pharmacia (Uppsala, Sweden), 2 l of buVer C (20 mM HEPES/KOH, 20% glycerol, 100 mM KCl, 0.5 mM PMSF, pH 7.9), 4 l of buVer F (20% Wcoll400, 100 mM HEPES/KOH, 300 mM KCl, 10 mM DTT, 0.5 mM PMSF, pH 7.9), and 20,000 cpm of a 32P-labeled probe that encoded the B consensus sequence (5⬘-CAG AGG GGA CTT TCC GAG AG-3⬘) or AP-1 (5⬘-GCA TGA GTC AGA CAC AC-3⬘) in a Wnal volume of 20 l. DNA–protein complexes were resolved at 180 V for 4 h in a native 4% polyacrylamide gel, dried, and visualized (with autography using a Fuji X-ray Wlm). 2.9. Transient transfection and luciferase activity assay For transient transfections, HMC-1 cells were seeded at 2 £ 106 in a 6-well plate 1 day before transient transfection. The expression vector containing NF- B luciferase reporter construct (pNF- B-LUC, plasmid containing NF- B binding site; STANTAGEN, Grand Island, NY) were transfected with serum- and antibiotics-free Iscove’s medium containing 8 l of Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA). After 5 h of incubation, medium was replaced with Iscove’s medium containing 10% FBS and antibiotics. Cells were allowed to recover at 37 °C for 20 h and subsequently were stimulated as indicated. For luciferase activity assay, cell lysates were prepared and assay for luciferase activity using Luciferase Assay System (Promega, Madison, WI), according to the manufacturer’s instructions.

3. Results 3.1. EVects of FPT on the pro-inXammatory cytokine expression We examined whether FPT could regulate pro-inXammatory cytokines such as TNF- and IL-6 in HMC-1 cells. HMC-1 cell line is a useful cell for studying cytokine activation pathway (Kim and Shin, 2005). At Wrst, we examined the cytotoxicity of FPT in HMC-1 cells using MTT assay. FPT did not show cytotoxic eVect up to 1 mg/ml (Fig. 1). Stimulation of HMC-1 cells with PMA (20 nM) and A23187 (1 M) during 4 h induced the gene expression of both cytokines. Pretreatment of FPT inhibited PMA and A23187-induced TNF- and IL-6 gene expression concentration dependently (Fig. 2). Culture supernatants were assayed for both cytokine levels using ELISA. As shown in Fig. 3, FPT decreased the secretion of PMA and A23187induced TNF- and IL-6 in a concentration dependent manner. In addition, the PMA and A23187-induced gene expression and secretion of both TNF- and IL-6 were blocked by PDTC, the potent NF- B inhibitor. 3.2. EVect of FPT on the NF- B activation To evaluate the mechanism of eVect of FPT on the TNF- and IL-6 gene expression, we examined the eVect of FPT on the NF- B activation. Expression of TNF- and IL-6 is regulated by a transcription factor, NF- B (Azzolina et al., 2003). We used PDTC as a positive control. Stimulation of HMC-1 cells with PMA and A23187 induced the degradation of I B and nuclear translocation of p65 NF- B after 2 h of incubation (Fig. 4A). FPT inhibited the PMA and A23187-induced degradation of I B and nuclear translocation of p65 NF- B. In order to inves-

2.10. Statistical analysis Statistical analyses were performed using SAS statistical software (SAS Institute, Cary, NC). Treatment eVects were analyzed using one-way ANOVA, followed by Duncan’s multiple range tests. p < 0.05 was used to indicate signiWcance.

Fig. 1. EVect of FPT on the cell viability in HMC-1 cells. Cells (1 £ 105 cells/ml) were incubated with FPT at 37 °C for 24 h as indicated concentrations. Viability was evaluated using MTT assay. Each data represents the mean § SEM of three independent experiments.

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Fig. 2. EVect of FPT on pro-inXammatory cytokine gene expressions in HMC-1 cells. Cells (1 £ 106 cells/ml) were pretreated with FPT for 30 min prior to PMA (20 nM) and A23187 (1 M) stimulation. Extraction and analysis of mRNA performed as described in Section 2, and TNF- and IL-6 mRNA levels were determined by RT-PCR. Band intensity for each gene expressions was normalized against -actin. Results are expressed as mean § SEM of three independent experiments. Representative gel photographs are shown in the same order. ¤ SigniWcantly diVerent from the PMA and A23187 value at p < 0.05.

tigate whether NF- B/DNA binding was inhibited by FPT, we performed EMSA. Cells pretreated with either FPT or PDTC (10 M) were subsequently stimulated by PMA and A23187, and the eVect of FPT and PDTC on binding activity of NF- B was examined. Treatment of PMA and A23187 caused a signiWcant increase in the DNA binding activity of NF- B within 4 h (Fig. 4B). FPT markedly suppressed the PMA and A23187-induced NF- B/DNA binding activity without altering the binding activity of AP-1. To further conWrm the inhibitory eVect of FPT on NF- B activation, we examined the eVect of FPT on the NF- B-dependent gene reporter assay. HMC-1 cells were transiently transfected with a NF- B-luciferase reporter construct or the empty vector. Exposure of cells to PMA and A23187 increased the luciferase activity in the cells transfected with the NF- B-luciferase reporter construct (Fig. 4C). FPT signiWcantly reduced the PMA and A23187induced luciferase activity.

Fig. 3. EVect of FPT on pro-inXammatory cytokine secretion in HMC-1 cells. Cells (1 £ 106 cells/ml) were pretreated with FPT or PDTC for 30 min prior to PMA (20 nM) and A23187 (1 M) stimulation and incubated additional 8 h. TNF- and IL-6 level in supernatant was measured using ELISA and represented as the mean § SEM of three independent experiments. ¤ SigniWcantly diVerent from the PMA and A23187 value at p < 0.05.

4. Discussion In response to diverse stimuli, mast cells release an array of mediators with the potential to cause allergic inXammation. Mast cell-derived pro-inXammatory cytokines, especially TNF- and IL-6 have a critical biological role in the allergic inXammation. Mast cells are a principal source of TNF- in human dermis, and degradation of mast cells in the dermal endothelium is abrogated by the anti-TNF- antibody (Walsh et al., 1991). IL-6 is also produced from mast cells and its local accumulation is associated with a local allergic reaction (Mican et al., 1992). We demonstrate here that FPT inhibits the TNF- and IL-6 expression in human mast cell line. FPT blocked the degradation of I B and nuclear translocation of NF- B, and therefore attenuated expression of pro-inXammatory cytokines TNF- and IL-6. TNF- promotes inXammation, leukocyte inWltration, granuloma formation and tissue Wbrosis and is thought to be an initiator of cytokine related inXammation states by stimulating cytokine production. TNF- is released as a prestored cytokine but is also newly synthesized upon mast cell activation (Hide et al., 1997; Jeong et al., 2002). These reports indicate that reduction of pro-inXammatory cyto-

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Fig. 4. EVect of FPT on the activation of NF- B in HMC-1 cells. Cells were pretreated with FPT or PDTC for 30 min prior to PMA (20 nM) and A23187 (1 M) stimulation. (A) I B degradation and NF- B translocation were assayed by Western blot (n-NF- B, nucleus NF- B; c-NF- B, cytoplasmic NF- B). (B) For EMSA analysis, nuclear extracts prepared and incubated with 32P-labeled oligonucleotides corresponding to NF- B or AP-1 was analyzed. (C) For NF- B luciferase assay, cells were transiently transfected with the NF- B luciferase reporter construct or empty vector. NF- B-dependent transcriptional activity was determined by luciferase activity assay. ¤ SigniWcantly diVerent from the PMA and A23187 value at p < 0.05.

kines from mast cell is a one of the key indicator of reduced inXammatory symptom. Our results showing the inhibition of TNF- and IL-6 expression by FTP support an antiinXammatory eVect resulting from the reduction of the generation of these mediators in mast cells. NF- B is a ubiquitous protein transcription factor that induces the transcription of a variety of genes. Many of these genes encode molecules important in inXammatory processes, such as cytokines and adhesion molecules (Sen and Baltimore, 1986; Siebenlist et al., 1994). The role of

NF- B activation and its regulation of cytokine production in allergic inXammation has been characterized (Blackwell et al., 1997). Expression of TNF- and IL-6 gene is dependent on the activation of transcription factor NF- B in mast cells. Activation of NF- B requires phosphorylation and proteolytic degradation of the inhibitory protein I B (Marquardt and Walker, 2000). In PMA and A23187-stimulated mast cells, FPT decreased the degradation of I B and nuclear translocation of p65 NF- B. In addition, the speciWc NF- B inhibitor, PDTC, reduced PMA and A23187-induced TNF- and IL-6 secretion. These data demonstrate that FPT attenuates activation of NF- B and downstream TNF- and IL-6 production. We previously reported eVects of FPT on the immediate type hypersensitivity through the inhibition of intracellular calcium and increase of cAMP (Lee et al., 1997, 1996). Taken together with our present data showing the decrease of pro-inXammatory cytokine expression by FPT in vitro, our results strongly supports a use of FPT in allergic inXammation. In addition, FPT may act as an inhibitor of NF- B activation in mast cells, which might explain its beneWcial eVect in the treatment of allergic inXammation. Because we used water extract of fruits of P. trifoliata not a puriWed component, the active components that are responsible for the biological eVect are still not fully known at this time. Recently, anti-anaphylactic eVect of Xavones from P. trifoliata has been reported (Park et al., 2005). Among the various components from P. trifoliata, naringenin, hesperetin, and ponciretin potently inhibited activation and degranulation of mast cells. It is known that other fruits such as Citrus aurantium, C. unshiu, and C. natsudaidai contain naringenin, hesperetin, and ponciretin. Hence comparing the eVect of P. trifoliata with other fruits is our goal for the future study. The eVorts in identiWcation of active components from FPT on the allergic inXammation are ongoing in our laboratory. Acknowledgement This work was supported by Wonkwang University grant in 2004. References Azzolina, A., Bongiovanni, A., Lampiasi, N., 2003. Substance P induces TNF-alpha and IL-6 production through NF kappa B in peritoneal mast cells. Biochimica et Biophysica Acta – Molecular Cell Research 1643, 75–83. Beaven, M.A., Metzger, H., 1993. Signal transduction by Fc receptors: the Fc epsilon RI case. Immunology Today 14, 222–226. Blackwell, T.S., Blackwell, T.R., Christman, J.W., 1997. Impaired activation of nuclear factor-kappa B in endotoxin-tolerant rats is associated with down-regulation of chemokine gene expression and inhibition of neutrophilic lung inXammation. Journal of Immunology 158, 5934–5940. Bradding, P., Feather, I.H., Wilson, S., Bardin, P.G., Heusser, C.H., Holgate, S.T., Howarth, P.H., 1993. Immunolocalization of cytokines in the nasal mucosa of normal and perennial rhinitic subjects. The mast cell as a source of IL-4, IL-5, and IL-6 in human allergic mucosal inXammation. Journal of Immunology 151, 3853–3865.

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