The effects of suplatast tosilate (IPD-1151T) on innate immunity and antigen-presenting cells

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Transplant Immunology 18 (2007) 108 – 114 www.elsevier.com/locate/trim

The effects of suplatast tosilate (IPD-1151T) on innate immunity and antigen-presenting cells Li-Wen Hsu a,c , Chin-Hsiang Yang a , Shigeru Goto a,b , Toshiaki Nakano a , Chia-Yun Lai a , Yu-Chun Lin a , Ying-Hsien Kao a , Shu-Hui Chen c , Yu-Fan Cheng d,g , Bruno Jawan e,g , King-Wah Chiu f,g , Fu-Kai Tsao h , Chao-Long Chen a,g,⁎ a

Liver Transplantation Program and Department of Surgery, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Kaohsiung, Taiwan b Iwao Hospital, Yufuin, Japan c Department of Chemistry, National Cheng Kung University, Tainan, Taiwan d Liver Transplantation Program and Department of Diagnostic Radiology, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Kaohsiung, Taiwan e Liver Transplantation Program and Department of Anesthesiology, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Kaohsiung, Taiwan f Liver Transplantation Program and Division of Hepatogastroenterology, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Kaohsiung, Taiwan g Chang Gung University College of Medicine, Tao-Yuan, Taiwan h Major Biological Science, University of California, Davis, CA, USA Received 16 February 2007; accepted 22 May 2007

Abstract We previously demonstrated that the anti-allergic drug, suplatast tosilate (IPD-1151T), prolonged rat survival after heterotopic heart transplantation (HHT) and suppressed mixed lymphocyte reaction (MLR). In the present study, we investigated the effects of suplatast on T cells, lipopolysaccharides (LPS), or peptidoglycan (PGN)-stimulated cells and dendritic cells (DCs). The addition of suplatast to concanavalin A (ConA) blasts inhibited the proliferation of cells in which the gene expression of T-helper-1 (Th1) and T-helper-2 (Th2) cytokines including interferon (IFN)-γ, interleukin (IL)-2, IL-4, and IL-10 were down-regulated with decreased concentration of the IFN-γ and IL-10 in the supernatants of ConA blast cells. Suplatast also showed down-regulation of the toll-like receptor (TLR)2, TLR4, and CD14 gene expressions on splenocytes stimulated by LPS and PGN, TLR2 or TLR4 agonist, respectively. DCs treated with suplatast expressed lower levels of CD40, CD80, and CD86 and reduced IL-12 production. These results suggest that suplatast may modulate the TLRs on antigen-presenting cells (APCs) and thus block the pathway of Th1/Th2 cytokine production. © 2007 Elsevier B.V. All rights reserved. Keywords: Suplatast tosilate; Dendritic cells; Toll-like receptor; Antigen-presenting cells; T-helper cytokines

1. Introduction Suplatast tosilate (IPD-1151T) is an immunoregulator that suppresses IgE production and eosinophil infiltration through selective inhibition of the synthesis of interleukins (IL)-4 and IL-5 by T-helper-2 (Th2)-like cells but not interferon (IFN)gamma production in T-helper-1 (Th1) cells [1]. It has also been reported that suplatast tosilate inhibits the production of IL-4 by a murine Th2 clone as well as by the human CD4+ T cell line ⁎ Corresponding author. Department of Surgery, Chang Gung Memorial Hospital-Kaohsiung Medical Center, 123 Ta-Pei Rd, Niao-Sung Hsiang, Kaohsiung Hsien 833, Taiwan Tel.: +886 7 731 7123x8097; fax: +886 7 733 6856. E-mail address: [email protected] (C.-L. Chen). 0966-3274/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.trim.2007.05.008

[2,3]. In our previous results, cell proliferation of mixed lymphocyte reaction (MLR) was suppressed by suplatast tosilate, and heart allograft survival was prolonged in recipient rats treated by this drug. This immunosuppressive mechanism cannot be explained by the inhibition of Th2 cytokines even though suplatast tosilate is designated as a Th2 inhibitor [4]. Innate immune responses that act via Toll-like receptors (TLRs) are actively involved in the development of diseases predominantly mediated by adaptive immune responses [5]. At least 11 TLR families have been identified, among which TLR4 and TLR2 signals have become recent topics of immunological interest. It is also true that these receptors play an essential role in antigen presentation and later in the development of immune response into pro-allergic (Th2), cellular (Th1), or regulatory

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(Tr1) responses [6] to allergic diseases. It is widely believed that the consequence of TLR activation is the induction of an adaptive immune response of the Th1 type, in which IFN-γ production by CD4+ T cells predominates [7]. Most studies have focused on the TLR signaling which leads to Th1 immunity, whereas Th2 responses may be controlled by unidentified pathogen recognition receptors or by known innate immune receptors acting in ways which are still unclear [8,9]. Recent attention has been focused on the concept that stimulation of innate immunity through the TLRs can modulate subsequent adaptive immune responses including antigen recognition, presentation, and further activation of T cells. The example most usually described is that the endotoxin ligate of TLR4 increases the co-stimulatory molecules CD80 and CD86 expressed on dendritic cells (DCs) [10,11]. DCs are the periphery capture and process antigens that migrate to lymphoid organs and secrete cytokines to initiate immune responses necessary for the activation of lymphocytes [12,13]. 2. Objective In the present study, the effects of suplatast tosilate on TLR4 and TLR2 stimulation and DCs maturation was investigated to elucidate the mechanisms of the immunosuppressive activity of suplatast tosilate. 3. Materials and Methods 3.1. Animals Male DA (major histocompatibility complex haplotype RT1a) rats were obtained from Japan SLC (Hamamatsu, Japan). All animals were maintained in specific pathogen-free animal facilities with water and commercial rat food provided ad libitum.

3.2. ConA stimulation assay Cell suspensions of lymphocytes were obtained from rat spleen. The cells were suspended at 2.5 × 106 cells/ml in culture medium (RPMI 1640 containing 10% FBS, 100 U/ml penicillin, and 100 μg/ml Streptomycin (GIBCO™ Invitrogen Corporation, Carlsbad, CA)) and then stimulated with 5 μg/ml of Concanavalin A (ConA) (Sigma, St. Louis, MO). An equal number of cells (5× 105 cells/ml) were prepared in a total volume of 200 μl per well in 96-well round-bottom microculture plates. Suplatast tosilate (IPD-1151) (suplatast: (±)-[2-[4-(3-ethoxy-2-hydroxypropoxy) phenylcarbamoyl] ethyl] dimethylsulfonium p-toluenesulfonate supplied by Taiho Pharmaceutical Co. Ltd. (Tokyo, Japan)) was supplemented into the ConA blast culture at different concentrations (10, 20, 50 and 100 μg/ml) and was cocultured for 3 days at 37 °C in a humidified atmosphere with 5% CO2. Cell proliferation was detected with Cell Proliferation Reagent WST-1 (Roche Applied Science, Mannheim, Germany) and measured using a MRX Microplate Reader (Dynatech Laboratories, Chantilly, VA) that reads the optical density at both 450 nm and 620 nm. The supernatants and cells were harvested and stored at − 70 °C until used. All experiments were repeated more than three times.

inactivated fetal bovine serum (FBS, GIBCO™ Invitrogen Corporation, Carlsbad, CA) to the sample. After 1 min, the CFSE-labeled cells were washed twice, recounted, and adjusted to a concentration of 5× 105 cells/ml in culture medium. The culture condition was described as above. Cultured cells from each well were harvested after 3 days and pre-incubated with mouse anti-rat CD32 (Fc γ II receptor) (BD Biosciences Pharmingen, San Diego, CA) to block nonantigen-specific binding of immunoglobulins. CFSE-labeled cells were incubated at 4 °C for 30 min with phycoerythrin (PE)-conjugated anti-CD3. Cells were suspended in PBS/BSA with 0.01% NaN3 and 2 μg/ml 7-aminoactinomycin D (7-AAD, GIBCO™ Invitrogen Corporation) to measure the population distributions of apoptotic/necrotic cell death. Three-color flow cytometery was performed on an Epics® ALTRA™ flow cytometer (Beckman Coulter, Fullerton, CA) using EXPO32 software.

3.4. Toll-like receptors (TLRs) in splenocytes cultured with LPS or PGN Homogenous splenic suspensions were cultured in round-bottom, 96-well plates (1× 106 cells/well) with 10 μg/ml lipopolysaccharides (LPS) (Sigma), 10 μg/ ml peptidoglycan (PGN) (Sigma), or no additions. Subsequently, suplatast was supplemented into the wells at different concentrations (10, 20, 50 and 100 μg/ml). After incubation at 37 °C in 5% CO2 for 3 days, the cells were harvested and stored at − 70 °C until used. All experiments were repeated more than three times.

3.5. RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR) RNA was extracted from ConA blasted of DA splenocytes or splenocytes cultured with LPS or PGN using TRIzol® reagent (GIBCO™ Invitrogen Corporation) according to the manufacturer's instructions. The total RNA (1.2 μg) was reverse-transcribed into cDNA using ImProm-II™ Reverse Transcriptase (Promega Biosciences, Inc., San Luis Obispo, CA). The cDNA was then amplified by polymerase chain reaction (PCR), in a total volume of 50 μl containing 3 μl of either cDNA, 5 μl of the 10-μM PCR primer, 5 μl of 25 mM dNTP, 5 μl of 10-fold PCR buffer, 34.5 μl of H2O, and 0.5 U Taq enzyme (Roche Applied Science). The sequences of rat-specific primers used in this study have been previously described [14]. The PCR reaction was hotstarted at 94 °C for 2 min in order to denature all cDNA samples. After an initial denaturation step, the cDNA samples were subjected to rounds of denaturation (94 °C for 40 s), annealing at 55 °C to 62 °C for 40 s, and extension at 72 °C for 40 s. Each reaction was completed by heating at 72 °C for the final 10 min. According to optimal amplification efficiencies, the samples were amplified at different cycle numbers: 30 (GAPDH, TLR4 and CD14), 25 (IFN-γ and IL-2), 40 (IL-4), and 25 (IL-10). The amplification products were separated by electrophoresis on 2% TBE agarose gels and stained with ethidium bromide. The gel profiles were visualized (photographed) using UVI gel documentation (UVItec, Cambridge, UK) and analyzed using UVI photo version 99 (UVItec) and TotalLab software version 1.00 (Nonlinear USA Inc., NC). PCR products were titrated to establish standard curves for documenting linearity and permitting semiquantitative analysis. Levels of gene expression were expressed as the ratios of densities between PCR products and GAPDH in the same sample. All experiments were repeated more than three times.

Table 1 Inhibition of ConA blast by suplatast Stimulator Responder a In vitro addition suplatast (μg/ml) ConA

3.3. 5-(6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) or 7-aminoactinomycin D (7-AAD) labeling of responder cells Lymphocytes obtained from DA spleen were labeled by CFSE (Sigma, St. Louis, MO) as previously described [14]. A 5-mM stock solution of CFSE in DMSO (Sigma) was thawed and diluted to 5 μM in a volume of PBS equal to that in which the responder cells (1 ×107 cells/ml in PBS) were suspended and incubated at 37 °C for 10 min. The labeling process was quenched by adding an equal volume of heat-

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a

DA

– 5 10 20 100

WST-1 (O.D ± SD)

Percentage inhibition (%) b

2.621 ± 0.061 2.033 ± 0.223 1.675 ± 0.092 1.094 ± 0.019 1.029 ± 0.120

– 32.0 51.5 83.1 86.7

Responder only DA: 0.784 ± 0.061. Percentage inhibition: (1 − [O.D of culture with added suplatast − O.D of culture with responder cells only / O.D of culture with stimulator − O.D of culture with responder cells only]) × 100%. b

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3.6. Real-time PCR (Quantitative PCR, Q-PCR) PCR of GAPDH and TLR-2 gene expression was carried out using the LC Fast Start DNA Master SYBR Green kit (Roche Applied Science) employing 5 μl of cDNA in a 20 μl final volume, with 4 mM MgCl2 and 0.5 μM of each primer (final concentration). Briefly, quantitative PCR was performed using LightCycler (Roche Applied Science) for 45 cycles at 95 °C for 5 s, and specific annealing temperatures of 60 °C for 5 s and 72 °C for 12 s. Amplification specificity was checked using the melting curve according to the manufacturer's instructions. Results were analyzed using LightCycler Software v.3.5 (Roche Applied Science). Quantification using standard curves was carried out using Real Quant Software (Roche Applied Science). Levels of gene expression were expressed as the ratios of concentration between PCR products and GAPDH in the same sample. All experiments were repeated more than three times.

3.7. Dendritic cells (DCs) culture from PBMC DCs are generated from human peripheral blood mononuclear cells (PBMCs), as we published previously, with some modifications [15]. Cultured PBMCs were

stimulated with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 (CytoLab Ltd., Rehovot, Israel). On day 7, tumor necrosis factorα(TNF-α) (CytoLab Ltd.) and suplatast were added to the culture. Cultured DCs were harvested by vigorous washing on day 9. Aliquots were taken for cell counting and vitality staining by trypan blue. Cell differentiation was monitored by light microscopy. In order to determine the effect of suplatast (20 μg/ml) on the maturation of DC cells, flow cytometric analysis was performed using EPICS ALTRA (Beckman Coulter, Fullerton, CA) employing antibody against mouse anti-human CD40, CD80, and CD86 antibody (BD Biosciences Pharmingen, San Diego, CA). FITC-conjugated rabbit anti-mouse IgG (BD Biosciences Pharmingen) was used as the second antibody. Analysis of fluorescence staining was performed using EXPO32 Software. The supernatants and cells were harvested and stored at − 70 °C until used.

3.8. Enzyme-linked immunosorbent Assay (ELISA) for culture supernatant Culture supernatant of ConA stimulation was used to measure IFN-γ and IL10 expressions. Undiluted samples (100 μl/well) were added into the plate and measured according to the procedures specified by the rat-IL10 ELISA kit and rat-IFN-γ ELISA kit (Pierce-Endogen, Rockford, IL). The IL-12 p70 level of the

Fig. 1. Histogram plots show the cell division associated with CFSE labeling of DA splenocytes. All plots were gated on CD3-positive cells (histograms not show), while the histograms of (A) were also gated to include both resting lymphocytes (R1) and blast (R2). Alterations in light scatter characteristics, CFSE labeling of DA splenocytes alone, ConA blast of DA splenocytes, ConA blast of DA splenocytes mixed with suplatast (5, 10, 20, 40, 50 or 100 μg/ml). (B) Expression of cell proliferation percentage by ConA blast with or without suplatast addition was determined by flow cytometric analysis. (C) 7-AAD staining of DA splenocytes alone, ConA blast of DA splenocytes, and ConA blast of DA splenocytes mixed with suplatast (5, 10, 20, 40, 50 or 100 μg/ml). (D) Expression of cell death percentage by ConA blast with or without suplatast addition was determined by flow cytometric analysis. Data are representatives of three independent experiments with essentially similar tendency.

L.-W. Hsu et al. / Transplant Immunology 18 (2007) 108–114 supernatants of the DC culture was measured using a human-IL12 p70 ELISA kit (Endogen Pierce). The plate was read at 450 nm in an MRX Microplate Reader (Dynatech Laboratories). All experiments were repeated more than three times.

3.9. Statistical analysis Descriptive statistics, mean, standard deviation, and range were used where appropriate. For comparison of groups, one-way ANOVA and the Duncan post hoc test were used where appropriate. The results are given as mean values ± the standard deviation of the mean. A p b 0.05 was considered to indicate statistical significance.

4. Results

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of suplatast to the culture system resulted in a dose-dependent suppression of the proliferation response. Further assays using CFSE fluorescence and flow cytometry demonstrated whether or not this dosedependent suppression by suplatast was related to cells death. CFSElabeled cells were stimulated by ConA and cultured with suplatast. All cells were harvested after 3 days, and CFSE fluorescence was detected by flow cytometry (Fig. 1). Daughter T cells, derived from the responder splenocytes following ConA stimulation, were differentiated from undivided T cells and identified by the intensity of CFSE staining (Fig. 1A). The addition of suplatast to CFSE-labeled cells stimulated with ConA suppressed lymphocyte proliferation (Fig. 1B), however high dosage of suplatast (more than 40 μg/ml) induced apoptotic/ necrotic cell death, as confirmed by the cell fluorescence measurements with 7-AAD (Fig. 1C and D).

4.1. Suppression of the lymphocyte proliferative response by suplatast 4.2. mRNA expression of cytokines suppressed by suplatast Suplatast was tested for its ability to suppress the in vitro proliferative response of ConA blast formation. The results of experiments in which various dosages of suplatast were added to the ConA blast systems at the initiation of cultures are shown in Fig. 1. The response of lymphocytes cultured with suplatast was compared to that of lymphocytes cultured without suplatast as shown in Table 1. Addition

We investigated mRNA expression for IFN-γ, IL-2, IL-4, and IL-10 on ConA blasts of rat splenic cells cultured with or without suplatast. The gene expression of not only Th2 cytokines (IL-4, IL-10) but also Th1 cytokines (IFN-γ, IL-2) dose-dependently decreased in ConAstimulated cells to which had been added various concentrations of

Fig. 2. Cytokine gene expression in ConA blasts splenocytes cultured with or without suplatast. mRNA was used to obtain first-strand cDNA, which was then used as a template in reverse transcriptase-polymerase chain reaction (RT-PCR) according to the protocols described in the Materials and methods Section. (A) Th1 and Th2 cytokine gene expression (IL-2, IFN-γ, IL-4, and IL-10). Lane 1: marker; Lane 2: splenocytes; Lane 3: ConA blast; Lane 4–Lane 7: ConA blast cultured with various dosage of suplatast (5, 10, 20, or 100 μg/ml); Lane 8: negative control. (B) The relative gene expression to GAPDH, which was quantified by densitometric analysis, is shown as a ratio, respectively. The bars indicate the average density values from three independent experiments.

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Fig. 3. (A) Expression of TLR4 and CD14 mRNA in splenocytes after LPS stimulation. In all experiments, mRNA was analyzed using RT-PCR as described in Materials and methods. Lane 1: rat splenocytes only; Lane 2: LPS stimulation; Lane 3–Lane 6: LPS stimulation mixed with various dosages of suplatast (10–100 μg/ ml); Lane 7: negative control. (B) The expression relative to GAPDH, which was quantified by TotalLab, is shown at the bottom respectively as a ratio. The bars indicate the average density values from three independent experiments. (C) Effect of suplatast on TLR2 gene expression after rat splenocytes were stimulated by TLR2 agonist. The expression relative to GAPDH, which was quantified by Q-PCR, is shown at the bottom respectively as a ratio. The bars indicate the average values from three independent experiments.

suplatast (Fig. 2). In fact, high dosage of suplatast caused cells death with Th1/Th2 down-regulation. 4.3. Suplatast restrained the mRNA expression of TLRs and CD14 The effect of suplatast on innate response was also investigated using a highly purified preparation of E. coli with LPS (a selective TLR4 agonist) and Staphylococcus aureuswith PGN (a TLR2 agonist). Splenic cells of DA rats were stimulated with optimal stimulatory doses of LPS or PGN. Accordingly, we assessed the mRNA expression of TLR2, TLR4, and CD14 in splenocytes stimulated by LPS or PGN, and subsequently cultured with suplatast. As shown in Fig. 3, TLR4 expression levels in splenocytes cultured with suplatast were down-regulated at 10 and 20 μg/ ml of suplatast without cells death as compared with those of control

splenocytes cultured without suplatast. However, suplatast did not affect CD14 expression. As the TLR2 expression is too weak to detect it by RTPCR, Q-PCR was applied to evaluate TLR2 expression. TLR2 mRNA Table 2 IFN-γ and IL-10 concentrations of supernatants by ConA blast Addition suplatast (μg/ml) a

IFN-γ concentration (pg/ml)

IL-10 concentration (pg/ml)

5 10 20 100

325.3 ± 5.5 276.0 ± 9.8 164.7 ± 6.4 139.7 ± 4.5

622 ± 7.5 558 ± 6.9 363 ± 10.1 307 ± 9.9

a Supernatant of DA only: IFN-γ: undetectable, IL-10: 559 ± 13.2 pg/ml. Supernatant of ConA blast: IFN-γ:527.9 ± 12.3 pg/ml, IL-10: 720 ± 9.1 pg/ml.

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Fig. 4. Inhibition of expression of co-stimulatory molecules on DCs. DCs were propagated from human PBMC in IL-4 + GM-CSF, and on day 7, TNF-α and suplatast (20 μg/ml) were added to the culture. Expression of the DC maturation surface markers CD40, CD 80, and CD86 was determined by flow cytometric analysis. The results are expressed as the mean fluorescence intensity ± SD of three independent experiments. Box plots show the median, interquartile range, outliers, and extreme cases of individual variables. (p b 0.05).

expression levels were down-regulated in splenocytes stimulated by PGN and subsequently cultured with suplatast at low dosage (Fig. 3(C)). 4.4. IFN-γ and IL-10 levels in supernatants of ConA blast with or without treatment of suplatast Table 2 shows levels of IFN-γ and IL-10 in supernatants of ConAstimulated splenocytes treated with various dosages of suplatast. Suplatast inhibited both IFN-γ and IL-10 production in a dose-dependent manner. 4.5. Phenotypic maturation of human PBMC-derived DCs treated with suplatast We determined whether or not the addition of suplatast to PBMCderived DC cultures would inhibit the maturation and function of human DCs. PBMC cells obtained from human whole blood were cultured with rGM-CSF and rIL-4. After 7 days, TNF-α together with or without suplatast was added to the culture. Flow cytometric analyses were performed to determine the properties of PBMC-derived DC maturation in the presence or absence of suplatast. It is well known that CD40, CD80, and CD86 are expressed mostly on mature dendritic cells. However, the mean fluorescence intensity of DCs treated with suplatast expressed significantly lower levels of CD40, CD80, and CD86 compared with that of untreated DCs (Fig. 4). Subsequently, we measured the IL-12 p70 concentration in the supernatants by using an ELISA kit. Fig. 5 represents the results of three independent experiments. IL-12 production by DCs cultured with suplatast obviously decreased compared to that of untreated DCs.

blast splenocytes in the presence or absence of suplatast. Both gene expressions of Th1 and Th2 cytokines were down-regulated when cultured with suplatast. IFN-γ and IL-10 levels of supernatants in ConA blasts were also restrained by suplatast. Cytokines play a central role in the orchestration of host immune response to the allograft. The classification of T-helper cells into Th1 and Th2 subsets on the basis of their cytokine arrays has provided an effective model with which to interpret the role of cytokines in allograft rejection and acceptance. However, our results clearly demonstrated that the Th1/Th2 pathway could not fully explain the mechanism of suplatast's inhibition of MLR and its prolongation of the survival of heart allografts. Therefore, we evaluated the effect of suplatast on PBMC-derived DCs. Suplatast downregulated CD40, CD80, and CD86 in DCs and decreased IL-12 concentration in the supernatant of suplatast-treated DCs, suggesting that DCs treated with suplatast do not mature or function well. DCs are potent antigen-presenting cells (APC) that play a central role in initiating adaptive and innate immune responses..

5. Discussion Our previous results showed that suplatast suppressed cell proliferation of MLR, while heart allograft survival was prolonged in recipient rats treated by suplatast [4]. We here explored the immunosuppressive mechanism of suplatast. Suplatast not only suppressed MLR activity but also inhibited the cell proliferation of ConA blasts. Subsequently, we assayed the Th1 and Th2 cytokines including IFN-γ, IL-2, IL-4, and IL-10 gene expression in ConA

Fig. 5. IL-12 p70 level in supernatants of DC cultured with or without suplatast (20 μg/ml). The results are expressed as the concentration ± SD of three independent experiments.

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Toll-like receptors, which are a group of transmembrane proteins expressed mainly on APC such as DCs or macrophages, sense the presence of infection through the recognition of pathogen-associated molecular patterns (PAMPs) and thus trigger adaptive immunity in higher organisms [16,17]. Recognition of PAMPs by TLRs on APC such as DCs promotes the maturation of DCs. The mature DCs presents the antigen to naïve T cells, in association with the induction of co-stimulatory molecules as well as cytokines. TLRs also induce cytokine expression, among which IL-12 is an important cytokine in the differentiation of naïve T cells into Th1 and Th2 cells, and induces the activation of these cells to produce cytokines such as IFN-γ or IL-4, IL-5, and IL-10 and IL-13, respectively [8,18]. We speculated that suplatast may contributed to interact with TLRs, and subsequently influence DCs maturation and cytokine production. In fact, we demonstrated the down-regulation of the gene expression of TLR2, TLR4, and CD14 in suplatast-terated splenocytes stimulated through a TLR4 agonist (LPS) or a TLR2 agonist (PGN). Several investigations have provided evidence that signaling via TLRs is crucial for the development of Th1dependent immune responses [19]. On the other hand, the role of TLRs and innate immunity in Th2 responses remains obscure. One study has shown that a low dose of LPS initiates Th2 immunity, whereas higher LPS doses cause Th1 immunity [20]. Another study provided evidence that TLR2 ligation leads to a Th2 immune response [21]. In our results, suplatast suppressed not only Th2 cytokines, but also inhibited innate immunity and DCs maturation. Our results showed that suplatast could down-regulate not only adaptive immunity but also innate response, which may contribute to the prolongation of HHT graft survival time. Inhibition of innate immunity through the TLRs by suplatast may inhibit subsequent adaptive immune responses including antigen recognition, presentation, and further activation and development of immune response into pro-allergic (Th2), cellular (Th1), or regulatory (Tr1) responses [6] in not only allergic diseases but also allograft rejection. We speculate that the anti-allergic drug suplatast could be applied to transplantation immunotherapy and find use as a new immunosuppressive drug for transplantation. Acknowledgements This work was supported by program project grant NHRIEX94-9228SP from the National Health Research Institute, and grants 95-2314-B-182A-089, 95-2314-B-182A-132 from the National Science Council, and Chang Gung Memorial Hospital (Chang Gung Medical Research Project; CMRPG850052, CMRPG850071 and CMRPG860181) of Taiwan.

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