Dendritic cell activation by polysaccharide isolated from Angelica dahurica

Food and Chemical Toxicology 55 (2013) 241–247

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Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox

Dendritic cell activation by polysaccharide isolated from Angelica dahurica Hyung Sook Kim a,1, Bo Ram Shin a,1, Hong Kyung Lee a, Yun Soo Park a, Qing Liu a, Sung Yeon Kim b, Mi Kyeong Lee a, Jin Tae Hong a, Youngsoo Kim a, Sang-Bae Han a,⇑ a b

College of Pharmacy, Chungbuk National University, Cheongju, Chungbuk 361-763, Republic of Korea College of Pharmacy, Wonkwang University, Chonbuk 570-749, Republic of Korea

a r t i c l e

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Article history: Received 12 September 2012 Accepted 5 December 2012 Available online 14 December 2012 Keywords: Angelica dahurica Dendritic cells TLR4 MAPK NF-jB

a b s t r a c t Angelica dahurica is used in functional foods for the prevention and treatment of various diseases, such as inflammation and cancer. In the present study, we examined the effect of A. dahurica polysaccharide (ADP) on dendritic cell (DC) maturation. ADP increased the expressions of CD86 and MHC-II molecules, the production of IL-12, IL-1b, and TNF-a, and allogeneic T cell activation ability of DCs, and reduced DC endocytosis. As a mechanism of action, the knockdown of TLR4 with small interfering RNA decreased the ADP-induced production of nitric oxide and IL-12 by DCs, suggesting the membrane receptor candidate of ADP. After binding to TLR4, ADP increased the phosphorylation of ERK, JNK, and p38 MAPKs, and the nuclear translocation of NF-jB p50/p65. These results indicate that ADP activates DCs through TLR4 and downstream signalings. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction An immunomodulating biological response modifier (BRM) is important for the treatment of infectious diseases and cancer. Plant-derived polysaccharides are one of the well-known BRMs traditionally used as oriental medicines and food substances (Leung et al., 2006). Angelica dahurica, as a BRM candidate, has been used as a traditional folk medicine in Korea, China, and Japan. The root is classified as a sweet-inducing drug able to counter harmful external influences on the skin, such as cold, heat, and dryness, and toothache, headache, and sinusitis (Kang et al., 2007). The active compounds derived from A. dahurica are coumarin, phellopterin, isoimperatorin, imperatorin, oxypeucedanin, byakangelicin, and pimpinellin, which have been reported to have anti-inflammatory, anti-mutagenic, anti-microbial, anti-tumor, and anti-oxidant activities (Ban et al., 2003; Kim et al., 1991, 2007c; Lechner et al., 2004). Recently, it has been reported that the polysaccharides isolated from the root of A. dahurica enhance the proliferation of rat skin cells. Polysaccharides also have been reported to possess anti-oxidant and anti-inflammatory activities (Ban et al., 2003; Abbreviations: ADP, Angelica dahurica polysaccharides; CR3, complement receptor 3; DCs, dendritic cells; ERK, extracellular signal-regulated kinase; IjB, inhibits NF-jB; JNK, C-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; siRNA, small interfering RNA; TLR, toll-like receptor. ⇑ Corresponding author. Address: College of Pharmacy, Chungbuk National University, 52 Naesoodong, Heungduk, Cheongju, Chungbuk 361-763, Republic of Korea. Tel.: +82 43 261 2815; fax: +82 43 268 2732. E-mail address: [email protected] (S.-B. Han). 1 These authors contributed equally to this work. 0278-6915/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2012.12.007

Xu et al., 2011a). A variety of biological activities of A. dahurica have been reported, but the effect of polysaccharides (ADP) isolated from A. dahurica on dendritic cells (DCs) are unknown. DCs are antigen-presenting cells that are continuously differentiated from hematopoietic stem cells in the bone marrow and are believed to possess immune sentinel properties (Dieu et al., 1998). DCs position at different portals of the human body, such as skin, mucosal surface, and blood, where they can encounter invading pathogens (Wu and Liu, 2007). Under normal conditions, DCs are termed immature DCs because they express very low levels of costimulatory molecules (CD40 and CD80/86) and major histocompatibility complex (MHC)-I/II on their surface, and are unable to activate T cells (Kim et al., 2007a). In tissues, by receiving micro-environmental signals, immature DCs can mature, capture and process antigen, migrate to secondary lymphoid organs, and sensitize naïve T cells. In the T cell-rich zones of the lymphoid organs, these mature DCs secrete substances and stimulate T cells specific for antigen (Pahuja et al., 2006). Mature DCs express high levels of cytokine, chemokine, adhesion molecules that include ICAM-1 and LFA-3, costimulatory molecules, and MHC-I/II (Lanzavecchia and Sallusto, 2000; Moser and Murphy, 2000). However, DCs are present in very low numbers in tumor-bearing hosts and the cells usually have the phenotype of immature DCs (Kim et al., 2010b). These immature DCs induce either T cell unresponsiveness or regulatory T cells (Tregs) (Harding et al., 1992). Accordingly, the maturation step is very important for DCs to initiate anti-tumor T cell responses. DC maturation can be induced by numerous factors that include pathogen-related molecules such as lipopolysaccharide (LPS) and CpG, and inflammatory cytokines

H.S. Kim et al. / Food and Chemical Toxicology 55 (2013) 241–247

such as tumor necrosis factor (TNF) and prostaglandins (PGs) (Aiba and Tagami, 1998; Hartmann et al., 1999). Although these mediators are potent stimuli of DC maturation, they are likely to have no medicinal benefit, since they may induce detrimental adverse effects. In this regard, many studies have been attempted to find new DC activators having no side effects. In this study, we examined the effect of ADP on DCs. Especially, we examined the inducing activity of ADP on phenotypic and functional maturation of DCs. We also investigated the underlying mechanism of action of ADP on DC activation with regard to the receptor of ADP and the related intracellular signalings in DCs.

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Female 6–8-week-old C57BL/6 and BALB/c mice were obtained from the Korea Research Institute of Bioscience and Biotechnology (Chungbuk, Korea). The mice were housed in specific pathogen-free conditions at 21–24 °C and 40–60% relative humidity under a 12 h light/dark cycle. All animals were acclimatized for at least 1 week prior to the experiments. All experimental procedures were approved by the Animal Experimentation Ethics Committee of Chungbuk National University. Anti-mouse antibodies against CD11c, CD40, CD86, and MHC-I/II were purchased from BD Pharmingen (San Diego, CA, USA) and those against extracellular signalregulated kinase (ERK), C-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinases (MAPKs), nuclear factor-kappa B (NF-jB) p65, and histone were purchased from Cell Signaling Technology (Beverly, MA, USA). Lipopolysaccharide (LPS), polymyxin B (PMB), and propidium iodide (PI) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Polysaccharide of A. dahurica was prepared as described previously (Kim et al., 2007b). Briefly, the dried fruit was pulverized into powder. The sample was then successively extracted twice with hot water (65–70 °C) each time for 10 h. The extract was combined and concentrated under reduced pressure to small volumes. The crude polysaccharide was precipitated by adding four volumes of ethanol. The precipitate was collected by centrifugation and washed twice with ethanol. The precipitate was then suspended in water and lyophilized to yield the polysaccharide, named as ADP. No endotoxin was detected at 100 lg/ml of ADP as determined by the LAL test (Wako Pure Chemicals, Osaka, Japan).

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ADP (μg/ml) Fig. 2. Effect of polymyxin B (PMB) on LPS- or ADP-induced DC maturation. Immature DCs were treated with LPS (1 lg/ml) or ADP (10–100 lg/ml) for 24 h. ADP and LPS were pre-treated with 2000 U/ml PMB for 1 h. DCs were harvested and stained with two antibodies: PE-conjugated CD11c antibody plus FITC-conjugated MHC-class II antibody (A). The nitrite levels in culture medium were determined using Griess reagent (B). Significance was determined using the Student’s t-test versus PMB-untreated groups (⁄p < 0.01). 2.2. Nitrite production assay Nitrite accumulation was used as an indicator of NO production in the medium as previously described (Han et al., 2001). Cells were plated at 5  105 cells/ml in 96-well culture plates and stimulated with LPS of ADP for 24 h. The isolated supernatants were mixed with an equal volume of Griess reagent (1% sulfanilamide, 0.1% naphthylethyl-enediamine dihydrochloride, and 2% phosphoric acid) and incubated at room temperature for 10 min. Using NaNO2 to generate a standard curve, the concentration of nitrite was measured at an optical density (O.D.) of 540 nm. 2.3. Lymphoproliferation assay

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Spleen cells were freed of red blood cells by treating with lysis buffer. Spleen cells were cultured in RPMI 1640 complete medium. Cells were seeded in a 96 well plate at a density of 2  105 cells/well and stimulated with LPS or ADP. Cell were pulsed with 3H-thymidine (113 Ci/nmol; NEN, Boston, MA, USA) at a concentration of 1 lCi/well for the last 18 h and harvested on day 3 using an automated cell harvester (Inotech, Dottikon, Switzerland). The amount of 3H-thymidine incorporated into the cells was measured using a Microbeta scintillation counter (Wallac, Turku, Finland) (Kim et al., 2010c).

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Fig. 1. Cell-type selectivity of ADP. RAW 264.7 macrophages were treated with ADP for 24 h and NO production was determined (A). Immature DCs were treated with ADP for 24 h and CD 86 and MHC-II expression level was determined with flow cytometer (B). Total spleen cells were treated with ADP for 72 h and lymphoproliferation was determined (C). Cell viability was measured using PI-staining method (D). Significance was determined using the student’s t-test versus untreated control groups (⁄p < 0.01).

DCs were generated from bone marrow (BM) cells (Kim et al., 2007b). Briefly, BM cells were flushed out from femurs and tibias. After lysing red blood cells, whole BM cells (2  105 cells/ml) were cultured in 100-mm2 culture dishes in 10 ml/dish of complete medium containing 2 ng/ml granulocyte macrophage colony-stimulating factor (GM-CSF). On culture day 3, another 10 ml of fresh complete medium containing 2 ng/ml GM-CSF was added, and on day 6 half of the medium was changed. On day 8 non-adherent and loosely adherent DCs were harvested by vigorous pipetting and used as immature DCs (iDCs). iDCs recovered from these cultures were generally >85% CD11c+, but not CD3+ and B220+. 2.5. Phenotype analysis Phenotypic maturation of DCs was analyzed by flow cytometry. Cell staining was performed using a combination of fluorescein isothiocynate (FITC)-conjugated anti-CD86 or anti-MHC plus phycoerythrin (PE)-conjugated CD11c antibodies. Cells

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H.S. Kim et al. / Food and Chemical Toxicology 55 (2013) 241–247 were analyzed using a FACSCalibur flow cytometer and data were analyzed using CellQuest Pro (BD Biosciences, San Jose, CA, USA). Propidium iodide was added to stain dead cells. 2.6. Endocytosis assay To analyze the endocytosis of DCs, 4  105 DCs were incubated at 37 °C for 1 h with 0.4 mg/ml FITC-dextran (42,000 Da, Sigma–Aldrich). After incubation, cells were washed twice with cold washing buffer (PBS containing 0.5% BSA) and stained using PE-conjugated anti-CD11c antibody. Double stained DCs were analyzed by flow cytometry. In addition, parallel experiments were performed at 4 °C to determine the nonspecific biding of FITC-dextran to DCs (Kim et al., 2011b). 2.7. Cytokine assay Total RNA was isolated using TRIZOL™ Reagent (Molecular Research Center, Cincinnati, OH, USA). For RT-PCR, single-strand cDNA was synthesized from 2 lg total RNA. The primer sequences used were as follows: interleukin (IL)-12, sense, 50 AGA GGT GGA CTG GAC TCC CGA-30 , antisense, 50 -TTT GGT GCT TCA CAC TTC AG-30 ; IL-1b, sense, 50 -ATG GCA ATG TTC CTG AAC TCA ACT-30 , antisense, 50 -CAG GAC AGG TAT AGA TTC TTT CCT TT-30 ; tumor necrosis factor-a TNF-a, sense, 50 -AGG TTC TGT CCC TTT CAC TCA CTG-30 , antisense, 50 -AGA GAA CCT GGG AGT CAA GGT A-30 ; IL-23, sense, 50 -AGC GGG ACA TAT GAA TCT ACT AAG AGA-30 , antisense, 50 -GTC CTA GTA GGG AGG TGT GAA GTT G-30 ; b-actin, sense, 50 -TGG AAT CCT GTG GCA TCC ATG AAA C-30 , and antisense 50 -TAA AAC GCA GCT CAG TAACAG TCC G-30 . PCR products were fractionated on 1% agarose gels and stained with 5 lg/ml ethidium bromide. After analyzing band areas using an Chemi Doc™ XRS+ (Bio-Rad, Hercules, CA, USA) and Image Lab Software. Cytokine levels of IL-1b, IL-12, TNF-a, IL-2, IL-17, and IFN-c in culture supernatants were measured using commercial immunoassay kits (R&D Systems, Minneapolis, MN, USA) (Kim et al., 2010c).

electrophoresis and transferred to pure nitrocellulose membranes. Membranes were blocked with 5% skim milk in Tween 20 plus Tris-buffered saline for 1 h and then incubated with an appropriate dilution of primary antibody in 5% bovine serum albumin (in Tris-buffered saline containing Tween 20) for 2 h. Blots were incubated with biotinylated antibody for 1 h and further incubated with horseradish peroxidase-conjugated streptavidin for 1 h. Signals were detected by enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ, USA). 2.9. Mixed leukocyte reaction (MLR) Responder T cells were purified from the spleen of BALB/c mice by negative depletion using biotinylated antibodies for B220, GR-1, and CD11c (BD Pharmingen) and Dynabeads M-280 streptoavidin (Invitrogen, Dynal, Oslo, Norway) as previously described (Kim et al., 2010c). Purity was typically more than 90%. DCs were generated from the BM cells of C57BL/6 mice and were treated with 40 lg/ml mitomycin C (MMC) for 1 h. MMC-treated DCs were added to 1  105 T cells in U-bottom 96-well plates. Allogenic T cells were pulsed with 3H-thymidine (113 Ci/nmol; NEN, Boston, MA, USA) at a concentration of 1 lCi/well for the last 18 h and harvested on days 3 and 5 using an automated cell harvester (Innotech, Dottikon, Switzerland). The amount of 3H-thymidine incorporated into cells was measured using a Microbeta scintillation counter (Wallac). 2.10. Small interfering RNA (siRNA) preparation and transfection siRNAs to mouse dectin-1, TLR2, TLR4, and CR3 consisting of 21 nucleotides were synthesized from Bioneer (Daejeon, Korea). The GeneBank accession numbers for mouse were Dectin-1 (NM020008.1), TLR2 (NM011905.2), TLR4 (NM 021297.1), and CR3 (NM008401.1). siRNAs (100 nM) were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) into DCs following the manufacturer’s protocol. Cells were incubated at 37 °C in a CO2 incubator for 48 h for gene knock-down.

2.8. Western blot

2.11. SEAP reporter assay

Lysates were prepared from total cells or nuclear as previously described (Kim et al., 2010c). Detergent-insoluble materials were removed, and equal amounts of protein were fractionated by 10% sodium dodecyl sulfate–polyacrylamide gel

RAW 264.7 cells containing pNF-jB-SEAP-neomycin phosphotransferase (NPT), an NF-jB-dependent (secetory alkaline phosphatase (SEAP) reporter construct (Moon et al., 2001), were stimulated with ADP or LPS for 20 h. Aliquots of culture

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ADP (μg/ml) Fig. 3. Functional activation of DCs by ADP. Immature DCs were treated with LPS (1 lg/ml) or ADP (10–100 lg/ml) for 48 h. Cells were harvested and treated with 0.4 mg/ml of FITC-dextran for 1 h on 37 °C and 4 °C. After washing, DCs were stained with PE-conjugated anti-CD11c Ab and double stained DCs were analyzed by flow cytometry. Parallel experiments were performed at 4 °C to confirm that uptake of dextran by DCs was inhibited at low temperatures. Numbers indicate the percentage of CD11c-PE and FITC-dextran positive cells (A). iDCs were treated with LPS (1 lg/ml) or ADP (10–100 lg/ml) for 4 h. The gene expression of IL-12, IL-1b, TNF-a, and IL-23 were analyzed by RT-PCR (B). iDCs were treated with LPS or ADP for 24 h and protein levels of cytokines in culture supernatants were measured using ELISA (C). Significance was determined using the student’s t-test versus untreated control groups (⁄p < 0.01).

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media were heated to 65 °C for 5 min and then reacted with 4-methylumbelliferyl phosphate (500 lM) in the dark. SEAP activity was measured as RFUs with emission at 449 nm and excitation at 360 nm (Roh et al., 2011).

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ADP might activate DCs via direct binding to membrane receptor due to the large molecular size. The receptor candidates of ADP might include dectin-1, TLR2/4, and complement receptor 3 (CR3), which are known as receptors of well-known glucans and polysaccharides (Kim et al., 2011a). To clarify ADP receptors in DCs, we reduced their expression using siRNA. Gene (Fig. 5A) and protein (Fig. 5B) expression of dectin-1, TLR2, TLR4, and CR3 were reduced 48 h after transfection with the respective siRNA. DCs were stimulated with ADP or LPS for 24 h. DCs transfected with TLR4 siRNA showed reduced production of NO (Fig. 5C) and IL-12 (Fig. 5D).

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3.2. Effect of ADP on DC functions In addition to the phenotypic changes of DCs, ADP activates DCs and changes immune functions of DCs, such as endocytosis, cytokine production, and allo-T cell activation. Immature DCs efficiently capture antigen and lose this capacity on activation and maturation. To examine the effect of ADP on DC endocytosis, we used the fluorescent marker FITC-dextran, which was mainly taken up via the mannose receptor. After incubation of DCs with ADP or LPS for 48 h, FITC-dextran was added to the culture medium for 1 h. Antigen uptake was reduced in LPS- or ADP-treated DCs (Fig. 3A). Parallel experiments were performed at 4 °C to examine nonspecific uptake of FITC-dextran to DCs. To examine the effect of ADP on cytokine production of DCs, we isolated RNA from DCs 4 h and culture medium 24 h after treatment. ADP strongly increased gene (Fig. 3B) and protein (Fig. 3C) expression of IL-12, IL-1b, and TNF-a by DCs. To test the effect of ADP on allo-T cell activation by DCs, we induced mixed leukocyte response using DCs originated from C57BL/ 6 mice (H-2b) and T cells from BALB/c mice (H-2d). After 3 days (Fig. 4A) or 5 days (Fig. 4B), ADP-treated DCs, compared to ADP-untreated DCs, strongly augmented allo-T cell proliferation. Although immature DCs weakly activated T cells to produce cytokines, ADPtreated DCs activated T cells to produce IL-2 (Fig. 4C), IFN-c (Fig. 4D), and IL-17 (Fig. 4E). Overall these results demonstrate that ADP activates DCs and changes their immune functions.

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3.1. Cell-type selectivity of ADP To examine the cell-type selectivity of ADP, we examined the effect of ADP on several immune cells. ADP increased NO production by RAW264.7 cells (Fig. 1A), the cell surface expression of CD86 and MHC-II in DCs (Fig. 1B), and proliferation of spleen cells (Fig. 1C). ADP or LPS did not affect cell viability during the incubation (Fig. 1D). To rule out LPS contamination of ADP, we treated ADP with PMB, a specific inhibitor of LPS. PMB effectively inhibited MHC-II expression (Fig. 2A) and NO production (Fig. 2B) by LPStreated DCs, but not by ADP-treated DCs. These results suggest that ADP can activate both monocytic and lymphocytic immune cells. In subsequent experiments, we investigated the stimulatory effect of ADP on DCs in greater detail.

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Data represent the mean ± STD of more than three samples and all experiments were performed more than three times. Standard deviations (STD) were calculated using the Student’s t-test and p values were calculated using ANOVA software (GraphPad Software, San Diego, CA).

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Fig. 4. Allo-T cell activation by ADP-treated DCs. Immature DCs were activated with ADP for 24 h and treated with 40 lg/ml mitomycin C (MMC) for 1 h. MMC-treated DCs were added to 1  105 T cells purified from BALB/c mice. After incubation for 3 (A) and 5 (B) days, cells were labeled with [3H]-thymidine and harvested using automated cell harvester. Culture medium was collected 48 h after mixing DCs and T cells. IL-2 (C), IFN-c (D), and IL-17 (E) levels were measured using ELISA. Significance was determined using the Student’s t-test versus only T cell groups (⁄p < 0.01).

However, siRNA of dectin-1, TLR2, and CR3 did not affect ADP activity. The role of TLR4 as the LPS receptor was also confirmed by reduced responsiveness of TLR4 siRNA-treated DCs to LPS. 3.4. Activation of MAPKs and NF-jB by ADP Upon binding with LPS, TLR4 activates both MyD88-dependent and TRIF-dependent signaling pathways. Although the signaling downstream of MyD88 and TRIF differ, MAPKs and NF-jB signaling are usually activated and play key roles in DC activation (Neves et al., 2009). Thus, we investigated the activation of MAPKs and NF-jB in ADP-treated DCs. ADP induced the phosphorylation of p38, JNK, and ERK in dose-dependent manner (Fig. 6A). ADP also increased the nuclear translocation of NF-jB p50/p65 (Fig. 6B). In addition, ADP increased SEAP expression of RAW264.7 cells containing pNF-jB-SEAP-NPT (Fig. 6C). These results suggest that ADP activates DCs through MAPKs and NF-jB signalings, which are downstream from TLR4. 4. Discussion DCs play an important role in initiating antitumor immune responses. Tumor progression usually induces defects in DC activation and, thus, tumor-bearing hosts exhibit immunosuppression, which is one of the problems in success of DC-based immunotherapy. Many studies have attempted to find an efficient way to induce DC activation. In particular, plant-derived polysaccharides are considered potent and safe DC activators (Song et al., 2011;

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Fig. 5. Characterization of membrane receptor of ADP in DCs. Immature DCs were transfected with siRNA of dectin-1, TLR4, TLR2, and CR3. Knockdown of each receptor expression was confirmed with RT-PCR (A) and Western blotting (B). After transfection, cells were further activated with LPS or ADP for 24 h. NO production was determined using Griess reagent (C). IL-12 production was determined using ELISA (D). Significance was determined using the student’s t-test versus negative siRNA transfected groups (⁄p < 0.01).

Xu et al., 2011b). In this study, we show that ADP isolated from A. dahurica induces phenotypic and functional activation of DCs. Several phenotypic and functional changes were observed to occur in ADP-treated DCs. These included increased expression of co-stimulatory molecule and MHC-II, decreased endocytosis, increased cytokine production, and an increased capacity to induce allo-T cell activation. The CD86/CD28 and MHC/TCR pathways play a crucial role in regulating T cell activation and differentiation (Linsley et al., 1991). ADP increases the expression of CD86 and MHC-II in DCs and then strongly enhances T cell activation (Grewal and Flavell, 1996). Then, activated T cells can produce IFN-c and IL-2. Furthermore, ADP can increase cytokine production, such as IL-12, TNF-a, and IL-1b, by DCs. IL-12 is the key cytokine that stimulates Th1 cells. Also, IL-12 mediates enhancement of the cytotoxic activity of natural killer (NK) cells and CD8+ cytotoxic T cells (Romani, 2011). In addition, inflammatory cytokines such as TNFa and IL-1b affect several aspects of the inflammatory process, including chemotaxis and activation of leukocytes, and upregulation of adhesion molecules on endothelial cells (Ming et al., 1987; Pober and Cotran, 1990). These results suggest that ADP activates DCs and may result in the activation of anti-tumor effector cells in a tumor environment. We also examined ADP receptor in DCs. Because ADP could not penetrate cells, DC maturation is caused by ADP binding to receptors expressed on DCs. The major receptors for polysaccharides might be dectin-1, TLRs, and CR3. Upon binding with polysaccha-

rides, dectin-1, TLR, and CR3 induce signaling cascades and activate immune cells (Kim et al., 2011a). Dectin-1 is the known C-type lectin receptor in DCs. Dectin-1 recognizes various b-glucan polysaccharides, such as curdlan from Alcaligenes faecalis, zymosan from Saccharomyces cerevisiae, and SCG from Sparassis crispa (Agrawal et al., 2010; Dillon et al., 2006; Harada et al., 2008). Binding of dectin-1 with ligand induces several signaling pathways to activate immune responses, such as phagocytosis, ROS production, and inflammatory cytokine production (Grunebach et al., 2002). For example, blocking dectin-1 with antibodies inhibits the induction of TNF-a production by SCG-treated DCs (Harada et al., 2008). TLRs are also expressed in DCs. Among TLRs, TLR2 and TLR4 are considered as major polysaccharide receptors and can bind to polysaccharides isolated from Cordyceps militaris, Phellinus linteus, and Platycodon grandiflorum (Han et al., 2001; Kim et al., 2004, 2010c). Especially, zymosan can bind both dectin-1 and TLR2 in macrophages and increase the cytokine production. This observation implies that dectin-1 and TLR2 signalings might closely collaborate in zymosan-treated macrophages (Dennehy et al., 2008; Kim et al., 2011a). CR3 is a heterodimeric integrin receptor and recognizes microbial cells and adhesion molecules. CR3 also possesses a lectin domain, which recognizes a variety of b-glucan polysaccharides (Ross, 2000). However, whether CR3 directly binds to polysaccharides is still unclear. To clarify ADP receptors, we used siRNA of dectin-1, TLR2/4, and CR3. TLR4 siRNA-transfected DCs were hyporesponsive to ADP, suggesting that ADP might induce

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ADP (μg/ml) Fig. 6. Activation of MAPK and NF-jB signaling in ADP-treated DCs. Immature DCs were treated with LPS (1 lg/ml) or ADP (10–100 lg/ml) for 15 min. Phosphorylated-p38, JNK, and -ERK were detected by Western blotting with specific antibodies (A). Nuclear extracts were blotted with anti-p65 and -50 antibodies (B). RAW 264.7 cells containing pNF-jB-SEAP-NPT construct were stimulated with ADP or LPS for 20 h. SEAP activity, as reported by NF-jB transcription activity, was measured as relative fluorescence units (RFUs) with emission at 449 nm and excitation at 360 nm (C).

DC activation through the TLR4 receptor. However, siRNA of dectin-1, TLR2, and CR3 did not affect ADP activity. TLR4, which is mainly expressed in monocytes, macrophages, DCs, and B cells (Leung et al., 2006), is considered as a main target of BRMs. Polysaccharides from Ganoderma lucidum, S. crispa, Angelican gigas, and P. linteus activate DCs through TLR4 (Kim et al., 2004, 2007b, 2010c; Shao et al., 2004). However, TLR4 siRNA did not completely block the ADP activity on DCs. It might be due to partial knockdown of TLR4 or possibly owing to the role of other receptor candidates with the exception of dectin-1, TLR2, and CR3. The present data show that the knockdown of TLR4 impairs ADP activity on DCs and restrictively suggests that TLR4 can be used as one of membrane receptor of ADP. Upon binding with ligands, TLR4 recruits adaptor proteins and activates both the MyD88- and TRIF-dependent signaling pathways. Although the downstream signalings of MyD88 and TRIF differ, MAPKs and NF-jB signaling are commonly activated (Kim et al., 2010a; Neves et al., 2009). MAPKs and NF-jB signaling pathways regulate phenotypic and functional maturation of DCs: (a) ERK activation, which regulates DCs survival and proliferation, (b) p38 activation, which induces DC maturation and migration, and (c) NF-jB activation, which is responsible for DC maturation (Kim et al., 2011c; Nakahara et al., 2006; Rescigno et al., 1998). The present study provides evidence that ADP activates DCs through MAPKs and NF-jB downstream from TLR4. Although the activity of ADP is quite similar to LPS, differences between LPS and ADP were also observed. PMB, a specific inhibitor of LPS, inhibited MHC-II expression in LPS-treated DCs, but not in ADP-treated DCs. Limulus Color KY Test showed no endotoxin contamination in up to 100 lg/ml of ADP. Since ADP was the crude polysaccharide, which was extracted from A. dahurica with hot water and ethanol, it might contain certain unidentified compounds as well as polysaccharide. It was already reported that A. dahurica mainly contained several coumarins, such as imperatorin, oxypeucedanin, furanocoumarins, and byakangelicin (Ban et al.,

2003; Kim et al., 1991, 2007c; Lechner et al., 2004). However, these compounds had anti-inflammatory activity, suggesting that immunostimulating activity of ADP might be due to polysaccharide component of this sample. In summary, our data show that ADP binds to the membrane receptors including TLR4 and activates MAPK and NF-jB. Finally, ADP activates DCs to produce cytokines, to express MHC/costimulatory molecules, and to increase all-T cell activation. However, we cannot clarify in this study the detailed signaling networks in ADPtreated DCs, which requires the continuous efforts to identify molecular mechanisms of ADP for DC activation. Conflict of Interest The authors declare that there are no conflicts of interest. Acknowledgments This research was supported by the MRC program (20100029480) and the Public Welfare & Safety research program (2012-0006548) through the National Research Foundation of Korea (NRF). References Agrawal, S., Gupta, S., Agrawal, A., 2010. Human dendritic cells activated via dectin1 are efficient at priming Th17, cytotoxic CD8 T and B cell responses. PLoS One 5, e13418. Aiba, S., Tagami, H., 1998. Dendritic cell activation induced by various stimuli, e.g. exposure to microorganisms, their products, cytokines, and simple chemicals as well as adhesion to extracellular matrix. J. Dermatol. Sci. 20, 1–13. Ban, H.S., Lim, S.S., Suzuki, K., Jung, S.H., Lee, S., Lee, Y.S., Shin, K.H., Ohuchi, K., 2003. Inhibitory effects of furanocoumarins isolated from the roots of Angelica dahurica on prostaglandin E2 production. Planta Med. 69, 408–412. Dennehy, K.M., Ferwerda, G., Faro-Trindade, I., Pyz, E., Willment, J.A., Taylor, P.R., Kerrigan, A., Tsoni, S.V., Gordon, S., Meyer-Wentrup, F., Adema, G.J., Kullberg, B.J., Schweighoffer, E., Tybulewicz, V., Mora-Montes, H.M., Gow, N.A., Williams,

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