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Rehmannia glutinosa polysaccharide induces maturation of murine bone marrow derived Dendritic cells (BMDCs)
International Journal of Biological Macromolecules 54 (2013) 136–143
Contents lists available at SciVerse ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Rehmannia glutinosa polysaccharide induces maturation of murine bone marrow derived Dendritic cells (BMDCs) Zhenjie Zhang a , Yiming Meng a , Yanxia Guo b , Xin He a , Qingrui Liu a , Xiufeng Wang a , Fengping Shan a,c,∗ a b c
Department of Immunology, School of Basic Medical Science, China Medical University, No. 92, North Second Road, Heping District, Shenyang 110001, PR China Department of Internal Medicine, Zibo Central Hospital, No. 54, Gong Qing Tuan Road, Zhangdian District, Zibo 255036, PR China Institute of Pathology and Pathophysiology, School of Basic Medical Science, China Medical University, No. 92, North Second Road, Heping District, Shenyang 110001, PR China
a r t i c l e
i n f o
Article history: Received 8 August 2012 Received in revised form 30 November 2012 Accepted 5 December 2012 Available online 12 December 2012 Keywords: Rehmannia glutinosa polysaccharide Bone marrow derived Dendritic cells Modulation Maturation Immune system
a b s t r a c t Purified Rehmannia glutinosa polysaccharide (RGP) is used as functional foods for the prevention and treatment of various diseases. In this study, we examined the effects of RGP on phenotypic and functional maturation of murine bone marrow derived Dendritic cells (BMDCs). Phenotypic maturation of BMDCs was confirmed by conventional scanning electron microscopy (SEM), flow cytometry (FCM) and functional maturation by transmission electron microscopy (TEM), cytochemistry assay, Acid phosphatase (ACP) activity, FITC-dextran, bio-assay and enzyme linked immunosorbent assay (ELISA).We found that RGP up-regulated the expression of CD40, CD80, CD83, CD86 and MHC II molecules of BMDCs, downregulated pinocytosis and phagocytosis activity, induced IL-12 and TNF-␣ production of BMDCs. It is therefore concluded that RGP can effectively promote the maturation of DCs. Our study provides evidence and rationale on using RGP in various clinical conditions to enhance host immunity and suggests RGP as a potent adjuvant for the design of DC-based vaccines. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Rehmannia glutinosa is a member of the Scrophulariaceae family. It is a traditional Chinese medicinal herb which is widely used in China, Japan, Korea and other Asian countries. Its steamed roots have been used for different medical purposes as traditional Chinese medicine (TCM) for thousands of years. It is often used as a primary herb in formulas to treat a great variety of diseases, include ameliorates the progressive renal failure [1], regulate immediate type allergic reaction [2], inhibit atopic dermatitis [3], enhance the bone metabolism in osteoporosis [4], inhibit liver inflammation and fibrosis [5], anti-tumor [6]. It also has Anti-fatigue [7], Anti-depressant [8], anti-diabetes [9], protect auditory cell [10], neuroprotective effect [11]. Modern researches have also indicated that polysaccharides are the main chemical components
Abbreviations: RGP, Rehmannia glutinosa polysaccharide; ACP, acidic phosphatase; BMDCs, bone marrow derived Dendritic cells; APCs, antigen presenting cells; LPS, lipopolysaccharide; SEM, scanning electron microscopy; TEM, transmission electron microscopy; DAB, 3*3 -diaminobenzidine; MTT, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; FCM, flow cytometry; MACS, magnetic activated cell sorting; CTL, cytotoxic lymphocyte. ∗ Corresponding author at: Department of Immunology, School of Basic Medical Science, China Medical University, No. 92, North Second Road, Heping District, Shenyang 110001, PR China. Tel.: +86 189 009 11546; fax: +86 24 86604452. E-mail address: [email protected] (F. Shan). 0141-8130/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2012.12.005
related to the bioactivities and pharmacological properties of the herb. Rehmannia glutinosa polysaccharide a, b were separated and identified. They are commonly composed of l-arabinose:dgalactose:l-rhamnose:d-galacturonic acid in the molar ratios of 10:10:1:1 (rehmannan SA) and 14:7:3:8 (rehmannan SB) [7,12]. DCs are the most powerful antigen-presenting cells (APCs) that serve as a link between the innate and adaptive immune system. DCs initiate the majority of immune responses. Its specific function is uptaking, processing, presenting antigen and activating naïve T cells [13]. Immature DCs patrol as sentinels in almost every non-lymphoid tissue and organ, have a highly developed capacity to capture antigens, but a poor antigen-presenting ability and T cell-stimulatory activity with low expression of surface markers such as CD40, CD80, CD83, CD86 and MHC II. Upon uptaking of pathogens, DCs undergo phenotypic and functional changes to differentiate into mature stage with higher expression of CD40, CD80, CD83, CD86 and MHC II, which accompany enhanced antigen-presenting ability and more production of cytokines [14]. However, so far little is known about the molecular mechanisms responsible for the regulation of DCs in their activation and maturation states by RGP and detailed mechanisms through which RGP exerts immunoregulation remain unclear. We endeavor to conduct the following experiments to try to answer the question.
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Fig. 1. The CD11c+ cells were purified by MACS. After co-cultured for 6 d, the purity of CD11c+ cells was examined and the percentage was over 80%. Followed by purification with MACS, the CD11c+ cells were enriched and the results of FCM showed that the purity of CD11c+ cells approached 95%.
2. Materials and methods 2.1. Reagents In this study, the purified RGP (≥98% purity) was obtained from Ci Yuan Biotechnology co. Ltd. Shanxi, China. LPS (10 ng/ml) as a positive control was purchased from Sigma–Aldrich. The mAbs were products of eBioscience (San Diego, CA) and BD Pharmingen (San Jose, CA). GM-CSF and IL-4 were purchased from PeproTech Inc (Rocky Hill, NJ). The ELISA assay kits for IL-12p70 and TNF-␣ analysis were products of eBioscience (San Diego, CA). All other chemicals frequently used in our lab were purchased from either Sigma–Aldrich or BD Pharmingen. 2.2. Bone marrow derived Dendritic cells (BMDCs) culture BMDCs were generated as previously described [15,16]. The overall steps were as followed: (1) The femurs and tibiae of mice (4–6 weeks old raised in pathogen free animal house, China Medical University) were removed and isolated from surrounding muscle tissue. Intact bones were left for disinfection in 70% ethanol for 1 min, washed twice in PBS, and transferred into a fresh dish with RPMI 1640. Cut off the both metaphysic to expose marrow cavity then blow out the cells in the cavity by RPMI 1640 with 1 ml syringe. Clusters within the marrow suspension were disintegrated by vigorous pipetting. Lyses buffer was used to eliminate red blood cells. (2) 106 cells were transferred into 24-well plates (Corning incorporated Costar) with RPMI1640 (1 ml) and supplements including recombinant murine granulocyte macrophage colony stimulating factor (GM-CSF) (10 ng/ml), IL-4 (10 ng/ml), L-glutamine (2 mM), fetal bovine serum (FBS) (10%), streptomycin (100 g/ml) and penicillin (100 units/ml) in a humidified atmosphere of 5% CO2 at 37 ◦ C; (3) After incubation for 4 h, the medium with non-adherent cells was replaced with a fresh medium, while the nutrient medium was replaced every three days; (4) On the day 6, MACS (Miltenyi Biotec, CA) was used under the instruction of the manufacturer to isolate cells with CD11c expression, which were later seeded into new wells with fresh medium; (5) The sorted cells were treated with or without RGP (0.2 mg/ml) for 24 h; (6) Flow cytometry (FCM) analysis was conducted to test the cell purity of separated BMDCs. 2.3. Effects of a range of RGP doses on BMDCs The BMDCs were cultured in 96-well micro plate and added varied concentrations of RGP, ranging from 3.2 mg/ml to
0.78 × 10−3 mg/ml to the wells, triplicate/conc. Then we conducted MTT analysis to assess the proliferation of BMDCs under different RGP concentrations to determine the optimal concentration. Adding MTT to the culture, then removed the supernatant and dissolved the precipitation with DMSO after 4 h. Finally the optical density was measured on each checking point at OD570 .
2.4. Morphology of BMDCs with inverted phase contrast microscope and SEM The cultured BMDCs were incubated with LPS and 0.2 mg/ml RGP, respectively, for 24 h before further morphology study under inverted phase contrast microscope. The cell samples for SEM were prepared per steps including glutaraldehyde fixation, cleaning, osmium tetroxide post-fixation, cleaning, replacement, critical point drying, and gold spraying, etc. Finally samples were checked with SEM (JEOL JSM-T300).
2.5. Analysis by flow cytometry The cultured cells from both testing group and control group were collected and stained with anti-CD40, anti-CD80, anti-CD83, anti-CD86 and anti-MHC II antibodies for 30 min, and then washed with PBS/2% FACS twice. The prepared cells were fixed in 4% paraformaldehyde and later collected using FACS Calibur (Becton Dickinson, San Diego, CA) for confirmation of surface molecules.
2.6. Analysis of pinocytotic vesicles within the BMDCs by TEM The cultured BMDCs treated with 0.2 mg/ml RGP for 24 h were digested with trypsin. Following steps included centrifugation at 1000 rpm for 10 min, glutaraldehyde fixation and slicing. The ready samples were checked for their intracellular pinocytotic vesicles with TEM (JEOL JEM-1200EX).
2.7. Pinocytosis assessment by BMDCs using peroxidase BMDCs with a concentration of 1 × 105 /ml were treated with LPS and 0.2 mg/ml RGP for 24 h. The above cells were processed with 0.08 mg/ml horseradish peroxidase, followed by fixed with methanol for 4 hr and staining with DAB kit. Finally, microscope was conducted to exam the prepared samples.
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Z. Zhang et al. / International Journal of Biological Macromolecules 54 (2013) 136–143
Fig. 2. The proliferation of BMDCs under a range of RGP doses (A) and at different time point under the optimal concentration (B). Results represent the mean ± sd of six samples. ** p < 0.01 or * p < 0.05 vs. that in RPMI 1640.
2.8. Assessment of phagocytosis
2.11. Statistical analysis
The phagocytosis activity of BMDCs was measured as cellular uptake of FITC-Dextran and was quantified by flow cytometry. The cultured BMDCs were tested after treatment with 0.2 mg/ml RGP for 24 h. Three steps were involved in the samples preparation: (1) Adding 100 l FITC-Dextran (40,000 Da) to the media; (2) Incubating at 4 ◦ C for 2 h; (3) Incubating at 37 ◦ C for 1 h. Finally, the samples were analyzed with FACS Calibur (Becton Dickinson, San Diego, CA) to assess the phagocytic activity.
The data of FCM were processed with WinMDI2.9 (Joseph Trotter, BD Biosciences). Data for all groups were evaluated with the statistical program SPSS (Statistical Package for Social Sciences, Version 16.0) for Windows and all variables were expressed as mean ± sd. ANOVA and the student t-test using Prism (Graph Pad Software) were adopted to assess the differences. When p < 0.05, significance was indicated. 3. Results
2.9. Acid phosphatase (ACP) activity detection 3.1. MACS study Firstly the BMDCs were treated with LPS and 0.2 mg/ml RGP for 24 h and adjusted the cell concentration to 1 × 106 /ml, and then the ACP activity was assessed with ACP testing kit (Jiancheng Bio-engineering Institute of the South) by phenol-4-AAP (amino antipyrine) method and measured at OD absorbance on 520 nm (A520). 2.10. Cytokine assay by ELISA The BMDCs treated with LPS and 0.2 mg/ml RGP for 24 h, and then collected supernatant for IL-12p70 and TNF-␣ assay per the instructions in the ELISA kit. The OD absorbance at 450 nm (A450) was determined with bichromatic microplate reader (BIO-TEK, USA).
The cells with CD11c+ were isolated by MACS. FCM analysis was conducted to test the cell purity of separated BMDCs as shown in Fig. 1. 3.2. Effects of a range of RGP doses on BMDCs The BMDCs proliferated into differential numbers when treated with various concentrations of RGP. A bilateral relationship between RGP concentration from 3.2 mg/ml to 0.78 × 10−3 mg/ml and cell proliferation could be seen. It was obvious that cell proliferation was inhibited at lower and higher concentrations, and the optimal concentration appeared to be 0.2 mg/ml (Fig. 2A). Under the optimal concentration, the cell proliferation at different time
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Fig. 3. The morphology of BMDCs before and after treatment with RGP under a light microscope (A) (×200) and SEM (B) (×5000).
points was also assessed by MTT, and the optimal time for cell proliferation was found to be 24 h after treatment with RGP (Fig. 2B). 3.3. Morphology of BMDCs treated with RGP The morphology of cultured BMDCs treated with GM-CSF and IL4 for 6 d was observed with an inverted phase contrast microscope (Fig. 3A). Large cells with oval or irregular nuclei and many small dendrites could be seen, indicating maturity. Simultaneously, The SEM photos of the BMDCs were compared before and after treatment with RGP. The treated ones showed more protrusions and greater number of cascading folds (Fig. 3B). 3.4. FCM analysis The BMDCs were cultured with GM-CSF and IL-4 for 6 d, and then FCM analysis was conducted on the BMDCs treated with 0.2 mg/ml RGP for 24 h. The results showed increased expression of CD40, CD80, CD83, CD86 and MHC-II which are important surface markers for mature DCs as reflected in Fig. 4.
3.6. Cytochemistry analysis of BMDCs pinocytosis Pinocytosis of BMDCs could be observed under light microscope after staining with DAB kit. Pinocytosis of horseradish peroxidase by BMDCs was shown in the photos. The less pinocytosis could be found in RGP group, indicating a weakened capability in antigen arresting while with a strengthened ability in antigen presenting (Fig. 6).
3.7. Analysis for down-regulation of the phagocytosis of BMDCs by RGP with FCM The phagocytosis reflects the digestion of antigen by immature DCs [17]. FITC-dextran is generally used to examine the intrinsic phagocytosis capacity. The uptaking of FITC-dextran is known to be maximal in the immature BMDCs and occurs by a combination of macropinocytosis and binding to the mannose receptor [18]. The results revealed that the phagocytosis of BMDCs had undergone a notable decrease after treatment with RGP (Fig. 7).
3.5. TEM on pinocytotic vesicles of BMDCs Immature BMDCs are normally associated with strong phagocytic ability and more pinocytotic vesicles while mature BMDCs are opposite, however, with increased antigen presenting ability. The TEM photo of the cells after treatment with RGP revealed a sharp decrease in the number of pinocytotic vesicles compared with that of BMDCs in RPMI 1640 group (Fig. 5).
3.8. Acid phosphatase activity detection ACP, as a major enzyme in the lysosome to digest antigen, is related to the DC maturity in an inverse manner. As can be seen from (Fig. 8), ACP activity in BMDCs was greatly decreased after treatment with RGP.
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Fig. 4. RGP up regulated the key surface molecules on BMDCs. The expressions of CD40, CD80, CD83, CD86 and MHC II were analyzed by FCM and displayed, respectively, by the single parameter diagram. The mean fluorescence intensity (MFI) and the gated % values are shown as the mean ±sd of six independent samples.
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Fig. 5. The changes of pinocytotic vesicles inside the BMDCs before and after treatment with RGP under TEM. The number of pinocytotic vesicles inside BMDCs which treating with RGP for 24 h had reduced significantly (arrows).
Fig. 6. Cytochemistry assay of BMDCs before and after treatment with RGP. The capability of BMDCs for pinocytosis stimulated with 0.2 mg/ml RGP decreased obviously compared with those in the RPMI 1640 group.
3.9. RGP increases production of IL-12p70 and TNF-˛ by BMDCs Usually matured BMDCs secreted higher level of IL-12p70 and TNF-␣. IL-12p70 and TNF-␣ were measured by ELISA of the BMDCs after treatment with 0.2 mg/ml RGP for 24 h. The results showed
up-regulation of production of IL-12p70 and TNF-␣ in mature cells (Fig. 9). 4. Discussion Recorded in Chinese medical classical Shennong’s Herba, RGP is considered as one of the top grade herbs in China. RGP enhanced
Fig. 7. The affect on the phagocytosis of BMDCs by RGP. The BMDCs stimulated with 0.2 mg/ml RGP for 24 h were incubated with FITC-dextran for check by FCM. The average percentages from six independent experiments showed that mature BMDCs reduced markedly in phagocytosis. ** p < 0.01 vs. that in RPMI 1640.
Fig. 8. ACP activity of BMDCs after treatment with RGP. ACP activity was determined after treatment with RGP for 24 h by the phenol-4-AAP method in conjunction with ACP testing kit. Results represent the mean ± sd of six samples. ** p < 0.01 or * p < 0.05 vs. that in RPMI 1640.
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Fig. 9. Effects of the 0.2 mg/ml RGP on the production of IL-12p70 (A), and TNF-␣ (B). After treatment with RGP the supernatant from cell cultures was collected and the secreted cytokines were detected by ELISA. Similar results were obtained in three independent experiments and a representative graph was shown. Results represent the mean ± sd of six samples. ** p < 0.01 or * p < 0.05 vs. that in RPMI 1640.
the proliferation of T, B lymphocytes in spleen by aged mice and phagocytosis of peritoneal macrophage [19]. It also suppressed phosphorylation of mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-B [20,21]. Moreover, the expression levels of adhesion molecules such as intercellular adhesion molecule1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1) were suppressed by treatment with RGP [3]. It also lowered plasma Creactive protein levels in diabetic rats [22].These results reveal that RGP has anti-inflammatory and enhance immune system activity. Our results showed that RGP fully induced the maturation of BMDCs by up-regulating phenotype and functions with the fact: (1) The morphology of BMDCs had more protrusions, rougher surface accompanying with up-regulated the expression of CD40, CD80, CD83, CD86, and MHC II. (2) RGP induced functional maturation of BMDCs, as characterized by decreasing phagocytosis ability and ACP activity which usually indicates the degree of maturation of BMDCs. (3) RGP also enhanced the production of IL-12 and TNF-␣ of BMDCs as they became more matured, resulting in initiation of T cell response [23]. The maturation of BMDCs depends on the maturation signal as well as the constraints imposed by ontogeny and environmental modifiers. Signal 1 is provided by the T cell receptor-recognizing antigen peptides bound to MHC molecules. Signal 1 alone inactivates T cells, causing T cell deletion or inducing regulatory T cell generation. Signal 2 is a co-stimulatory signal. When combined with Signal 1, it will induce T cell responses. The T cells expand clonal and differentiate into effector or memory cells. Furthermore, by binding CD80 or CD86 on DCs to its receptor, Signal 2 promotes antigen-specific immunity strongly [24]. By binding CD80 or CD86 on DCs to cytotoxic T-lymphocyte antigen 4 (CTLA4) on regulatory T cells, Signal 2 can also induce tolerogenic signals. Signal 3,
which is also delivered by DCs, can determine the differentiation of Th1 cells, Th2 cells, and cytotoxic lymphocyte (CTLs). One Signal 3 molecule is IL-12, which can induce Th1 cells and CTLs differentiation. The Notch ligand promotes the development of Th2 cells as Signal 3. It is helpful in inducing efficient anti-tumor immunity by controlling the interactions between DCs and T cells on multiple levels [25,26]. As the immunosuppressive conditions in cancer patients have been well-characterized, the clinical significance of DCs in cellular therapy cannot be overlooked [27]. The mild adjuvants are necessary for the maturation of DCs. Most of the adjuvants for current experiment use can provide signals which the innate immune system vigorously reacts to, resulting in the potentiating of the immune system so that the Ag co-administrated with the adjuvant can be uptaked, processed and presented in a more effective manner by the activated DCs [28,29]. This work can therefore contribute to a better understanding of RGP’s modulating effects on immune system, and a better understanding of the complicated mechanisms at molecular level, through which purified herbal extract is working [30]. Overall, the data in this study indicates that RGP induces the maturation of BMDCs in both phenotypic and functional aspects. RGP is a good supplement for people with poor immunity, such as those with cancer, chronic disease, and aging. The improvement of immunity in these cases can be achieved by simply oral administration [31]. Furthermore, this work also provides a meaningful mode of action for RGP and highlights the clinical significance of RGP as an immune enhancer which may play a important role in combating cancer [32,33]. Finally, although RGP has ever been used as an adjuvant in stimulation of tetanus toxoid-specific immune responses and identification of a novel vaccine adjuvant that stimulates and maintains diphtheria toxoid immunity as well as an agent in therapy of several cancers like esophageal cancer clinically, we may think of RGP as a possible adjuvant in DC-based vaccine preparations in future clinical practice [34,35]. Conflict of interest The authors have no financial conflicts of interest with any party. Acknowledgment This work was supported financially by China Liaoning Provincial Foundation for Education No. L2010576 (to Fengping SHAN). References [1] B.C. Lee, J.B. Choi, H.J. Cho, Y.S. Kim, Journal of Ethnopharmacology 122 (2009) 131–135. [2] H. Kim, E. Lee, S. Lee, T. Shin, Y. Kim, J. Kim, International Journal of Immunopharmacology 20 (1998) 231–240. [3] Y.Y. Sung, T. Yoon, J.Y. Jang, S.J. Park, H.K. Kim, Journal of Ethnopharmacology 134 (2011) 37–44. [4] K.O. Oh, S.W. Kim, J.Y. Kim, S.Y. Ko, H.M. Kim, J.H. Baek, H.M. Ryoo, J.K. Kim, Clinica Chimica Acta 334 (2003) 185–195. [5] P.S. Wu, S.J. Wu, Y.H. Tsai, Y.H. Lin, J.C.J. Chao, American Journal of Chinese Medicine 39 (2011) 1173–1191. [6] X.L. Wei, X.B. Ru, Zhongguo Yao Li Xue Bao 18 (1997) 471–474. [7] W. Tan, K.Q. Yu, Y.Y. Liu, M.Z. Ouyang, M.H. Yan, International Journal of Biological Macromolecules 50 (2012) 59–62. [8] D. Zhang, X.S. Wen, X.Y. Wang, M. Shi, Y. Zhao, Journal of Ethnopharmacology 123 (2009) 55–60. [9] R. Zhang, J. Zhou, Z. Jia, Y. Zhang, G. Gu, Journal of Ethnopharmacology 90 (2004) 39–43. [10] H.H. Yu, S.J. Seo, Y.H. Kim, H.Y. Lee, R.K. Park, H.S. So, S.ll Jang, Y.O. You, Journal of Ethnopharmacology 107 (2006) 383–388. [11] X. Zhang, A. Zhang, B. Jiang, Y. Bao, J. Wang, L. An, Phytomedicine 15 (2008) 484–490. [12] M. Tmoda, H. Miyamoto, N. Shimizu, Chemical and Pharmaceutical Bulletin 42 (1994) 1666–1668.
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Contents lists available at SciVerse ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Rehmannia glutinosa polysaccharide induces maturation of murine bone marrow derived Dendritic cells (BMDCs) Zhenjie Zhang a , Yiming Meng a , Yanxia Guo b , Xin He a , Qingrui Liu a , Xiufeng Wang a , Fengping Shan a,c,∗ a b c
Department of Immunology, School of Basic Medical Science, China Medical University, No. 92, North Second Road, Heping District, Shenyang 110001, PR China Department of Internal Medicine, Zibo Central Hospital, No. 54, Gong Qing Tuan Road, Zhangdian District, Zibo 255036, PR China Institute of Pathology and Pathophysiology, School of Basic Medical Science, China Medical University, No. 92, North Second Road, Heping District, Shenyang 110001, PR China
a r t i c l e
i n f o
Article history: Received 8 August 2012 Received in revised form 30 November 2012 Accepted 5 December 2012 Available online 12 December 2012 Keywords: Rehmannia glutinosa polysaccharide Bone marrow derived Dendritic cells Modulation Maturation Immune system
a b s t r a c t Purified Rehmannia glutinosa polysaccharide (RGP) is used as functional foods for the prevention and treatment of various diseases. In this study, we examined the effects of RGP on phenotypic and functional maturation of murine bone marrow derived Dendritic cells (BMDCs). Phenotypic maturation of BMDCs was confirmed by conventional scanning electron microscopy (SEM), flow cytometry (FCM) and functional maturation by transmission electron microscopy (TEM), cytochemistry assay, Acid phosphatase (ACP) activity, FITC-dextran, bio-assay and enzyme linked immunosorbent assay (ELISA).We found that RGP up-regulated the expression of CD40, CD80, CD83, CD86 and MHC II molecules of BMDCs, downregulated pinocytosis and phagocytosis activity, induced IL-12 and TNF-␣ production of BMDCs. It is therefore concluded that RGP can effectively promote the maturation of DCs. Our study provides evidence and rationale on using RGP in various clinical conditions to enhance host immunity and suggests RGP as a potent adjuvant for the design of DC-based vaccines. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Rehmannia glutinosa is a member of the Scrophulariaceae family. It is a traditional Chinese medicinal herb which is widely used in China, Japan, Korea and other Asian countries. Its steamed roots have been used for different medical purposes as traditional Chinese medicine (TCM) for thousands of years. It is often used as a primary herb in formulas to treat a great variety of diseases, include ameliorates the progressive renal failure [1], regulate immediate type allergic reaction [2], inhibit atopic dermatitis [3], enhance the bone metabolism in osteoporosis [4], inhibit liver inflammation and fibrosis [5], anti-tumor [6]. It also has Anti-fatigue [7], Anti-depressant [8], anti-diabetes [9], protect auditory cell [10], neuroprotective effect [11]. Modern researches have also indicated that polysaccharides are the main chemical components
Abbreviations: RGP, Rehmannia glutinosa polysaccharide; ACP, acidic phosphatase; BMDCs, bone marrow derived Dendritic cells; APCs, antigen presenting cells; LPS, lipopolysaccharide; SEM, scanning electron microscopy; TEM, transmission electron microscopy; DAB, 3*3 -diaminobenzidine; MTT, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; FCM, flow cytometry; MACS, magnetic activated cell sorting; CTL, cytotoxic lymphocyte. ∗ Corresponding author at: Department of Immunology, School of Basic Medical Science, China Medical University, No. 92, North Second Road, Heping District, Shenyang 110001, PR China. Tel.: +86 189 009 11546; fax: +86 24 86604452. E-mail address: [email protected] (F. Shan). 0141-8130/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2012.12.005
related to the bioactivities and pharmacological properties of the herb. Rehmannia glutinosa polysaccharide a, b were separated and identified. They are commonly composed of l-arabinose:dgalactose:l-rhamnose:d-galacturonic acid in the molar ratios of 10:10:1:1 (rehmannan SA) and 14:7:3:8 (rehmannan SB) [7,12]. DCs are the most powerful antigen-presenting cells (APCs) that serve as a link between the innate and adaptive immune system. DCs initiate the majority of immune responses. Its specific function is uptaking, processing, presenting antigen and activating naïve T cells [13]. Immature DCs patrol as sentinels in almost every non-lymphoid tissue and organ, have a highly developed capacity to capture antigens, but a poor antigen-presenting ability and T cell-stimulatory activity with low expression of surface markers such as CD40, CD80, CD83, CD86 and MHC II. Upon uptaking of pathogens, DCs undergo phenotypic and functional changes to differentiate into mature stage with higher expression of CD40, CD80, CD83, CD86 and MHC II, which accompany enhanced antigen-presenting ability and more production of cytokines [14]. However, so far little is known about the molecular mechanisms responsible for the regulation of DCs in their activation and maturation states by RGP and detailed mechanisms through which RGP exerts immunoregulation remain unclear. We endeavor to conduct the following experiments to try to answer the question.
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Fig. 1. The CD11c+ cells were purified by MACS. After co-cultured for 6 d, the purity of CD11c+ cells was examined and the percentage was over 80%. Followed by purification with MACS, the CD11c+ cells were enriched and the results of FCM showed that the purity of CD11c+ cells approached 95%.
2. Materials and methods 2.1. Reagents In this study, the purified RGP (≥98% purity) was obtained from Ci Yuan Biotechnology co. Ltd. Shanxi, China. LPS (10 ng/ml) as a positive control was purchased from Sigma–Aldrich. The mAbs were products of eBioscience (San Diego, CA) and BD Pharmingen (San Jose, CA). GM-CSF and IL-4 were purchased from PeproTech Inc (Rocky Hill, NJ). The ELISA assay kits for IL-12p70 and TNF-␣ analysis were products of eBioscience (San Diego, CA). All other chemicals frequently used in our lab were purchased from either Sigma–Aldrich or BD Pharmingen. 2.2. Bone marrow derived Dendritic cells (BMDCs) culture BMDCs were generated as previously described [15,16]. The overall steps were as followed: (1) The femurs and tibiae of mice (4–6 weeks old raised in pathogen free animal house, China Medical University) were removed and isolated from surrounding muscle tissue. Intact bones were left for disinfection in 70% ethanol for 1 min, washed twice in PBS, and transferred into a fresh dish with RPMI 1640. Cut off the both metaphysic to expose marrow cavity then blow out the cells in the cavity by RPMI 1640 with 1 ml syringe. Clusters within the marrow suspension were disintegrated by vigorous pipetting. Lyses buffer was used to eliminate red blood cells. (2) 106 cells were transferred into 24-well plates (Corning incorporated Costar) with RPMI1640 (1 ml) and supplements including recombinant murine granulocyte macrophage colony stimulating factor (GM-CSF) (10 ng/ml), IL-4 (10 ng/ml), L-glutamine (2 mM), fetal bovine serum (FBS) (10%), streptomycin (100 g/ml) and penicillin (100 units/ml) in a humidified atmosphere of 5% CO2 at 37 ◦ C; (3) After incubation for 4 h, the medium with non-adherent cells was replaced with a fresh medium, while the nutrient medium was replaced every three days; (4) On the day 6, MACS (Miltenyi Biotec, CA) was used under the instruction of the manufacturer to isolate cells with CD11c expression, which were later seeded into new wells with fresh medium; (5) The sorted cells were treated with or without RGP (0.2 mg/ml) for 24 h; (6) Flow cytometry (FCM) analysis was conducted to test the cell purity of separated BMDCs. 2.3. Effects of a range of RGP doses on BMDCs The BMDCs were cultured in 96-well micro plate and added varied concentrations of RGP, ranging from 3.2 mg/ml to
0.78 × 10−3 mg/ml to the wells, triplicate/conc. Then we conducted MTT analysis to assess the proliferation of BMDCs under different RGP concentrations to determine the optimal concentration. Adding MTT to the culture, then removed the supernatant and dissolved the precipitation with DMSO after 4 h. Finally the optical density was measured on each checking point at OD570 .
2.4. Morphology of BMDCs with inverted phase contrast microscope and SEM The cultured BMDCs were incubated with LPS and 0.2 mg/ml RGP, respectively, for 24 h before further morphology study under inverted phase contrast microscope. The cell samples for SEM were prepared per steps including glutaraldehyde fixation, cleaning, osmium tetroxide post-fixation, cleaning, replacement, critical point drying, and gold spraying, etc. Finally samples were checked with SEM (JEOL JSM-T300).
2.5. Analysis by flow cytometry The cultured cells from both testing group and control group were collected and stained with anti-CD40, anti-CD80, anti-CD83, anti-CD86 and anti-MHC II antibodies for 30 min, and then washed with PBS/2% FACS twice. The prepared cells were fixed in 4% paraformaldehyde and later collected using FACS Calibur (Becton Dickinson, San Diego, CA) for confirmation of surface molecules.
2.6. Analysis of pinocytotic vesicles within the BMDCs by TEM The cultured BMDCs treated with 0.2 mg/ml RGP for 24 h were digested with trypsin. Following steps included centrifugation at 1000 rpm for 10 min, glutaraldehyde fixation and slicing. The ready samples were checked for their intracellular pinocytotic vesicles with TEM (JEOL JEM-1200EX).
2.7. Pinocytosis assessment by BMDCs using peroxidase BMDCs with a concentration of 1 × 105 /ml were treated with LPS and 0.2 mg/ml RGP for 24 h. The above cells were processed with 0.08 mg/ml horseradish peroxidase, followed by fixed with methanol for 4 hr and staining with DAB kit. Finally, microscope was conducted to exam the prepared samples.
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Fig. 2. The proliferation of BMDCs under a range of RGP doses (A) and at different time point under the optimal concentration (B). Results represent the mean ± sd of six samples. ** p < 0.01 or * p < 0.05 vs. that in RPMI 1640.
2.8. Assessment of phagocytosis
2.11. Statistical analysis
The phagocytosis activity of BMDCs was measured as cellular uptake of FITC-Dextran and was quantified by flow cytometry. The cultured BMDCs were tested after treatment with 0.2 mg/ml RGP for 24 h. Three steps were involved in the samples preparation: (1) Adding 100 l FITC-Dextran (40,000 Da) to the media; (2) Incubating at 4 ◦ C for 2 h; (3) Incubating at 37 ◦ C for 1 h. Finally, the samples were analyzed with FACS Calibur (Becton Dickinson, San Diego, CA) to assess the phagocytic activity.
The data of FCM were processed with WinMDI2.9 (Joseph Trotter, BD Biosciences). Data for all groups were evaluated with the statistical program SPSS (Statistical Package for Social Sciences, Version 16.0) for Windows and all variables were expressed as mean ± sd. ANOVA and the student t-test using Prism (Graph Pad Software) were adopted to assess the differences. When p < 0.05, significance was indicated. 3. Results
2.9. Acid phosphatase (ACP) activity detection 3.1. MACS study Firstly the BMDCs were treated with LPS and 0.2 mg/ml RGP for 24 h and adjusted the cell concentration to 1 × 106 /ml, and then the ACP activity was assessed with ACP testing kit (Jiancheng Bio-engineering Institute of the South) by phenol-4-AAP (amino antipyrine) method and measured at OD absorbance on 520 nm (A520). 2.10. Cytokine assay by ELISA The BMDCs treated with LPS and 0.2 mg/ml RGP for 24 h, and then collected supernatant for IL-12p70 and TNF-␣ assay per the instructions in the ELISA kit. The OD absorbance at 450 nm (A450) was determined with bichromatic microplate reader (BIO-TEK, USA).
The cells with CD11c+ were isolated by MACS. FCM analysis was conducted to test the cell purity of separated BMDCs as shown in Fig. 1. 3.2. Effects of a range of RGP doses on BMDCs The BMDCs proliferated into differential numbers when treated with various concentrations of RGP. A bilateral relationship between RGP concentration from 3.2 mg/ml to 0.78 × 10−3 mg/ml and cell proliferation could be seen. It was obvious that cell proliferation was inhibited at lower and higher concentrations, and the optimal concentration appeared to be 0.2 mg/ml (Fig. 2A). Under the optimal concentration, the cell proliferation at different time
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Fig. 3. The morphology of BMDCs before and after treatment with RGP under a light microscope (A) (×200) and SEM (B) (×5000).
points was also assessed by MTT, and the optimal time for cell proliferation was found to be 24 h after treatment with RGP (Fig. 2B). 3.3. Morphology of BMDCs treated with RGP The morphology of cultured BMDCs treated with GM-CSF and IL4 for 6 d was observed with an inverted phase contrast microscope (Fig. 3A). Large cells with oval or irregular nuclei and many small dendrites could be seen, indicating maturity. Simultaneously, The SEM photos of the BMDCs were compared before and after treatment with RGP. The treated ones showed more protrusions and greater number of cascading folds (Fig. 3B). 3.4. FCM analysis The BMDCs were cultured with GM-CSF and IL-4 for 6 d, and then FCM analysis was conducted on the BMDCs treated with 0.2 mg/ml RGP for 24 h. The results showed increased expression of CD40, CD80, CD83, CD86 and MHC-II which are important surface markers for mature DCs as reflected in Fig. 4.
3.6. Cytochemistry analysis of BMDCs pinocytosis Pinocytosis of BMDCs could be observed under light microscope after staining with DAB kit. Pinocytosis of horseradish peroxidase by BMDCs was shown in the photos. The less pinocytosis could be found in RGP group, indicating a weakened capability in antigen arresting while with a strengthened ability in antigen presenting (Fig. 6).
3.7. Analysis for down-regulation of the phagocytosis of BMDCs by RGP with FCM The phagocytosis reflects the digestion of antigen by immature DCs [17]. FITC-dextran is generally used to examine the intrinsic phagocytosis capacity. The uptaking of FITC-dextran is known to be maximal in the immature BMDCs and occurs by a combination of macropinocytosis and binding to the mannose receptor [18]. The results revealed that the phagocytosis of BMDCs had undergone a notable decrease after treatment with RGP (Fig. 7).
3.5. TEM on pinocytotic vesicles of BMDCs Immature BMDCs are normally associated with strong phagocytic ability and more pinocytotic vesicles while mature BMDCs are opposite, however, with increased antigen presenting ability. The TEM photo of the cells after treatment with RGP revealed a sharp decrease in the number of pinocytotic vesicles compared with that of BMDCs in RPMI 1640 group (Fig. 5).
3.8. Acid phosphatase activity detection ACP, as a major enzyme in the lysosome to digest antigen, is related to the DC maturity in an inverse manner. As can be seen from (Fig. 8), ACP activity in BMDCs was greatly decreased after treatment with RGP.
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Fig. 4. RGP up regulated the key surface molecules on BMDCs. The expressions of CD40, CD80, CD83, CD86 and MHC II were analyzed by FCM and displayed, respectively, by the single parameter diagram. The mean fluorescence intensity (MFI) and the gated % values are shown as the mean ±sd of six independent samples.
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Fig. 5. The changes of pinocytotic vesicles inside the BMDCs before and after treatment with RGP under TEM. The number of pinocytotic vesicles inside BMDCs which treating with RGP for 24 h had reduced significantly (arrows).
Fig. 6. Cytochemistry assay of BMDCs before and after treatment with RGP. The capability of BMDCs for pinocytosis stimulated with 0.2 mg/ml RGP decreased obviously compared with those in the RPMI 1640 group.
3.9. RGP increases production of IL-12p70 and TNF-˛ by BMDCs Usually matured BMDCs secreted higher level of IL-12p70 and TNF-␣. IL-12p70 and TNF-␣ were measured by ELISA of the BMDCs after treatment with 0.2 mg/ml RGP for 24 h. The results showed
up-regulation of production of IL-12p70 and TNF-␣ in mature cells (Fig. 9). 4. Discussion Recorded in Chinese medical classical Shennong’s Herba, RGP is considered as one of the top grade herbs in China. RGP enhanced
Fig. 7. The affect on the phagocytosis of BMDCs by RGP. The BMDCs stimulated with 0.2 mg/ml RGP for 24 h were incubated with FITC-dextran for check by FCM. The average percentages from six independent experiments showed that mature BMDCs reduced markedly in phagocytosis. ** p < 0.01 vs. that in RPMI 1640.
Fig. 8. ACP activity of BMDCs after treatment with RGP. ACP activity was determined after treatment with RGP for 24 h by the phenol-4-AAP method in conjunction with ACP testing kit. Results represent the mean ± sd of six samples. ** p < 0.01 or * p < 0.05 vs. that in RPMI 1640.
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Fig. 9. Effects of the 0.2 mg/ml RGP on the production of IL-12p70 (A), and TNF-␣ (B). After treatment with RGP the supernatant from cell cultures was collected and the secreted cytokines were detected by ELISA. Similar results were obtained in three independent experiments and a representative graph was shown. Results represent the mean ± sd of six samples. ** p < 0.01 or * p < 0.05 vs. that in RPMI 1640.
the proliferation of T, B lymphocytes in spleen by aged mice and phagocytosis of peritoneal macrophage [19]. It also suppressed phosphorylation of mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-B [20,21]. Moreover, the expression levels of adhesion molecules such as intercellular adhesion molecule1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1) were suppressed by treatment with RGP [3]. It also lowered plasma Creactive protein levels in diabetic rats [22].These results reveal that RGP has anti-inflammatory and enhance immune system activity. Our results showed that RGP fully induced the maturation of BMDCs by up-regulating phenotype and functions with the fact: (1) The morphology of BMDCs had more protrusions, rougher surface accompanying with up-regulated the expression of CD40, CD80, CD83, CD86, and MHC II. (2) RGP induced functional maturation of BMDCs, as characterized by decreasing phagocytosis ability and ACP activity which usually indicates the degree of maturation of BMDCs. (3) RGP also enhanced the production of IL-12 and TNF-␣ of BMDCs as they became more matured, resulting in initiation of T cell response [23]. The maturation of BMDCs depends on the maturation signal as well as the constraints imposed by ontogeny and environmental modifiers. Signal 1 is provided by the T cell receptor-recognizing antigen peptides bound to MHC molecules. Signal 1 alone inactivates T cells, causing T cell deletion or inducing regulatory T cell generation. Signal 2 is a co-stimulatory signal. When combined with Signal 1, it will induce T cell responses. The T cells expand clonal and differentiate into effector or memory cells. Furthermore, by binding CD80 or CD86 on DCs to its receptor, Signal 2 promotes antigen-specific immunity strongly [24]. By binding CD80 or CD86 on DCs to cytotoxic T-lymphocyte antigen 4 (CTLA4) on regulatory T cells, Signal 2 can also induce tolerogenic signals. Signal 3,
which is also delivered by DCs, can determine the differentiation of Th1 cells, Th2 cells, and cytotoxic lymphocyte (CTLs). One Signal 3 molecule is IL-12, which can induce Th1 cells and CTLs differentiation. The Notch ligand promotes the development of Th2 cells as Signal 3. It is helpful in inducing efficient anti-tumor immunity by controlling the interactions between DCs and T cells on multiple levels [25,26]. As the immunosuppressive conditions in cancer patients have been well-characterized, the clinical significance of DCs in cellular therapy cannot be overlooked [27]. The mild adjuvants are necessary for the maturation of DCs. Most of the adjuvants for current experiment use can provide signals which the innate immune system vigorously reacts to, resulting in the potentiating of the immune system so that the Ag co-administrated with the adjuvant can be uptaked, processed and presented in a more effective manner by the activated DCs [28,29]. This work can therefore contribute to a better understanding of RGP’s modulating effects on immune system, and a better understanding of the complicated mechanisms at molecular level, through which purified herbal extract is working [30]. Overall, the data in this study indicates that RGP induces the maturation of BMDCs in both phenotypic and functional aspects. RGP is a good supplement for people with poor immunity, such as those with cancer, chronic disease, and aging. The improvement of immunity in these cases can be achieved by simply oral administration [31]. Furthermore, this work also provides a meaningful mode of action for RGP and highlights the clinical significance of RGP as an immune enhancer which may play a important role in combating cancer [32,33]. Finally, although RGP has ever been used as an adjuvant in stimulation of tetanus toxoid-specific immune responses and identification of a novel vaccine adjuvant that stimulates and maintains diphtheria toxoid immunity as well as an agent in therapy of several cancers like esophageal cancer clinically, we may think of RGP as a possible adjuvant in DC-based vaccine preparations in future clinical practice [34,35]. Conflict of interest The authors have no financial conflicts of interest with any party. Acknowledgment This work was supported financially by China Liaoning Provincial Foundation for Education No. L2010576 (to Fengping SHAN). References [1] B.C. Lee, J.B. Choi, H.J. Cho, Y.S. Kim, Journal of Ethnopharmacology 122 (2009) 131–135. [2] H. Kim, E. Lee, S. Lee, T. Shin, Y. Kim, J. Kim, International Journal of Immunopharmacology 20 (1998) 231–240. [3] Y.Y. Sung, T. Yoon, J.Y. Jang, S.J. Park, H.K. Kim, Journal of Ethnopharmacology 134 (2011) 37–44. [4] K.O. Oh, S.W. Kim, J.Y. Kim, S.Y. Ko, H.M. Kim, J.H. Baek, H.M. Ryoo, J.K. Kim, Clinica Chimica Acta 334 (2003) 185–195. [5] P.S. Wu, S.J. Wu, Y.H. Tsai, Y.H. Lin, J.C.J. Chao, American Journal of Chinese Medicine 39 (2011) 1173–1191. [6] X.L. Wei, X.B. Ru, Zhongguo Yao Li Xue Bao 18 (1997) 471–474. [7] W. Tan, K.Q. Yu, Y.Y. Liu, M.Z. Ouyang, M.H. Yan, International Journal of Biological Macromolecules 50 (2012) 59–62. [8] D. Zhang, X.S. Wen, X.Y. Wang, M. Shi, Y. Zhao, Journal of Ethnopharmacology 123 (2009) 55–60. [9] R. Zhang, J. Zhou, Z. Jia, Y. Zhang, G. Gu, Journal of Ethnopharmacology 90 (2004) 39–43. [10] H.H. Yu, S.J. Seo, Y.H. Kim, H.Y. Lee, R.K. Park, H.S. So, S.ll Jang, Y.O. You, Journal of Ethnopharmacology 107 (2006) 383–388. [11] X. Zhang, A. Zhang, B. Jiang, Y. Bao, J. Wang, L. An, Phytomedicine 15 (2008) 484–490. [12] M. Tmoda, H. Miyamoto, N. Shimizu, Chemical and Pharmaceutical Bulletin 42 (1994) 1666–1668.
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