Transcriptional microarray analysis reveals suppression of histamine signaling by Kujin alleviates allergic symptoms through down-regulation of FAT10 expression

International Immunopharmacology 11 (2011) 1504–1509

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Transcriptional microarray analysis reveals suppression of histamine signaling by Kujin alleviates allergic symptoms through down-regulation of FAT10 expression Shrabanti Dev a, Hiroyuki Mizuguchi a, Asish Kumar Das a, Yoshinobu Baba b, Hiroyuki Fukui a,⁎ a b

Department of Molecular Pharmacology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan Department of Applied Chemistry, Graduate School of Engineering, Nagoya University, Furo-cho, Nagoya 464–8603, Japan

a r t i c l e

i n f o

Article history: Received 9 March 2011 Received in revised form 29 March 2011 Accepted 3 May 2011 Available online 19 May 2011 Keywords: Allergic rhinitis Kujin FAT10 Histamine H1 receptor Microarray analysis NF-κB Transcription

a b s t r a c t Previously, we have shown that hot water extract from Kujin, the dried roots of Sophora flavescens alleviates allergic symptoms by suppressing histamine signaling at the transcription level in toluene 2,4-diisocyanate (TDI)-sensitized rats. To know more insights into the mechanism of the anti-allergic action of Kujin, we carried out the microarray analysis to explore genes that were up-regulated by treatment with TDI and also were suppressed these up-regulated gene expression by Kujin. Microarray analysis revealed the substantial up-regulation of FAT10 (also called UbD) mRNA due to TDI sensitization and Kujin extract significantly suppressed this up-regulation. FAT10 is an ubiquitin like protein having an active role in the immune system and is induced by proinflammatory cytokines. Activation of NF-κB by FAT10 also has been reported. However, the role of FAT10 in allergic pathogenesis remains unknown. Here we investigated the correlation of FAT10NF-κB signaling with histamine signaling in TDI-sensitized rats. Real time RT-PCR analysis confirmed that treatment with TDI up-regulated FAT10 mRNA expression in the nasal mucosa of TDI-sensitized rats and Kujin extract suppressed this elevation. Treatment with H1-antihistamines suppressed the TDI-induced upregulation of FAT10 mRNA expression in TDI-sensitized rats. Direct administration of histamine into the nasal cavity of non-TDI-treated normal rats up-regulated the expression of FAT10 mRNA. Our data suggest that Kujin might alleviate allergic symptoms by inhibition of NF-κB activation through suppression of histamineinduced up-regulation of FAT10 mRNA expression. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Allergy is defined as hypersensitivity or hyperactivity of the immune system towards undefined foreign objects including toluene 2, 4-diisocyanate (TDI) [1]. Intranasal application of TDI led to the development of nasal allergy-like symptoms such as sneezing and watery rhinorrhea in sensitized rats and also display many of the characteristic features of allergic rhinitis in human including infiltration of eosinophils and mast cells [2], increase in the levels of Th2 cytokines [1,3–7], elevations of histamine H1 receptor (H1R) mRNA and protein level [6], and NF-κ B [8]. Histamine is an important mediator in the initiation and the development of allergic reactions. Many studies have shown that activation of H1R by histamine is responsible for the symptoms of allergic rhinitis. Our recent data suggest that H1R gene is an allergic

Abbreviations: H1R, histamine H1 receptor; TDI, toluene 2,4-diisocyanate; NF-κB, nuclear factor-kappa B. ⁎ Corresponding author at: Department of Molecular Pharmacology, Institute of Health Biosciences, The University of Tokushima Graduate School, 1-78-1 Shyomachi, Tokushima 770-8505, Japan. Tel.: + 81 88 633 7263; fax: + 81 633 7264. E-mail address: [email protected] (H. Fukui). 1567-5769/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2011.05.004

diseases-sensitive gene whose expression level affects severity of allergic symptoms [9,10], and compounds that suppress H1R gene expression should be good therapeutics [11–13]. The allergic reaction is also characterized by a disruption of Th1/Th2 balance toward a pronounced Th2 profile. Th1/Th2 imbalance in the immune system towards the Th2 responses results in the clinical expression of nasal allergy and asthma [14]. NF-κB is a pleiotropic transcription factor and plays a critical role in Th2 cell differentiation and is therefore required for induction of allergic airway inflammation [15]. Histamine was shown to activate NF-κB through H1R [16–18]. Over expression of H1R by histamine led to a significant induction of NF-κB activities and this activity was inhibited by H1-antihistamines [19]. Recent study demonstrated the contribution of FAT 10 on NF-κB activation and subsequent expression of NF-κB-regulated genes [20]. FAT10 belongs to the UBL family of proteins and contains two ubiquitin-like moieties fused in tandem [21]. It was originally discovered through the identification of expressed genes covering the HLA-F genomic locus [22]. FAT10 is constitutively expressed in mature dendritic cells and B cells [23], and is induced in many tissues by the proinflammatory cytokines, IFNγ and TNFα [24]. Overexpression of the FAT10 gene has been observed in several epithelial cancers and high FAT10 expression can increase chromosome

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instability by reducing kinetochore localization of MAD2 during the prometaphase stage of cell-cycle [25]. Root of Sophora radix (Kujin) is a widely used traditional herbal medicine for asthma, sores, gastrointestinal hemorrhage, diarrhea, allergy and inflammation [26–28]. In our previous study, we showed the anti allergic effect of Kujin mediated through the suppression of histamine signaling and Th2 cytokines [7]. Inhibition of NF-κB activation by Kujin was also reported [29,30]. All these reports and observations prompt us to know more insight about the mechanism of anti-allergic effect of Kujin and to identify the other genes involved. For this we performed transcriptional microarray analysis and found that TDI-induced up-regulation of FAT10 mRNA. This finding led a working hypothesis that the anti-allergic effect of Kujin is due to suppression of histamine signaling which intern suppress the FAT10 induced NF-κB activation. In the present study we observed the significant up-regulation of FAT10 mRNA in TDI-sensitized allergy model rats. This up-regulation of FAT10 mRNA expression was suppressed by the pre-treatment with H1-antihistamines and also by Kujin. Transcriptional microarray analysis showed that Kujin suppressed the transcription of NF-κBdependent genes. These observations together with the knowledge that FAT10 mediates NF-κB activation suggest the involvement of FAT10 in TDI-induced allergic rhinitis and Kujin inhibits FAT10induced NF-κB–Th2 cytokine signaling through suppression of histamine-induced FAT 10 mRNA up-regulation.

2. Materials and methods 2.1. Animals Six weeks old male Brown Norway rats (200–250 g; Japan SLC, Hamamatsu) were used. Rats were allowed free access to water and food and kept in a room maintained at 25 ± 2 °C and 55 ± 10% humidity with a 12-h light/dark cycle. The animals were divided into 3 groups with 4 rats in each; a control group, a group sensitized with TDI (Wako Pure Chemical, Osaka), and a test group. All procedures involving the animals were conducted in accordance with the Guidelines for Animal Experiments approved by the Ethical Committee for Animal Studies, School of Medicine, The University of Tokushima.

2.2. TDI sensitization and provocation Sensitization with TDI was performed by the method described previously [7]. Briefly, 10 μl of a 10% solution of TDI in ethyl acetate (Wako) was applied bilaterally on the nasal vestibule of each rat once a day for five consecutive days. This sensitization procedure was then repeated after 2 days interval. Nine days after the second sensitization, 10 μl of 10% TDI solution was again applied to the nasal vestibule to provoke nasal allergy-like symptoms. The control group was sensitized and provocated with 10 μl of ethyl acetate only by the same procedure.

2.3. Preparation and administration of Kujin extract Kujin extract was prepared and administered as described previously [7]. Root of S. radix was procured and authenticated by a local expert. Sixty grams of Kujin (“Kojima Kujin M”, Lot 902607; Kojima Kampo, Osaka) was boiled in 1 l of distilled water for 1.5 h, and filtered twice to remove insoluble materials. Then, the extract was concentrated and used for this study. The yield of freeze-dried extract was 20% (w/w) with respect to the dried root. The extract was administered orally once a day at a dose of 300 mg/kg for 3 weeks.

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2.4. Treatment with drugs Epinastine (30 mg/kg/day), d-chlorpheniramine (30 mg/kg/day) and olopatadine (10 mg/kg/day) were administered orally 1 h before provocation. Histamine was given into the nostril at different dose of 1, 2, and 4 mg/kg for 4 h before collection of nasal mucosa. 2.5. Collection of nasal mucosa and isolation of total RNA Rats were sacrificed and nasal mucosa was collected in RNAlater® (Applied Biosystems, Foster City, CA, USA) 4 h after provocation, and stored at − 80 °C until use. Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer's instructions. Briefly, nasal mucosa was homogenized using a Polytron (Model PT-K; Kinematica AG, Littau/Luzern, Switzerland) in 10 volumes of ice-cold TRIzol reagent. The homogenates were mixed with chloroform and centrifuged at 15,000 rpm for 15 min at 4 °C. The aqueous phase containing RNA was separated and then precipitated by the addition of isopropanol. After centrifugation RNA pellet was washed with 70% ice-cold ethanol, air-dried, and dissolved in 20 μl of diethylpyrocarbonatetreated water. The purity and the yield of total RNA were determined spectrophotometrically. 2.6. Hybridization and microarray scanning The reverse transcription labeling and hybridization for Agilent microarray analysis were performed following the protocol recommended by Agilent Technologies Inc. [31]. Briefly, RNA isolated from the nasal mucosa of all four rats in each group were mixed together and 20 μg aliquot of RNA sample from each group was reverse transcribed into a cDNA probe with oligo(dT) primer and labeled nucleotides. To check the gene over expression in TDI-sensitized rat nasal mucosa, the cDNA from normal rat nasal mucosa were labeled with Cy3-CTP and that of TDI-sensitized rats with Cy5-CTP. In the other case to check the suppressive effect of Kujin on the elevation of gene expression in TDI-sensitized rat, we labeled the cDNA from TDIsensitized group with Cy3-CTP and Kujin-treated group with Cy5-CTP. The reaction was carried out in a solution containing 50 mM dATP/dGTP/dTTP, 25 mM dCTP, 25 mM Cy3-dCTP or Cy5-dCTP (Enzo Diagnostics, Inc.) and 400 U of MMLV reverse transcriptase at 42 °C for 1 h. The labeling reaction was terminated by incubating the reaction mixture at 70 °C for 10 min. The RNA was then degraded by adding 0.05 mg RNase I, followed by incubation at 37 °C for 30 min. Degraded RNA and unicorporated nucleotides were removed using a QIAquick PCR Purification Kit (Qiagen Inc.) according to the instructions of Agilent Technologies Inc. Hybridization was carried out in 22 μl of a hybridization mixture containing cDNA probes, the labeled orientation marker (Deposition Control SP300; Operon Technologies Inc.) and mouse Cot-1 DNA (Invitrogen Corporation) at 65 °C for 17 h. The glass slides were then washed with 0.5× SSC and 0.01% SDS at room temperature for 5 min, and with 0.06× SSC at room temperature for 2 min. After immediate removal of the wash buffer by centrifugation, the glass slides were scanned using GenePix 4000B (Axon Instruments, Inc.) containing a 532 nm laser for Cy3 measurement and a 635 nm laser for Cy5 measurement. Scans were made with a pixel resolution of 5 mm, a laser power of 100%, and a photomultiplier tube voltage of 600 V for the 532 nm laser and 520 V for the 635 nm laser. 2.7. Normalization and analysis of microarray data Data analysis was done following the instruction described by Unami et al. [31]. Sixteen-bit TIFF images produced by the Axon scanner were analyzed using the GenePixPro 3.0 (Axon Instruments, Inc.) software package. After obtaining Cy3 and Cy5 grayscale images,

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each pseudo-color image was overlaid, and all spots in the ratio image were defined by accessing the gene list file that described the location of each gene on the microarray. The average of the signal intensity was subtracted from the median of background intensity and outputted with the UniGene and GenBank descriptors to a Microsoft Excel data spreadsheet. Relative expression levels were calculated by global normalization between two samples using all detected genes, after the exclusion of spots annotated as “Agilent QC”, “Agilent Blank”, and “Buffer”. 2.8. Measurement of mRNA expression level in the nasal mucosa by real-time quantitative reverse transcriptase polymerase chain reaction (real-time RT-PCR) For measuring mRNA expression, RNA samples from each rat were reverse transcribed to cDNA using SuperScript II reverse transcriptase (Invitrogen). TaqMan primers and probe were designed using Primer Express software (Applied Biosystems). The nucleotide sequences of the primers and probes for FAT10 are as follows; Sense primer; 5′GGAAAGAGGCTGGAAGATGGA-3′; Anti sense primer, 5′-GCGCTGTGAGAAAGAGCAAAC-3′; Probe, FAM-TCATGGCCGACTACAACATCAAGAGTGG-TAMRA. Real-time PCR was conducted using a GeneAmp 7300 sequence detection system (Applied Biosystems). Amplicon size and reaction specificity were confirmed by agarose gel electrophoresis. The identity of the PCR products was verified by sequencing using a genetic analysis system (Beckman CEQ 8000; Beckman Coulter). To measure the differences in starting material, GAPDH primers and probe reagents (Applied Biosystems) were used. 2.9. Statistical analysis The results are presented as means ± S.E.M. Data were analyzed using GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA, USA). The unpaired T-test or one-way ANOVA followed by Dunnett's multiple comparison tests were used for statistical analysis. Values of P b 0.05 were considered to be statistically significant. 3. Results 3.1. Changes of gene expression level in rat's nasal mucosa as observed by micro array analysis To gain insights into the molecular mechanism of anti-allergic effect of Kujin, we performed gene expression experiments using microarray technology. The RNA used for microarray analysis was derived from the nasal mucosa of three groups of rats. To check the up-regulation of gene expression in TDI-sensitized rats, we compared gene expression level between the control and the TDI-sensitized group. In a view to check the effect of Kujin on this up-regulation, we compared gene expression level between the TDI-sensitized and the Kujin-treated TDI group. Among the 20,500 genes represented on the array, we selected 20 genes which were upregulated in TDI-sensitized group compared to control as well as down-regulated in Kujin-treated group compared to TDI-sensitized group (Table 1). We selected those genes that showed significant change in expression of more than 2 fold. Among these genes IL-1B mRNA level was up-regulated mostly (N25 fold compared to control) and suppressed by Kujin (~ 3 fold) and FAT10 was up-regulated about 11.7-fold compared to the control group but suppressed by Kujin mostly (~ 4.5 fold). 3.2. Effect of TDI on FAT10 gene expression in rat nasal mucosa To confirm the result of microarray assay, we checked the effect of TDI on FAT10 gene expression by real-time RT-PCR. This analysis

Table 1 Changes of gene expression level in TDI-sensitized and Kujin-treated rat's nasal mucosa. Gene name

Description

295048_Rn FAT10 NM_053687 Schlafen 4 (Slfn4) 205792_Rn Interleukin 1 beta (Il1b) NM_012881 Secreted phosphoprotein 1 (Spp1) CB546432 Unnamed protein product. NM_134361 Small inducible cytokine subfamily C, member 1 (lymphotactin) (Scyc1) NM_057151 Small inducible cytokine subfamily A (Cys–Cys), member 17 (Scya17) NM_053843 Fc receptor, IgG, low affinity III (Fcgr3) 292212_Rn Small inducible gene JE (Scya2) 261613_Rn Transporter 2, ABC (ATP binding cassette) (Tap2) NM_022214 CXC chemokine LIX (LOC60665) NM_012591 Interferon regulatory factor 1 (Irf1) NM_031514 Janus kinase 2 (a protein tyrosine kinase) (Jak2) 221045_Rn Proteasome (prosome, macropain) subunit, beta type, 8 (low molecular mass polypeptide 7) (Psmb8) NM_022634 Leucocyte specific transcript 1 (Lst1) 221782_Rn CSF-1 290822_Rn Interleukin 4 receptor (Il4r) 295616_Rn similar to Mouse BCL3 (Bcl3) NM_013114 Selectin, platelet (Selp), NM_017104 Colony stimulating factor 3 (granulocyte) (Csf3)

Folds upregulated by TDI (vs control)

Folds upregulated by TDI + Kujin (vs control)

Folds downregulated by Kujin (vs TDI)

11.74 2.03 25.8

2.64 0.67 9.74

4.45 3.01 2.65

11.43

4.51

2.53

2.86

1.14

2.51

3.92

1.68

2.33

2.58

1.24

2.07

4.5

2.21

2.04

13.37

6.62

2.02

2.41

1.2

2

3.15

1.81

1.74

4.7

2.79

1.69

2.38

1.48

1.61

2.22

1.4

1.59

2.45

1.68

1.46

2.14 2.22

1.54 1.6

1.39 1.39

7.45

5.34

1.4

5.79 3.9

4.46 3.07

1.3 1.27

showed more than 10 times up-regulation of FAT10 mRNA in TDIsensitized rat nasal mucosa compared to the control (Fig. 1). 3.3. Effect of Kujin on TDI-induced FAT10 gene up-regulation in rat nasal mucosa To investigate the effect of Kujin on TDI-induced up-regulation of FAT10 gene expression, we performed real-time RT-PCR experiments with RNA separated from the nasal mucosa of TDI-sensitized rats and Kujin-treated TDI-sensitized rats. Kujin was treated for 1 week or for 3 weeks 1 h prior to TDI treatment. As shown in Fig. 2, 1-week treatment with Kujin showed no effect on the up-regulation of FAT10 gene expression, while 3-week treatment with Kujin showed significant inhibitory effect on TDI-induced up-regulation of FAT10 gene expression (Fig. 2).

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FAT10 mRNA/GAPDH mRNA

15

*

*

12

9 #

#

#

6

#

#

#

3

0

Control

TDI

Fig. 1. Effect of TDI on FAT10 gene expression in rat nasal mucosa. Rats were sensitized with TDI for two weeks, after 1 week interval rats were provocated with TDI and sacrificed 4 h after provocation. Nasal mucosa was collected, RNA were extracted and mRNA level was determined by reverse transcription followed by real-time RT PCR. Data are presented as mean ± standard error. For statistical analysis, unpaired T-test was used. *p b 0.05 vs. control (n = 4).

Fig. 3. Effect of H1-antihistamines on TDI-induced up-regulation of FAT10 mRNA. Rats were sensitized with TDI for two weeks, after 1-week interval, rats were provocated with TDI and sacrificed 4 h after provocation. Nasal mucosa was collected, RNA were extracted and mRNA level was determined by reverse transcription followed by real time RT PCR. H1-antihistamines were administered for one week (1 W) and just once (1D) 1 h before provocation. Data are presented as mean ± standard error. For statistical analysis, one-way ANOVA followed by Dunnett's multiple comparison tests was used. *p b 0.05 vs. control; #p b 0.05 vs. TDI (n = 4).

3.4. Effects of H1-antihistamines on TDI-induced FAT10 gene expression in the nasal mucosa of TDI-sensitized rats We investigated the effect of antihistamines on TDI-induced upregulation of FAT10 gene expression. Real-time RT-PCR analysis showed that treatment with H1-antihistamines significantly suppressed TDI-induced up-regulation of FAT10 gene expression (Fig. 3). 3.5. Effects of histamine on FAT10 gene expression in the nasal mucosa of normal rats As our previous data showed that Kujin extract inhibited histamine signaling (7), we investigated the effect of histamine on the expression of FAT10 mRNA in nasal mucosa from normal (i.e. nonTDI-sensitized) rats. Direct administration with histamine to the nasal

16

4. Discussion It was reported that repeated intranasal application of TDI induced release of histamine from mast cells via neurogenic inflammation and led to the development of nasal allergy-like symptoms such as sneezing and watery rhinorrhea in TDI-sensitized guinea pigs and rats [32,33]. Allergic rhinitis (AR) is defined as an IgE-mediated disease, so it is different from TDI-induced non-IgE-mediated rhinitis. However, nasal allergy-like symptoms induced by TDI are similar to those observed in AR patients [34,35]. TDI-sensitized rats also display many of the characteristic features of AR in humans, including infiltration of eosinophils and mast cells [2], increase in the level of cytokines [1,4,36,37], elevation of H1R mRNA and protein level [6], increase in the HDC mRNA level, HDC activity, and histamine content [38].

12

3

8

# 4

0 control

TDI

1 week

3 weeks

TDI + Kujin Fig. 2. Effect of Kujin extract on TDI-induced up-regulation of FAT10 mRNA. Rats were sensitized with TDI for two weeks, after 1-week interval, rats were provocated with TDI and sacrificed 4 h after provocation. Nasal mucosa was collected, RNA were extracted and mRNA level was determined by reverse transcription followed by real time RT PCR. Kujin was administered 1 h before provocation for one week or 3 weeks. Data are presented as mean ± standard error. For statistical analysis, one-way ANOVA followed by Dunnett's multiple comparison tests was used. *p b 0.05 vs. control; #p b 0.05 vs. TDI (n = 4).

FAT10 mRNA/GAPDH mRNA

FAT10 mRNA/GAPDH mRNA

*

cavity of normal rats increased FAT10 mRNA expression in a dosedependent manner (Fig. 4).

2.5

* 2 1.5 1 0.5 0

control

1

2

4

histamine (mg) Fig. 4. Effect of histamine on FAT10 mRNA. Rats were treated with histamine for 4 h. Nasal mucosa was collected, RNA were extracted and mRNA level was determined by reverse transcription followed by real time RT-PCR. Data are presented as mean ± standard error. For statistical analysis, one-way ANOVA followed by Dunnett's multiple comparison tests was used. *p b 0.05 vs. control (n = 4).

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Furthermore, the expression of IL-4 and IL-5 mRNAs was also upregulated in the nasal mucosa of TDI-sensitized rats after provocation with TDI [11–13]. We have demonstrated that repeated pretreatment of antihistamines 1–5 weeks before provocation were more effective in suppressing both nasal symptoms and the expression of H1R mRNA in the nasal mucosa of the TDI-sensitized rats than their single administration [9]. Furthermore, we have shown that there was a significant correlation between the nasal symptoms and the expression level of H1R mRNA in patients with pollinosis [10]. These findings indicate that the expression level of H1R gene affects severity of the nasal symptoms and drugs that suppress up-regulation of H1R gene expression could be a good therapeutics. Kujin is a widely used traditional herbal medicine for asthma, sores, gastrointestinal hemorrhage, diarrhea, allergy and inflammation [26–28]. We previously reported that extract from Kujin inhibited the TDI-induced nasal allergy-like symptoms by suppressing histamine signaling including suppression of TDI-induced up-regulation of H1R mRNA [7]. However, molecular basis of the anti-allergic activity of Kujin has not yet been fully understood. In the present study, we performed microarray analysis to identify genes that were upregulated by TDI treatment in TDI-sensitized rats and also were suppressed by Kujin. Among 20,500 genes investigated, we found that expression of FAT10 mRNA was significantly increased (about 12fold) in TDI-sensitized rats compared to the normal rats, and also significantly suppressed by treatment with Kujin extract (about 4.5fold vs. TDI-sensitized rats). However, physiological roles of FAT10 in allergic rhinitis are unknown. FAT10 encodes an ubiquitin-like protein that consists of two ubiquitin-like domains initially identified as one of the genes at the major histocompatibility complex locus in human chromosome 6 [22]. It was reported that FAT10 mRNA is expressed constitutively in some lymphoblastoid cells and dendritic cells and is induced by IFN-γ or TNF-α [23,24]. It was also reported that overexpression of the FAT10 gene was observed in several epithelial cancers [25]. It is well known that NF-κB is a transcription factor that controls the expression and function of Th2 cytokines including IL-4, IL-5, and IL-13 [39]. Therefore, NF-κB is one of the therapeutic targets for allergic diseases. Gong et al. reported that FAT10 mediates TNFαinduced NF-κB activation in renal tubular epithelial cells [20]. Although no mechanistic explanation was described, suppression of NF-κB by Kujin was reported [29,30]. Activation of NF-κB by histamine through H1R was also reported [19]. We have not examined the effect of Kujin extract on NF-κB activation in the nasal mucosa from TDIsensitized rats. However, as shown in Table 1, expression of NF-κBdependent genes including IL-1β, Spp1, Irf1, JAk2, and Bcl3 were also up-regulated with TDI treatment and suppressed their up-regulation by Kujin pretreatment. This result supports that Kujin extract suppresses NF-κB activation in this model rats, although it is unclear whether this suppression is direct or indirect (i.e. via FAT10 downregulation) effect. Our previous study demonstrated that Kujin suppressed TDIinduced up-regulation of Th2 cytokines including IL-4, IL-5, and IL-13 as well as histamine signaling [7]. These reports and our data prompt us to investigate the effect of Kujin extract on FAT10 mRNA expression. Treatment with TDI caused up-regulation of FAT10 mRNA expression in the nasal mucosa of TDI-sensitized rats (Fig. 1). As shown in Fig. 2, Kujin extract significantly suppressed TDI-induced up-regulation of FAT10 expression in TDI-sensitized rats. This TDIinduced up-regulation of FAT10 mRNA expression was also inhibited by pre-treatment with H1-antihistamines (Fig. 3) suggesting the involvement of H1R activation in the FAT10 gene up-regulation. This was confirmed by the data that direct administration of histamine into the nasal cavity of non-TDI-treated normal rats increased FAT10 mRNA expression in the nasal mucosa dose-dependently (Fig. 4). In conclusion, our data suggest that suppression of histamine signaling by Kujin down-regulates TDI-induced FAT10 gene expres-

sion and this may result in suppression of the expression of Th2 cytokine genes through inhibition of NF-κB activation. To our best knowledge this study is the first to establish a role of FAT10 in TDIinduced allergic pathogenesis.

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