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Immunomodulatory activity of the seeds of Plantago asiatica L.
Journal of Ethnopharmacology 124 (2009) 493–498
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Immunomodulatory activity of the seeds of Plantago asiatica L. Dan-Fei Huang a , Ming-Yong Xie a,∗ , Jun-Yi Yin a , Shao-Ping Nie a , Yong-Fu Tang a , Xiao-Mei Xie b , Chao Zhou a a b
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, Jiangxi 330047, China Key Laboratory of Modern Preparation of TCM of Ministry of Education, Jiangxi Chinese Medical University, Nanchang, Jiangxi 330004, China
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Article history: Received 7 June 2008 Received in revised form 1 April 2009 Accepted 15 May 2009 Available online 23 May 2009 Keywords: Antigen presenting ability Dendritic cells Mixed lymphocyte culture The seeds of Plantago asiatica L.
a b s t r a c t Ethnopharmacological relevance: The seeds of Plantago asiatica L. were often used as a traditional Chinese medicine for some immunologically weak patients suffering from chronic illness. These uses could be related to immunomodulatory properties of the plant. Aim of the study: In this study, effects of extract of the seeds of Plantago asiatica L. (ES-PL) were investigated on the maturation of dendritic cells (DCs), which play significant role in primary immune system. Materials and methods: The phenotypes of DCs were analyzed by using flow cytometry while phagocytosis was assessed by the uptake of FITC-dextran. Antigen presenting ability to allogeneically naïve or syngeneically primed T lymphocytes was examined by the lymphocyte proliferation of mixed lymphocyte reaction (MLR). In addition, the level of chemokine receptor CCR7 mRNA was determined by RT-PCR. Results: DCs treated with ES-PL expressed higher levels of MHC class II molecules and major costimulatory molecules such as CD80 and CD86. Functional maturation of DCs treated with ES-PL was confirmed by decreased mannose receptor-mediated endocytosis and increased antigen presenting abilities to allogeneically naïve or syngeneically primed T lymphocytes. The CCR7 mRNA expression in DCs treated with ES-PL was also enhanced. Conclusions: These results indicated that ES-PL could induce the maturation of murine DCs. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Many biological activities in different species of the family Plantaginaceae have been reported. Traditionally, Plantago asiatica L. has long been used for the treatment of many diseases including wounds, bronchitis, cholesterolemia, chronic constipation and diarrhea (Marlett and Fischer, 2003; Ramkumar and Rao, 2005; Singh, 2007). This herbal medicine has also been shown to possess antileukemia, anticarcinoma and antiviral activities, as well as activities which modulate cell-mediated immunity (Chiang et al., 2003). The seeds of Plantago asiatica L. were regarded as orthodox product in ancient Chinese medical literature. Its chemical components, including phenylethanoid glycosides (Miyase et al., 1991), phenolics (Ravn et al., 1990) and various polysaccharides (Samuelsen et al., 1999) have been widely studied. In our previous study, it was found that the seeds of Plantago asiatica L. could enhance the immune function of the immunosuppressant mice (Xie et al., 2006). Therefore, it is very interesting to deeply study its immunological activity.
Dendritic cells (DCs) are the most potent professional antigen presenting cells (APCs) with distinct abilities to stimulate naïve T lymphocytes and to initiate primary immune responses (Steinman, 1991). Previous studies have shown that activation and migration of DCs are critical for the induction of primary immune responses (Banchereau et al., 2000; Lanzavecchia and Sallusto, 2000). This process is regulated by various stimuli, such as microorganisms, cytokines, membrane-bound ligands and simple chemicals like haptens (Usluoglu et al., 2007). However, a severe drawback possibly associated with these various stimuli is to induce detrimental adverse effects (Lehner et al., 2001). Thus, it was focused on finding new DCs maturation activators without side effects in many studies. Recently, it has been shown that some of the plants used as traditional medicines possess various biological activities including immunomodulatory activity (Kim et al., 2007). From this point of view, effects of extract of the seed of Plantago asiatica L. (ES-PL) on the maturation of dendritic cells were investigated in this study. 2. Materials and methods 2.1. Mice
∗ Corresponding author. Tel.: +86 791 3969009; fax: +86 791 3969009. E-mail address: [email protected] (M.-Y. Xie). 0378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2009.05.017
Female 4- to 6-week-old BALB/c (H-2Kd and I-Ad ) and C57BL/6 (H-2Kb and I-Ab ) mice were purchased from Shanghai SIPPR-BK
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Laboratory Animal Co. Ltd. (Shanghai, China). They were housed in a specific pathogen-free environment at 24 ◦ C and 40–60% relative humidity under a 12-h light/dark cycle. All conditions and handling of the animals were conducted according to the international guiding principles for biomedical research involving animals.
were used as controls. The possibility of LPS contamination in ESPL was eliminated by treating ES-PL with 5 g/ml polymyxin B (PB: LPS inhibitor; Sigma), which was sufficient to inhibit endotoxin but had no effect on DCs in each of our assays. 2.6. Flow cytometric analysis
2.2. Preparation of ES-PL The seeds of Plantago asiatica L. were supplied by Jiangxi Medicines & Health Products Import & Export Co., China. It was authenticated by Dr. Cui-sheng Fan, Jiangxi Chinese Medical University, as the seeds of Plantago asiatica L. The type specimen is deposited in the herbarium of Jiangxi Chinese Medical University (Serial number: 101568). 100 g of the dried seeds of Plantago asiatica L. were soaked in 10 volumes of 80% ethanol for 24 h. After filtration, the residues were dried at room temperature and then extracted twice with distilled water with a volume ratio of 10:1 at 50 ◦ C for 3 h. The extracts were filtered and centrifuged to separate the supernatant and the residue. The combined supernatant was then concentrated to a small volume at 55 ◦ C and proteins were removed by using enzymatic and Sevage method. Then 2.0 g of ES-PL were obtained from the deproteinized portion by dialysis, ethanol precipitation and lyophilization (Yin et al., 2008). Endotoxin was assayed under endotoxin-free experimental conditions by using Endotoxin-specific Tachypleus Amebocyte Lysate (TAL) (Chinese Horseshoe Crab Reagent Manufactory, CO. Ltd.). The experiments were conducted according to the manufacturer’s protocol: 100 l of ES-PL or controls were mixed with 100 l of TAL reagent and incubated for 1 h at 37 ◦ C. Each tube was then examined for gelation. The quantity of endotoxin was estimated to be ≤0.0625 endotoxin unit (EU) per mg of ES-PL. 2.3. Ovalbumin (OVA) immunization H-2Kd BALB/c mice were subcutaneously immunized three times at 1-week intervals with OVA (10 mg/animal). In the first immunization, OVA was mixed with complete Freund’s adjuvant. In the second and last immunization, OVA was mixed with incomplete Freund’s adjuvant. 3 days later, primed T lymphocytes were purified from the spleen by using Ficoll–Urografin density gradient and used in antigen-presentation assays.
DCs were harvested, washed with PBS, and resuspended in staining buffer (2% FBS and 0.1% sodium azide in PBS). Cells were first blocked with 10% (v/v) normal goat serum for 15 min at 4 ◦ C and stained with PE-conjugated anti-mouse CD11c (clone HL3), FITCconjugated anti-mouse MHC II (clone M5/114.15.2), CD80 (clone 16-10A1), and CD86 (clone GL1) monoclonal antibodies (all from BD Pharmingen) for 30 min at 4 ◦ C in the dark. Appropriate isotypematched monoclonal antibodies served as negative controls. After incubation, cells were washed three times and resuspended in 400 l of staining buffer. The stained cells were then analyzed by using a flow cytometer (Coulter Epics XL, Beckman Coulter). 2.7. Endocytosis assay 5 × 105 DCs were incubated at 37 ◦ C for 1 h with 1 mg/ml FITCdextran (40,000 Da, Sigma). After incubation, cells were washed twice with cold staining buffer and stained with PE-conjugated anti-mouse CD11c antibody. Double stained DCs were analyzed by flow cytometry. To evaluate the specificity of the uptake of dextran by DCs, parallel experiments were performed at 4 ◦ C. 2.8. Allogenic mixed lymphocyte reaction (MLR) Spleens from H-2Kb C57BL/6 mice were aseptically removed and placed in complete RPMI medium. Mononuclear lymphocytes were then obtained by using Ficoll–Urografin density gradient. Mature H-2Kd BALB/c DCs (matured in the presence of ES-PL or LPS) were pretreated with 25 g/ml mitomycin C (Sigma) for 30 min at 37 ◦ C. After being thoroughly washed, these cells were added to responder lymphocytes (1 × 106 cells/ml, 100 l/well) at ratios of 1:50 and 1:100 in U-bottomed 96-well culture plates for 3 days. Cell proliferation was determined by MTT assay, which is based on the ability of live but not dead cells to reduce a tetrazolium-based compound (MTT) to a blue formazan product that can be measured spectrophotometrically (Carmichael et al., 1987).
2.4. Generation of murine bone marrow (BM)-derived DCs 2.9. Antigen-presentation assay DCs were generated from murine bone marrow cells following the method of Inaba et al. (1992) with minor modifications. Briefly, bone marrow was harvested from the femur and tibiae of BALB/c (H2Kd and I-Ad ) mice and erythrocytes were depleted by lysing with Tris–NH4 Cl (139.6 mM NH4 Cl, 16.96 mM Tris, and adjust the pH to be 7.2 by using 1 mM HCl). The remaining cells were resuspended at a concentration of 2 × 106 cells/ml in RPMI media 1640 and cultured at 37 ◦ C, 5% CO2 for 2.5 h. Non-adherent cells were aspirated off and then complete medium (RPMI media 1640 supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS, hcyclone), 2 mM l-glutamine, 100 U/ml penicillin G, 100 U/ml streptomycin, 10 mM HEPES, 1 mM sodium pyruvate, 50 M 2-mercaptoethanol, 10 ng/ml recombinant mouse (rm) GM-CSF and 10 ng/ml rmIL-4 (R&D system)) was added. Every other day, 50% of the media was removed and replenished with fresh complete medium. 2.5. Stimulation of DCs by ES-PL On day 7, non-adherent and loosely adherent DCs were harvested by vigorous pipetting and incubated with the indicated concentration of ES-PL for 48 h. RPMI media 1640 and lipoplysaccharides (LPS, Escherichia coli serotype 0111: B4; Sigma) groups
Primed T lymphocytes were collected from immunized H-2Kd BALB/c mice as described in Section 2.3. Mature H-2Kd BALB/c DCs (matured in the presence of ES-PL or LPS) were added to primed T lymphocytes at ratios of 1:50 and 1:100. The co-cultures were stimulated with OVA (5 g/well) for 3 days, proliferation of cells was estimated by MTT assay. 2.10. Semi-quantitative RT-PCR Total RNA was extracted from DCs using Trizol Reagent (Invitrogen) according to the manufacturer’s instructions. The isolated total RNA was reverse transcribed into cDNA using an RT-PCR kit (TaKaRa RNA PCR kit Ver.3.0, TaKaRa Biotechnology Co., Dalian, China). The resulting cDNAs were then subject to 25 cycles of PCR amplification under the following conditions: denaturation at 94 ◦ C for 30 s, annealing at 60 ◦ C for 30 s, and extension at 72 ◦ C for 2 min with a final extension at 72 ◦ C for 10 min. The CCR7 (GenBank accession no. NM 007719) primers used were the forward primer, 5 -GCCTTCCTGTGTGATTTCTACAG-3 , and the reverse primer, 5 -TCACCTTCTCTCCTTTCTGTCAC-3 . The -actin (GenBank accession no. XR 032297) primers used were the forward primer,
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Fig. 1. Phenotypic analysis of DCs stimulated with ES-PL. (A) The expression of MHC II, CD80 and CD86 on DCs was analyzed by flow cytometer after treatment of ES-PL (10, 50, 100, or 200 g/ml) or 1 g/ml LPS (positive controls) for 48 h. Cells were gated on CD11c+ . (B) The up-regulation effect of ES-PL on DCs was dose-dependent. The results shown were from one representative experiment of three independent experiments performed.
5 -TGGCACCACACCTTCTACAATG-3 , and the reverse primer, 5 CCTGCTTGCTGATCCACATCTG-3 . Amplified cDNA products were resolved on 1.1% agarose gel by electrophoresis. Transcripts of actin served as internal controls. The two co-amplified bands were quantified and expressed as a ratio of intensity (CCR7 to -actin) using Quantity One software (BIO-RAD).
2.11. Statistical analysis The results were expressed as mean ± standard deviation (S.D.) of the indicated number of experiments. The statistical significance was estimated using a Student’s t-test. P < 0.05 were considered as statistically significant, P < 0.01 as highly significant.
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Fig. 2. Effect of ES-PL on endocytotic activity. Immature DCs were cultured either with 100 g/ml ES-PL or with 1 g/ml LPS (positive controls) for 48 h and then treated with FITC-dextran for 1 h at 37 ◦ C. Data are from one representative experiment out of three performed.
3. Results 3.1. ES-PL up-regulate the expression of MHC class II and costimulatory molecules on DCs To evaluate ES-PL influences the phenotypic maturation of DCs, immature DCs were cultured with ES-PL or LPS for 48 h and then analyzed for surface expression of CD80, CD86, and MHC class II molecules by flow cytometry. As shown in Fig. 1A, compared with untreated control, DCs treated with ES-PL exhibited increased surface expression of CD80, CD86, as well as MHC class II molecules on the surface of CD11c+ DCs. The concentration-dependent relationship was shown in Fig. 1B with the four doses (from 10 g/ml to 200 g/ml) tested. Similar phenotypic maturation of DCs was also observed after stimulation with 1 g/ml LPS. 3.2. ES-PL downregulate mannose receptor-mediated endocytosis of DCs Immature DCs capture and process antigens via their high endocytic capacity, and they lose this activity and mature into potent immunostimulatory APCs during differentiation (Sallusto et al., 1995). In this study, endocytic activity was measured in DCs treated with ES-PL, by monitoring the uptake of FITC-dextran. After incubation of BM-derived DCs with ES-PL or LPS, FITC-dextran was added to the culture medium for 1 h. As shown in Fig. 2, untreated DCs were able to incorporate FITC-dextran (30.71%), and ES-PL significantly decreased the uptake of FITC-dextran (15.20%) by DCs. A similar downregulation of endocytosis was observed in LPS-treated cells (16.87%). At 4 ◦ C, the percentage of CD11c+ cells that were pos-
itive for FITC-dextran was less than 10% which showed that the uptake of FITC-dextran by DCs was a specific activation process rather than a non-specific binding. 3.3. ES-PL enhance the capacity of DCs to induce proliferative response of allogeneic naïve T cells To test whether ES-PL induces the maturation of DCs to fully functional APCs, DCs were tested for their capacity to stimulate allogeneic T cells. In allogeneic mixed lymphocyte reaction, DCs were co-cultured with T cells at ratios of 1:50 and 1:100. As shown in Fig. 3A, DCs treated with ES-PL showed a high allostimulatory response compared with untreated DCs. At higher concentrations of ES-PL (100 and 200 g/ml), the MLR were significantly improved (RPMI 1640 control defined as 100%). In this study, all the DCs were first treated with mitomycin C (a selective proliferation inhibitor which abrogates DNA synthesis), followed by a through washing to remove this drug. Thus, the effects of ES-PL treatment were not likely to be attributed to the mitogenic activity of ES-PL on DCs. 3.4. ES-PL enhance the antigen-presentation capacity of DCs An important function of DCs is to present specific antigens to T cells and initiate adaptive immune response. To assess the effect of ES-PL on this functional property, we tested the capacity of DCs to prime the responses of syngeneic T cells toward OVA. As shown in Fig. 3B, T-cell proliferative responses stimulated by DCs treated with ES-PL were higher than the T cells incubated with untreated DCs. The inducing activity of ES-PL on DCs to activate syngeneic T cells operates in a dose-dependent manner. These results were
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Fig. 3. Effect of ES-PL on antigen presenting ability of DCs to T lymphocytes. (A) Effect of ES-PL on antigen presenting ability of DCs to allogeneically naïve T lymphocytes. (B) Effect of ES-PL on antigen presenting ability of DCs to syngeneically primed T lymphocytes. The proliferation of T cells was estimated by MTT assay. The absorption values of untreated DCs served as control values in the calculation of % proliferation. n = 6, *P < 0.05 compared to the RPMI-1640 group; **P < 0.01 compared to the RPMI1640 group. Data are from one representative experiment out of two performed.
Fig. 4. Effect of ES-PL on mRNA expression of CCR7 in DCs. Immature DCs were cultured either with 100 g/ml ES-PL or with 1 g/ml LPS (positive controls) for 48 h. The expression of CCR7 mRNA was performed by RT-PCR, and -actin was used as internal control. A representative gel graph from two experiments was shown.
compatible with those obtained in the phagocytosis analysis, since an increase in the antigen-presenting ability is matched with a decrease in phagocytosis. 3.5. ES-PL up-regulate CCR7 mRNA expression in DCs To examine whether the stimulatory effects of ES-PL on DCs are attributable to its influence on the expression of respective genes, semi-quantitative RT-PCR were performed for the mRNA of CCR7. As shown in Fig. 4, DCs treated with ES-PL expressed higher levels of CCR7 mRNA than untreated DCs. These data suggested that ESPL induced differentiation of DCs leading to elevated CCR7 mRNA expression. 4. Discussion Previous research results have demonstrated that the seeds of Plantago asiatica L. are especially effective in increasing the phagocytosis of murine peritoneal macrophage, inhibiting complement
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activation (Yamada et al., 1986), strengthening reticuloendothelial system-potentiating and alkaline phosphatase-inducing activities (Tomoda et al., 1991), and enhancing the transformation function of lymphocyte. Due to the differences of study models, how the seeds of Plantago asiatica L. affect the immune cells and its relative potency still remains largely unknown. Herein, the in vitro effects of extract of the seeds of Plantago asiatica L. on the phenotypic and functional maturation of BM-DCs, induced by rmGM-CSF and rmIL-4, were firstly investigated. The extract of the seed of Plantago asiatica L. was obtained by repeating hot water extraction. The procedures were ensured to carry out in safe and quality processes, to avoid contamination. They were repeatable. In our previous study, the primary phytochemical study of the extract has been performed by using high performance gel permeation chromatography (HPGPC), gas chromatography–mass spectrometry (GC–MS), UV spectroscopic techniques. It was found that the crude extract of the seed of Plantago asiatica L. mainly consisted of polysaccharides constituents (Yin et al., 2008). It was also reported that polysaccharides from other plants significantly induced the maturation of DCs as adjuvant of immunotherapy (Kim et al., 2005; Shao et al., 2006; Zhu et al., 2007). Therefore, in this study polysaccharide portion should play a central role for immunoregulation in vitro. ES-PL was showed no cytotoxicity to DCs for the reason that no decline of survival rate was observed. In the experiments, ES-PL was used at concentrations ≤200 g/ml. DCs maturation is accompanied by the changes in their morphological, phenotypic, and functional properties. In our study, ES-PL increased the expression of membrane molecules, including MHC class II, CD80 and CD86 in DCs. Also, ES-PL markedly reduced the endocytosis of DCs and augmented their capacity to promote the proliferation of T cells. Several researchers have demonstrated the immunopotentiating effects of polysaccharides purified from some plants on the maturation and function of murine DCs (Park et al., 2003; Kim et al., 2006). Our finding in ES-PL on murine DCs are similar to their results. Therefore, it was suggested that the adjuvant activity shown by the seeds of Plantago asiatica L. should be due in part to the differentiation-inducing activity on immature DCs. To elicit an effective immune response, naïve T cells need to be activated and subsequently differentiated into effector cells. In this study, the one-way MLR depleted the stimulation of T cells on DCs, and made it possible to focus on T cells proliferation induced by DCs. Our results showed that ES-PL could promote the proliferation of MLR induced by DCs, which indicated the modulating effects of ES-PL on innate immune response primed by mature DCs. As the most important professional APCs, a crucial role of DCs is to present processed antigens to T cells so that T cells can develop a specific immune response to particular antigens (Ren et al., 2008). Based on the above immunological study, many researchers have being engaged in the early application of DCs vaccination strategies (Banchereau and Palucka, 2005). Thus, it remains to be determined whether DCs treated with ES-PL are also better at stimulating T cell responses to antigens. In this study, T cells from BALB/c mice immunized by antigen OVA were used as responder T cells. After DCs treated with ES-PL in the presence of OVA were cultured together with allogeneic T cells, a significantly higher expansion of allogeneic T cells as indicated by enhanced cell proliferation was found. This result suggested that ES-PL can potentially affect antigen-specific T cell activation through modulating DCs maturation. In such a case, DCs treated with ES-PL could be used in the vaccination models based in DCs against tumor or viral antigens. To fulfill their sentinel function, DCs traffic from the blood to the tissues where they capture and process antigens as immature DCs. They then leave the tissues and migrate to the draining lymphoid organs where they prime naïve T cells, if they are converted into mature DCs. (Rot and von Andrian, 2004). In vivo, DCs migration
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is crucially directed by chemokines and their receptors expressed on DCs. CCR7, which directs migration of maturing DCs to lymphatic through the CCR7 ligand CCL21 and then to draining lymph nodes through another CCR7 ligand MIP-3, plays a particularly crucial role in this process (Liu, 2001; Jin et al., 2007). Here, ES-PL was shown to enhance the CCR7 mRNA expression in DCs, which suggested that the treatment with ES-PL for the activation and maturation of DCs may be crucial and the treated DCs could function as potent stimulatory cells in inducing primary T-cell immune responses to antigens. In summary, the results obtained clearly showed that extracts of seeds of Plantago asiatica L. have immunoregulatory activity and provided theoretical support for the use of this plant species in folk medicine. Acknowledgements This study is financially supported by the National Natural Science Foundation of China (No.: 30660226), and National Key Technology R&D Program (No.: 2006BAIO6A20-06), and Objective-Oriented Project of State Key Laboratory of Food Science and Technology (No.: SKLF-MB-200806), and Research Fund for the Doctoral Program of Higher Education of China (No.: 200804030001). References Banchereau, J., Briere, F., Caux, C., Davoust, J., Lebecque, S., Liu, Y.J., Pulendran, B., Palucka, K., 2000. Immunobiology of dendritic cells. Annual Review of Immunology 18, 767–811. Banchereau, J., Palucka, A.K., 2005. Dendritic cells as therapeutic vaccines against cancer. Nature Reviews Immunology 5, 296–306. Carmichael, J., DeGraff, W.G., Gazdar, A.F., Minna, J.D., Mitchell, J.B., 1987. Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Research 47, 936–942. Chiang, L.C., Chiang, W., Chang, M.Y., Lin, C.C., 2003. In vitro cytotoxic, antiviral and immunomodulatory effects of Plantago major and Plantago asiatica. American Journal of Chinese Medicine 31, 225–234. Inaba, K., Inaba, M., Romani, N., Aya, H., Deguchi, M., Ikehara, S., Muramatsu, S., Steinman, R.M., 1992. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colonystimulating factor. Journal of Experimental Medicine 176, 1693–1702. Jin, Y., Shen, L., Chong, E.M., Hamrah, P., Zhang, Q., Chen, L., Dana, M.R., 2007. The chemokine receptor CCR7 mediates corneal antigen-presenting cell trafficking. Molecular Vision 13, 626–634. Kim, G.Y., Ko, W.S., Lee, J.Y., Lee, J.O., Ryu, C.H., Choi, B.T., Park, Y.M., Jeong, Y.K., Lee, K.J., Choi, K.S., Heo, M.S., Choi, Y.H., 2006. Water extract of Cordyceps militaris enhances maturation of murine bone marrow-derived dendritic cells in vitro. Biological & Pharmaceutical Bulletin 29, 354–360. Kim, G.Y., Lee, M.Y., Lee, H.J., Moon, D.O., Lee, C.M., Jin, C.Y., Choi, Y.H., Jeong, Y.K., Chung, K.T., Lee, J.Y., Choi, I.H., Park, Y.M., 2005. Effect of water-soluble proteoglycan isolated from Agaricus blazei on the maturation of murine bone marrow-derived dendritic cells. International Immunopharmacology 5, 1523–1532. Kim, J.Y., Yoon, Y.D., Ahn, J.M., Kang, J.S., Park, S.K., Lee, K., Song, K.B., Kim, H.M., Han, S.B., 2007. Angelan isolated from Angelica gigas Nakai induces dendritic cell maturation through toll-like receptor 4. International Immunopharmacology 7, 78–87.
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Immunomodulatory activity of the seeds of Plantago asiatica L. Dan-Fei Huang a , Ming-Yong Xie a,∗ , Jun-Yi Yin a , Shao-Ping Nie a , Yong-Fu Tang a , Xiao-Mei Xie b , Chao Zhou a a b
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, Jiangxi 330047, China Key Laboratory of Modern Preparation of TCM of Ministry of Education, Jiangxi Chinese Medical University, Nanchang, Jiangxi 330004, China
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Article history: Received 7 June 2008 Received in revised form 1 April 2009 Accepted 15 May 2009 Available online 23 May 2009 Keywords: Antigen presenting ability Dendritic cells Mixed lymphocyte culture The seeds of Plantago asiatica L.
a b s t r a c t Ethnopharmacological relevance: The seeds of Plantago asiatica L. were often used as a traditional Chinese medicine for some immunologically weak patients suffering from chronic illness. These uses could be related to immunomodulatory properties of the plant. Aim of the study: In this study, effects of extract of the seeds of Plantago asiatica L. (ES-PL) were investigated on the maturation of dendritic cells (DCs), which play significant role in primary immune system. Materials and methods: The phenotypes of DCs were analyzed by using flow cytometry while phagocytosis was assessed by the uptake of FITC-dextran. Antigen presenting ability to allogeneically naïve or syngeneically primed T lymphocytes was examined by the lymphocyte proliferation of mixed lymphocyte reaction (MLR). In addition, the level of chemokine receptor CCR7 mRNA was determined by RT-PCR. Results: DCs treated with ES-PL expressed higher levels of MHC class II molecules and major costimulatory molecules such as CD80 and CD86. Functional maturation of DCs treated with ES-PL was confirmed by decreased mannose receptor-mediated endocytosis and increased antigen presenting abilities to allogeneically naïve or syngeneically primed T lymphocytes. The CCR7 mRNA expression in DCs treated with ES-PL was also enhanced. Conclusions: These results indicated that ES-PL could induce the maturation of murine DCs. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Many biological activities in different species of the family Plantaginaceae have been reported. Traditionally, Plantago asiatica L. has long been used for the treatment of many diseases including wounds, bronchitis, cholesterolemia, chronic constipation and diarrhea (Marlett and Fischer, 2003; Ramkumar and Rao, 2005; Singh, 2007). This herbal medicine has also been shown to possess antileukemia, anticarcinoma and antiviral activities, as well as activities which modulate cell-mediated immunity (Chiang et al., 2003). The seeds of Plantago asiatica L. were regarded as orthodox product in ancient Chinese medical literature. Its chemical components, including phenylethanoid glycosides (Miyase et al., 1991), phenolics (Ravn et al., 1990) and various polysaccharides (Samuelsen et al., 1999) have been widely studied. In our previous study, it was found that the seeds of Plantago asiatica L. could enhance the immune function of the immunosuppressant mice (Xie et al., 2006). Therefore, it is very interesting to deeply study its immunological activity.
Dendritic cells (DCs) are the most potent professional antigen presenting cells (APCs) with distinct abilities to stimulate naïve T lymphocytes and to initiate primary immune responses (Steinman, 1991). Previous studies have shown that activation and migration of DCs are critical for the induction of primary immune responses (Banchereau et al., 2000; Lanzavecchia and Sallusto, 2000). This process is regulated by various stimuli, such as microorganisms, cytokines, membrane-bound ligands and simple chemicals like haptens (Usluoglu et al., 2007). However, a severe drawback possibly associated with these various stimuli is to induce detrimental adverse effects (Lehner et al., 2001). Thus, it was focused on finding new DCs maturation activators without side effects in many studies. Recently, it has been shown that some of the plants used as traditional medicines possess various biological activities including immunomodulatory activity (Kim et al., 2007). From this point of view, effects of extract of the seed of Plantago asiatica L. (ES-PL) on the maturation of dendritic cells were investigated in this study. 2. Materials and methods 2.1. Mice
∗ Corresponding author. Tel.: +86 791 3969009; fax: +86 791 3969009. E-mail address: [email protected] (M.-Y. Xie). 0378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2009.05.017
Female 4- to 6-week-old BALB/c (H-2Kd and I-Ad ) and C57BL/6 (H-2Kb and I-Ab ) mice were purchased from Shanghai SIPPR-BK
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Laboratory Animal Co. Ltd. (Shanghai, China). They were housed in a specific pathogen-free environment at 24 ◦ C and 40–60% relative humidity under a 12-h light/dark cycle. All conditions and handling of the animals were conducted according to the international guiding principles for biomedical research involving animals.
were used as controls. The possibility of LPS contamination in ESPL was eliminated by treating ES-PL with 5 g/ml polymyxin B (PB: LPS inhibitor; Sigma), which was sufficient to inhibit endotoxin but had no effect on DCs in each of our assays. 2.6. Flow cytometric analysis
2.2. Preparation of ES-PL The seeds of Plantago asiatica L. were supplied by Jiangxi Medicines & Health Products Import & Export Co., China. It was authenticated by Dr. Cui-sheng Fan, Jiangxi Chinese Medical University, as the seeds of Plantago asiatica L. The type specimen is deposited in the herbarium of Jiangxi Chinese Medical University (Serial number: 101568). 100 g of the dried seeds of Plantago asiatica L. were soaked in 10 volumes of 80% ethanol for 24 h. After filtration, the residues were dried at room temperature and then extracted twice with distilled water with a volume ratio of 10:1 at 50 ◦ C for 3 h. The extracts were filtered and centrifuged to separate the supernatant and the residue. The combined supernatant was then concentrated to a small volume at 55 ◦ C and proteins were removed by using enzymatic and Sevage method. Then 2.0 g of ES-PL were obtained from the deproteinized portion by dialysis, ethanol precipitation and lyophilization (Yin et al., 2008). Endotoxin was assayed under endotoxin-free experimental conditions by using Endotoxin-specific Tachypleus Amebocyte Lysate (TAL) (Chinese Horseshoe Crab Reagent Manufactory, CO. Ltd.). The experiments were conducted according to the manufacturer’s protocol: 100 l of ES-PL or controls were mixed with 100 l of TAL reagent and incubated for 1 h at 37 ◦ C. Each tube was then examined for gelation. The quantity of endotoxin was estimated to be ≤0.0625 endotoxin unit (EU) per mg of ES-PL. 2.3. Ovalbumin (OVA) immunization H-2Kd BALB/c mice were subcutaneously immunized three times at 1-week intervals with OVA (10 mg/animal). In the first immunization, OVA was mixed with complete Freund’s adjuvant. In the second and last immunization, OVA was mixed with incomplete Freund’s adjuvant. 3 days later, primed T lymphocytes were purified from the spleen by using Ficoll–Urografin density gradient and used in antigen-presentation assays.
DCs were harvested, washed with PBS, and resuspended in staining buffer (2% FBS and 0.1% sodium azide in PBS). Cells were first blocked with 10% (v/v) normal goat serum for 15 min at 4 ◦ C and stained with PE-conjugated anti-mouse CD11c (clone HL3), FITCconjugated anti-mouse MHC II (clone M5/114.15.2), CD80 (clone 16-10A1), and CD86 (clone GL1) monoclonal antibodies (all from BD Pharmingen) for 30 min at 4 ◦ C in the dark. Appropriate isotypematched monoclonal antibodies served as negative controls. After incubation, cells were washed three times and resuspended in 400 l of staining buffer. The stained cells were then analyzed by using a flow cytometer (Coulter Epics XL, Beckman Coulter). 2.7. Endocytosis assay 5 × 105 DCs were incubated at 37 ◦ C for 1 h with 1 mg/ml FITCdextran (40,000 Da, Sigma). After incubation, cells were washed twice with cold staining buffer and stained with PE-conjugated anti-mouse CD11c antibody. Double stained DCs were analyzed by flow cytometry. To evaluate the specificity of the uptake of dextran by DCs, parallel experiments were performed at 4 ◦ C. 2.8. Allogenic mixed lymphocyte reaction (MLR) Spleens from H-2Kb C57BL/6 mice were aseptically removed and placed in complete RPMI medium. Mononuclear lymphocytes were then obtained by using Ficoll–Urografin density gradient. Mature H-2Kd BALB/c DCs (matured in the presence of ES-PL or LPS) were pretreated with 25 g/ml mitomycin C (Sigma) for 30 min at 37 ◦ C. After being thoroughly washed, these cells were added to responder lymphocytes (1 × 106 cells/ml, 100 l/well) at ratios of 1:50 and 1:100 in U-bottomed 96-well culture plates for 3 days. Cell proliferation was determined by MTT assay, which is based on the ability of live but not dead cells to reduce a tetrazolium-based compound (MTT) to a blue formazan product that can be measured spectrophotometrically (Carmichael et al., 1987).
2.4. Generation of murine bone marrow (BM)-derived DCs 2.9. Antigen-presentation assay DCs were generated from murine bone marrow cells following the method of Inaba et al. (1992) with minor modifications. Briefly, bone marrow was harvested from the femur and tibiae of BALB/c (H2Kd and I-Ad ) mice and erythrocytes were depleted by lysing with Tris–NH4 Cl (139.6 mM NH4 Cl, 16.96 mM Tris, and adjust the pH to be 7.2 by using 1 mM HCl). The remaining cells were resuspended at a concentration of 2 × 106 cells/ml in RPMI media 1640 and cultured at 37 ◦ C, 5% CO2 for 2.5 h. Non-adherent cells were aspirated off and then complete medium (RPMI media 1640 supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS, hcyclone), 2 mM l-glutamine, 100 U/ml penicillin G, 100 U/ml streptomycin, 10 mM HEPES, 1 mM sodium pyruvate, 50 M 2-mercaptoethanol, 10 ng/ml recombinant mouse (rm) GM-CSF and 10 ng/ml rmIL-4 (R&D system)) was added. Every other day, 50% of the media was removed and replenished with fresh complete medium. 2.5. Stimulation of DCs by ES-PL On day 7, non-adherent and loosely adherent DCs were harvested by vigorous pipetting and incubated with the indicated concentration of ES-PL for 48 h. RPMI media 1640 and lipoplysaccharides (LPS, Escherichia coli serotype 0111: B4; Sigma) groups
Primed T lymphocytes were collected from immunized H-2Kd BALB/c mice as described in Section 2.3. Mature H-2Kd BALB/c DCs (matured in the presence of ES-PL or LPS) were added to primed T lymphocytes at ratios of 1:50 and 1:100. The co-cultures were stimulated with OVA (5 g/well) for 3 days, proliferation of cells was estimated by MTT assay. 2.10. Semi-quantitative RT-PCR Total RNA was extracted from DCs using Trizol Reagent (Invitrogen) according to the manufacturer’s instructions. The isolated total RNA was reverse transcribed into cDNA using an RT-PCR kit (TaKaRa RNA PCR kit Ver.3.0, TaKaRa Biotechnology Co., Dalian, China). The resulting cDNAs were then subject to 25 cycles of PCR amplification under the following conditions: denaturation at 94 ◦ C for 30 s, annealing at 60 ◦ C for 30 s, and extension at 72 ◦ C for 2 min with a final extension at 72 ◦ C for 10 min. The CCR7 (GenBank accession no. NM 007719) primers used were the forward primer, 5 -GCCTTCCTGTGTGATTTCTACAG-3 , and the reverse primer, 5 -TCACCTTCTCTCCTTTCTGTCAC-3 . The -actin (GenBank accession no. XR 032297) primers used were the forward primer,
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Fig. 1. Phenotypic analysis of DCs stimulated with ES-PL. (A) The expression of MHC II, CD80 and CD86 on DCs was analyzed by flow cytometer after treatment of ES-PL (10, 50, 100, or 200 g/ml) or 1 g/ml LPS (positive controls) for 48 h. Cells were gated on CD11c+ . (B) The up-regulation effect of ES-PL on DCs was dose-dependent. The results shown were from one representative experiment of three independent experiments performed.
5 -TGGCACCACACCTTCTACAATG-3 , and the reverse primer, 5 CCTGCTTGCTGATCCACATCTG-3 . Amplified cDNA products were resolved on 1.1% agarose gel by electrophoresis. Transcripts of actin served as internal controls. The two co-amplified bands were quantified and expressed as a ratio of intensity (CCR7 to -actin) using Quantity One software (BIO-RAD).
2.11. Statistical analysis The results were expressed as mean ± standard deviation (S.D.) of the indicated number of experiments. The statistical significance was estimated using a Student’s t-test. P < 0.05 were considered as statistically significant, P < 0.01 as highly significant.
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Fig. 2. Effect of ES-PL on endocytotic activity. Immature DCs were cultured either with 100 g/ml ES-PL or with 1 g/ml LPS (positive controls) for 48 h and then treated with FITC-dextran for 1 h at 37 ◦ C. Data are from one representative experiment out of three performed.
3. Results 3.1. ES-PL up-regulate the expression of MHC class II and costimulatory molecules on DCs To evaluate ES-PL influences the phenotypic maturation of DCs, immature DCs were cultured with ES-PL or LPS for 48 h and then analyzed for surface expression of CD80, CD86, and MHC class II molecules by flow cytometry. As shown in Fig. 1A, compared with untreated control, DCs treated with ES-PL exhibited increased surface expression of CD80, CD86, as well as MHC class II molecules on the surface of CD11c+ DCs. The concentration-dependent relationship was shown in Fig. 1B with the four doses (from 10 g/ml to 200 g/ml) tested. Similar phenotypic maturation of DCs was also observed after stimulation with 1 g/ml LPS. 3.2. ES-PL downregulate mannose receptor-mediated endocytosis of DCs Immature DCs capture and process antigens via their high endocytic capacity, and they lose this activity and mature into potent immunostimulatory APCs during differentiation (Sallusto et al., 1995). In this study, endocytic activity was measured in DCs treated with ES-PL, by monitoring the uptake of FITC-dextran. After incubation of BM-derived DCs with ES-PL or LPS, FITC-dextran was added to the culture medium for 1 h. As shown in Fig. 2, untreated DCs were able to incorporate FITC-dextran (30.71%), and ES-PL significantly decreased the uptake of FITC-dextran (15.20%) by DCs. A similar downregulation of endocytosis was observed in LPS-treated cells (16.87%). At 4 ◦ C, the percentage of CD11c+ cells that were pos-
itive for FITC-dextran was less than 10% which showed that the uptake of FITC-dextran by DCs was a specific activation process rather than a non-specific binding. 3.3. ES-PL enhance the capacity of DCs to induce proliferative response of allogeneic naïve T cells To test whether ES-PL induces the maturation of DCs to fully functional APCs, DCs were tested for their capacity to stimulate allogeneic T cells. In allogeneic mixed lymphocyte reaction, DCs were co-cultured with T cells at ratios of 1:50 and 1:100. As shown in Fig. 3A, DCs treated with ES-PL showed a high allostimulatory response compared with untreated DCs. At higher concentrations of ES-PL (100 and 200 g/ml), the MLR were significantly improved (RPMI 1640 control defined as 100%). In this study, all the DCs were first treated with mitomycin C (a selective proliferation inhibitor which abrogates DNA synthesis), followed by a through washing to remove this drug. Thus, the effects of ES-PL treatment were not likely to be attributed to the mitogenic activity of ES-PL on DCs. 3.4. ES-PL enhance the antigen-presentation capacity of DCs An important function of DCs is to present specific antigens to T cells and initiate adaptive immune response. To assess the effect of ES-PL on this functional property, we tested the capacity of DCs to prime the responses of syngeneic T cells toward OVA. As shown in Fig. 3B, T-cell proliferative responses stimulated by DCs treated with ES-PL were higher than the T cells incubated with untreated DCs. The inducing activity of ES-PL on DCs to activate syngeneic T cells operates in a dose-dependent manner. These results were
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Fig. 3. Effect of ES-PL on antigen presenting ability of DCs to T lymphocytes. (A) Effect of ES-PL on antigen presenting ability of DCs to allogeneically naïve T lymphocytes. (B) Effect of ES-PL on antigen presenting ability of DCs to syngeneically primed T lymphocytes. The proliferation of T cells was estimated by MTT assay. The absorption values of untreated DCs served as control values in the calculation of % proliferation. n = 6, *P < 0.05 compared to the RPMI-1640 group; **P < 0.01 compared to the RPMI1640 group. Data are from one representative experiment out of two performed.
Fig. 4. Effect of ES-PL on mRNA expression of CCR7 in DCs. Immature DCs were cultured either with 100 g/ml ES-PL or with 1 g/ml LPS (positive controls) for 48 h. The expression of CCR7 mRNA was performed by RT-PCR, and -actin was used as internal control. A representative gel graph from two experiments was shown.
compatible with those obtained in the phagocytosis analysis, since an increase in the antigen-presenting ability is matched with a decrease in phagocytosis. 3.5. ES-PL up-regulate CCR7 mRNA expression in DCs To examine whether the stimulatory effects of ES-PL on DCs are attributable to its influence on the expression of respective genes, semi-quantitative RT-PCR were performed for the mRNA of CCR7. As shown in Fig. 4, DCs treated with ES-PL expressed higher levels of CCR7 mRNA than untreated DCs. These data suggested that ESPL induced differentiation of DCs leading to elevated CCR7 mRNA expression. 4. Discussion Previous research results have demonstrated that the seeds of Plantago asiatica L. are especially effective in increasing the phagocytosis of murine peritoneal macrophage, inhibiting complement
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activation (Yamada et al., 1986), strengthening reticuloendothelial system-potentiating and alkaline phosphatase-inducing activities (Tomoda et al., 1991), and enhancing the transformation function of lymphocyte. Due to the differences of study models, how the seeds of Plantago asiatica L. affect the immune cells and its relative potency still remains largely unknown. Herein, the in vitro effects of extract of the seeds of Plantago asiatica L. on the phenotypic and functional maturation of BM-DCs, induced by rmGM-CSF and rmIL-4, were firstly investigated. The extract of the seed of Plantago asiatica L. was obtained by repeating hot water extraction. The procedures were ensured to carry out in safe and quality processes, to avoid contamination. They were repeatable. In our previous study, the primary phytochemical study of the extract has been performed by using high performance gel permeation chromatography (HPGPC), gas chromatography–mass spectrometry (GC–MS), UV spectroscopic techniques. It was found that the crude extract of the seed of Plantago asiatica L. mainly consisted of polysaccharides constituents (Yin et al., 2008). It was also reported that polysaccharides from other plants significantly induced the maturation of DCs as adjuvant of immunotherapy (Kim et al., 2005; Shao et al., 2006; Zhu et al., 2007). Therefore, in this study polysaccharide portion should play a central role for immunoregulation in vitro. ES-PL was showed no cytotoxicity to DCs for the reason that no decline of survival rate was observed. In the experiments, ES-PL was used at concentrations ≤200 g/ml. DCs maturation is accompanied by the changes in their morphological, phenotypic, and functional properties. In our study, ES-PL increased the expression of membrane molecules, including MHC class II, CD80 and CD86 in DCs. Also, ES-PL markedly reduced the endocytosis of DCs and augmented their capacity to promote the proliferation of T cells. Several researchers have demonstrated the immunopotentiating effects of polysaccharides purified from some plants on the maturation and function of murine DCs (Park et al., 2003; Kim et al., 2006). Our finding in ES-PL on murine DCs are similar to their results. Therefore, it was suggested that the adjuvant activity shown by the seeds of Plantago asiatica L. should be due in part to the differentiation-inducing activity on immature DCs. To elicit an effective immune response, naïve T cells need to be activated and subsequently differentiated into effector cells. In this study, the one-way MLR depleted the stimulation of T cells on DCs, and made it possible to focus on T cells proliferation induced by DCs. Our results showed that ES-PL could promote the proliferation of MLR induced by DCs, which indicated the modulating effects of ES-PL on innate immune response primed by mature DCs. As the most important professional APCs, a crucial role of DCs is to present processed antigens to T cells so that T cells can develop a specific immune response to particular antigens (Ren et al., 2008). Based on the above immunological study, many researchers have being engaged in the early application of DCs vaccination strategies (Banchereau and Palucka, 2005). Thus, it remains to be determined whether DCs treated with ES-PL are also better at stimulating T cell responses to antigens. In this study, T cells from BALB/c mice immunized by antigen OVA were used as responder T cells. After DCs treated with ES-PL in the presence of OVA were cultured together with allogeneic T cells, a significantly higher expansion of allogeneic T cells as indicated by enhanced cell proliferation was found. This result suggested that ES-PL can potentially affect antigen-specific T cell activation through modulating DCs maturation. In such a case, DCs treated with ES-PL could be used in the vaccination models based in DCs against tumor or viral antigens. To fulfill their sentinel function, DCs traffic from the blood to the tissues where they capture and process antigens as immature DCs. They then leave the tissues and migrate to the draining lymphoid organs where they prime naïve T cells, if they are converted into mature DCs. (Rot and von Andrian, 2004). In vivo, DCs migration
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is crucially directed by chemokines and their receptors expressed on DCs. CCR7, which directs migration of maturing DCs to lymphatic through the CCR7 ligand CCL21 and then to draining lymph nodes through another CCR7 ligand MIP-3, plays a particularly crucial role in this process (Liu, 2001; Jin et al., 2007). Here, ES-PL was shown to enhance the CCR7 mRNA expression in DCs, which suggested that the treatment with ES-PL for the activation and maturation of DCs may be crucial and the treated DCs could function as potent stimulatory cells in inducing primary T-cell immune responses to antigens. In summary, the results obtained clearly showed that extracts of seeds of Plantago asiatica L. have immunoregulatory activity and provided theoretical support for the use of this plant species in folk medicine. Acknowledgements This study is financially supported by the National Natural Science Foundation of China (No.: 30660226), and National Key Technology R&D Program (No.: 2006BAIO6A20-06), and Objective-Oriented Project of State Key Laboratory of Food Science and Technology (No.: SKLF-MB-200806), and Research Fund for the Doctoral Program of Higher Education of China (No.: 200804030001). References Banchereau, J., Briere, F., Caux, C., Davoust, J., Lebecque, S., Liu, Y.J., Pulendran, B., Palucka, K., 2000. Immunobiology of dendritic cells. Annual Review of Immunology 18, 767–811. Banchereau, J., Palucka, A.K., 2005. Dendritic cells as therapeutic vaccines against cancer. Nature Reviews Immunology 5, 296–306. Carmichael, J., DeGraff, W.G., Gazdar, A.F., Minna, J.D., Mitchell, J.B., 1987. Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Research 47, 936–942. Chiang, L.C., Chiang, W., Chang, M.Y., Lin, C.C., 2003. In vitro cytotoxic, antiviral and immunomodulatory effects of Plantago major and Plantago asiatica. American Journal of Chinese Medicine 31, 225–234. Inaba, K., Inaba, M., Romani, N., Aya, H., Deguchi, M., Ikehara, S., Muramatsu, S., Steinman, R.M., 1992. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colonystimulating factor. Journal of Experimental Medicine 176, 1693–1702. Jin, Y., Shen, L., Chong, E.M., Hamrah, P., Zhang, Q., Chen, L., Dana, M.R., 2007. The chemokine receptor CCR7 mediates corneal antigen-presenting cell trafficking. Molecular Vision 13, 626–634. Kim, G.Y., Ko, W.S., Lee, J.Y., Lee, J.O., Ryu, C.H., Choi, B.T., Park, Y.M., Jeong, Y.K., Lee, K.J., Choi, K.S., Heo, M.S., Choi, Y.H., 2006. Water extract of Cordyceps militaris enhances maturation of murine bone marrow-derived dendritic cells in vitro. Biological & Pharmaceutical Bulletin 29, 354–360. Kim, G.Y., Lee, M.Y., Lee, H.J., Moon, D.O., Lee, C.M., Jin, C.Y., Choi, Y.H., Jeong, Y.K., Chung, K.T., Lee, J.Y., Choi, I.H., Park, Y.M., 2005. Effect of water-soluble proteoglycan isolated from Agaricus blazei on the maturation of murine bone marrow-derived dendritic cells. International Immunopharmacology 5, 1523–1532. Kim, J.Y., Yoon, Y.D., Ahn, J.M., Kang, J.S., Park, S.K., Lee, K., Song, K.B., Kim, H.M., Han, S.B., 2007. Angelan isolated from Angelica gigas Nakai induces dendritic cell maturation through toll-like receptor 4. International Immunopharmacology 7, 78–87.
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