International Immunology Advance Access published online on July 2, 2007
International Immunology, doi:10.1093/intimm/dxm061
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Response of human dendritic cells to different immunomodulatory polysaccharides derived from mushroom and barley
1 Department of Paediatrics and Adolescent Medicine, Hong Kong Jockey Club Clinical Research Center, Faculty of Medicine, University of Hong Kong, Queen Mary Hospital, 102 Pokfulam Road, Hong Kong, China
2 Department of Pharmacology, Peking University Health Science Center, School of Basic Medical Sciences, Beijing, China
Correspondence to: Correspondence to: G. C. Chan; E-mail: gcfchan{at}hkucc.hku.hk
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Abstract |
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Polysaccharides derived from fungi and plants have been increasingly used as dietary supplement with therapeutic intention for cancer. However, whether these polysaccharides from different sources and structures can elicit similar immunological effects remain unknown. This study aims to investigate and compare the effects of selected groups of purified and crude polysaccharides on human dendritic cells (DCs), the most potent antigen-presenting cells. The selected polysaccharides were from Ganoderma lucidum [(GL) Lingzhi, Reishi], a medicinal mushroom commonly used by oriental; and barley glucan, a purified polysaccharide with known in vivo immunomodulating effect. We found that purified polysaccharides from GL mycelium could induce human PBMC proliferation and phenotypic and functional maturation of DCs with significant IL-12 and IL-10 production. Polysaccharides of GL spore and barley were both rather weak immunostimulator in vitro. In general, all these polysaccharides did not polarize T cells into either Th1 or Th2 or regulatory T cells, except for crude spore polysaccharides-treated DCs which could suppress T cell proliferation with IL-10 production. This study revealed the polysaccharides of different sources have different immune potency and effect on human immune cells including DCs. Our study also provides a reproducible biological platform for comparing the potential therapeutic effects of different herbal-derived polysaccharides in the future.
Keywords: barley, Ganoderma lucidum, human dendritic cell, immunomodulation, polysaccharides
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Introduction |
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Polysaccharides extracted from mushroom have been advocated for their potential anti-cancer effect for the past two decades (1–4). Previous studies suggested that they exert their anti-cancer effect indirectly via immunomodulation rather than having direct cytotoxic activity. Ganoderma lucidum (GL) (also known as Lingzhi in Chinese or Reishi in Japanese) is a kind of Basidiomycetes mushroom and has been widely used and studied for its anti-cancer potential (5). The active components of the GL differ with its forms. For example, an immunopotent polysaccharide (1
3), (1 6)-ß-glucan is mainly found in the mycelium of GL-fruiting body whereas triterpenes are the main components of the GL spores (3, 6). Aware of the differences in their compositions (7), it could be hypothesized that polysaccharides from either GL mycelium or spore might induce a different immune response or with different immune potency.
Recent studies have demonstrated the structure–activity relationship of polysaccharides to our immune system especially in the form of ß-glucan. ß-Glucan is a carbohydrate polymer with chain of glucose molecules linked together by ß-glycosidic linkages (8). It is one of the main components of the cell wall in most fungi and plants. It has diverse structural variability including molecular weight, linkage pattern, degree of branching, triple helical conformation and water solubility (1, 3, 4, 8). All these are reported to be crucial determinants for the immunomodulating function of ß-glucans. In terms of immune potency for anti-cancer purpose, mushroom polysaccharides were proposed to be more effective due to its (1 3)-ß-glucan units with certain degree of branching. For instance, lentinan from Lentinula edodes, which has been approved for anti-cancer in Japan, is a linear (1 3)-ß core backbone with (1 6)-linked branches in 1:10 ratio, forming a comb-like structure (1, 3). In contrast, polysaccharide from barley is a linear (1 3), (1 4)-ß-glucan and believed to be less potent than (1 3), (1 6)-ß-glucans. However, recent reports showed that orally administered barley (1 3), (1 4)-ß-glucan can synergize with anti-tumor mAbs such as anti-CD20, anti-HER2/neu and anti-EGFR1 via the activation of complement activity (9, 10). Barley glucan taken orally was also found to enhance the anti-tumor effect of anti-GD2 ganglioside (3F8) mAbs in children with neuroblastoma (11). Due to the differences in the study models, how these polysaccharides affect the human immune cells and their relative potency still remains largely unknown.
Dendritic cells (DCs) are professional antigen-presenting cells which form a link between innate and adaptive immunity (12, 13). They can guide the naive T cells into either immunogenic or immune-tolerant direction when exposed to a particular antigen. DCs have been studied on their use as an adjuvant in tumor vaccine for cancer immunotherapy (14). However, monocytes-derived DCs from cancer patients were found to be defective in terms of phenotypic and functional maturation clinically (15, 16). How one can enhance or manipulate the intrinsic DCs for treatment purpose is currently under intense investigations (14, 17). One of the approaches is to identify substances that can induce DCs to differentiate when exposed to targeted antigens. In clinical study, use of tumor necrosis factor-
(TNF-), IL-6, IL-1ß and prostaglandin E2 can induce DC differentiation ex vivo and this will subsequently generate both CD8+ and CD4+ tumor-specific T cell responses in melanoma patients (18). When stimulated by inflammatory mediators or microbial pathogens, DCs become mature during their migration to the lymphoid tissues. The surface and co-stimulatory molecules, in particular CD40, CD80, CD86 and MHC class II will be up-regulated. All these molecules are important for priming the T cells and assist in mounting an effective anti-tumor immune response. However, the immunological role of DCs can be multidirectional since recent findings have shown that there are different subsets of regulatory DCs (19). The mature DCs might induce either immunogenic Th or immunotolerogenic regulatory T cells (Tregs). Thus, the successfulness of recent clinical trials using DCs as an anti-cancer therapy will depend on whether Th or Treg cells are induced by the targeted antigens.
DCs percept and respond to extracellular signals including pathogens and pathogen-associated molecular patterns (PAMPs) through various receptors. Toll-like receptors (TLRs) play an important role in the innate recognition of PAMPs and initiation of immune responses (20). The specific recognitions of different PAMPs are achieved by up to now 10 mammalian TLRs 1–10, for example, TLR2 recognizes gram-positive bacterial lipoteichoic acid, peptidoglycan and fungal-derived zymosan. Recently, dectin-1 was found to be the major receptor responsible for the recognition of ß-glucan and subsequent TNF- production in macrophages (21–23). It is a type II transmembrane C-type lectin receptor (CLR) with a single extracellular carbohydrate recognition domain and an immunoreceptor tyrosine-based activation motif in the cytoplasmic domain. It is predominately expressed on the surface of macrophages, neutrophils and DCs. Dectin-1, like other CLRs, plays a regulatory role on the immune responses such as phagocytosis and cytokine productions including IL-2 and IL-10. Activations of TLRs or dectin-1 drive the maturation of DCs and efforts have been put on finding natural ligands mimicking PAMPs for therapeutic uses.
Some recent data have shown that GL mycelium-derived polysaccharides can induce DC differentiation (24, 25). However, there is still no consensus regarding the relative immunological potency and effects of polysaccharides derived from different sources on human DCs. We thus compared the potency of both crude and purified GL and barley polysaccharides (MSK-BG) on human immune cells including PBMCs and DCs. Our findings would provide more insights on how they can affect the immune responses and guide us on the use of immunopotent polysaccharides in the DC-based tumor vaccine in future.
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Methods |
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Sources and preparation of polysaccharides
Crude mycelium polysaccharides (GL-M) and Crude spore polysaccharides (GL-S) were extracted from commercially available GL products (YorkBest International, Hong Kong) as previously described (26). Briefly, GL-M was prepared by boiling 1 g of mycelium powder in 10 ml filtered sterile water for 1.5 h. Supernatant was saved and the pellet was re-boiled with 10 ml water for another 1.5 h. GL-S was obtained by extracting 1 g of spore powder in 10 ml absolute ethanol for 3 days. The pellet after centrifugation was also re-extracted with same volume of absolute ethanol for another 3 days. The physical, chemical and pharmacological properties were tested by the Department of Pharmacology, The University of Hong Kong and the products fingerprinting pattern was performed by the Department of Chemistry, The University of Hong Kong. The ratio of polysaccharides to peptides in GL-M was 16.8:20.1% while that in GL-S was 7.3: 5.2%.
Pure mycelium polysaccharides (GL-PS) and pure spore polysaccharides (GL-SG) were prepared and analyzed as previously described (27). Briefly, GL-PS was extracted by hot water from the fruiting body of GL. The yield of GL-PS was 0.82% (w/w) in terms of the fruiting body of GL. It is a polysaccharide peptide with molecular weight of 584 900 and 17 amino acids. The ratio of polysaccharides to peptides is 93.51:6.49%. The polysaccharides consist of glucose, galactose, arabinose, xylose and mannose with molar ratios of 0.793:0.964:2.944:0.167:0.384:7.94 and linked by ß-glycosidic linkages. It is hazel and water-soluble powder. The powders were dissolved in RPMI 1640 (Gibco, Invitrogens) and filtered (0.22 µm filter) before used.
MSK-BG, a pure (1 3), (1 4)-ß-glucan, was kindly provided by Cheung V. N. K. (Department of Paediatrics, Memorial Sloan-Kettering Cancer Center, NY, USA). It is a linear (1 3), (1 4)-ß-glucan and was easily dissolved by boiling for 10 min in normal saline. Endotoxin levels in GL polysaccharides were measured by using Pyrochrome chromogenic limulus amoebocyte lysate (LAL) assay kit (Associates of Cape Cod, Inc., East Falmouth, MA, USA) according to the manufacturer's instructions. GlucashieldTM ß-glucan inhibiting buffer (Associates of Cape Cod, Inc.) was used to exclude the glucan interference to the LAL reagent. The endotoxin levels of GL-M and GL-S were <0.1 EU µg–1 of GL polysaccharides [equivalent to <1 pg ml–1 LPS (Escherichia coli derived)], which was used as positive control throughout the study.
Cell isolation
Mononuclear cells were isolated from buffy coat of healthy adult donors (Red Cross, Hong Kong SAR, China) by Ficoll-Paque Plus density gradient (Amersham Biosciences, Uppsala, Sweden). Monocytes were then isolated from PBMCs by positive selection using anti-CD14-conjugated magnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). CD3+ T cells were isolated with the same method except using anti-CD3-conjugated magnetic microbeads (Miltenyi Biotec). Isolated cells were cultured at a density of 1 x 106 cells per milliliter in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 50 IU ml–1 penicillin and 50 IU ml–1 streptomycin (Life Technologies, Grand Island, NY, USA). The purity of isolated monocytes was consistently >85% while that of T cells was consistently >98% as determined by flow cytometry.
Cell proliferation assay
The effects of polysaccharides on cell proliferation were measured using the Cell Proliferation Kit II XTT assay (Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturer’s instructions. XTT [2,3-Bis(2-methoxy-4-nitro-5- sulfophenyl)-2H-tetrazolium-5-carboxanilide] assay is based on the fact that live cells reduce tetrazolium salts into colored soluble formazan compounds. Briefly, 1 x 105 PBMCs were grown in flat-bottom 96-well plates in a final volume of 100 µl culture medium per well for 24 h. Cells were then exposed to crude and purified polysaccharides at different concentrations (1 µg ml–1 to 1 mg ml–1) for 24, 48 and 72 h. After the fixed time of incubation, 50 µl of the XTT-labeling mixture was added to each well and incubated for 8 h at 37°C in a humidified atmosphere with 5% CO2. The formation of formazan dyes in XTT-labeling mixture by metabolically active cells was detected spectrophotometrically at 490 nm. To minimize the inter-experimental variation, cell proliferation measured as optical density was expressed as relative cell proliferation percentage compared with the negative control. We used LPS as our positive control in each of our cell proliferation assay on PBMCs.
Generation of immature DCs in vitro
Monocytes at the density of 1 x 106 per well were cultured in the presence of granulocyte macrophage colony-stimulating factor (40 ng ml–1; Novartis Pharma A6, Basel, Switzerland) and IL-4 (40 ng ml–1; R&D Systems, Inc., Minneapolis, MN, USA) at 37°C under 5% CO2. On day 3, 90% of the medium was replaced with fresh medium and cytokines. Polysaccharides were added on day 5 and remained in the medium for the last 2 days of the culture. DCs were then harvested on day 7 and washed for further assays. Based on flow cytometry analysis, the immature DCs on day 5 were 98.3% CD11c+CD1a+ and 99.8% lineage negative (CD3–CD14–CD16–CD19–CD20–CD56–).
Flow cytometry analysis of DCs and T cells
On day 7, DCs were harvested, washed and labeled with fluorochrome-conjugated antibodies. After labeling, the cell suspension was washed and re-suspended in 300 µl of 1% PFA for flow cytometry. FITC, PE and PE–cyanin 5.1 (PC5)-conjugated isotype controls and CD14–PE, CD40–FITC, CD80–FITC, CD86–FITC, CD11c–PE and HLA-DR–PC5 antibodies were purchased from PharMingen (San Diego, CA, USA). After co-cultured with DCs, T cells were stained by CD4–PE (Beckman Coulter, Inc., Fullerton, CA, USA) and CD25–FITC antibodies (PharMingen). Flow cytometric analysis was performed with a Coulter Epics Flow Cytometer (Coulter Corporation, Miami, FL, USA) and analyzed with WINMDI version 2.8 flow cytometry analysis software (Purdue University, West Lafayette, IN, USA). The DCs were gated to the standard forward-scatter and side-scatter profiles for large cells. To compare the effect of GL on the surface antigen expression level, the mean fluorescence intensities for different CD markers were normalized with that of RPMI-treated negative control as relative fluorescence intensity.
FITC–dextran endocytosis assay
Polysaccharide-treated DCs were harvested and re-suspended in RPMI with 10% FBS. FITC–dextran (molecular weight 40 kDa; Sigma, St Louis, USA) was added at a final concentration of 1 mg ml–1. Cells were then incubated at 37 or 4°C for 1 h. Thereafter, the cells were washed four times with cold PBS and then analyzed with flow cytometer.
ELISA assay for cytokines
The supernatants from DCs cultures and DC:T cell co-cultures were collected after harvesting the cells and stored at –80°C until assayed for cytokines. The levels of IL-12p70, IL-10 and tumor growth factor-ß1 (TGF-ß1) were then measured in duplicate with human Duoset® ELISA Kit (R&D Systems, Inc.). The detection ranges for IL-12, IL-10 and TGF-ß1 were 31.25–2000, 62.5–4000 and 31.25–2000 pg ml–1, respectively.
Allogeneic mixed lymphocyte reaction
All polysaccharide-treated DCs were irradiated with a gamma-irradiator (Gammacell 1000 Elite, MDS Nordion Inc., Canada) at 30 Gy and added in graded doses to 1 x 105 allogeneic responder CD3+ T cells in flat-bottom 96-well microtiter plates. Bromodeoxyuridine (BrdU) was added into the wells 16 h before the end of 5-day culture. Cell proliferation during the last 16 h of the 5-day culture was then quantified by the Cell Proliferation ELISA, BrdU (colorimetric) kit (Roche Molecular Biochemicals).
Polarization of T cells by polysaccharide-treated DCs
On day 7, polysaccharide-treated DCs were harvested, irradiated at 30 Gy and co-cultured with CD3+ T cells at a ratio of 1:100 (DC to T cell). Seven days later, the cultures were re-stimulated with phorbol myristate acetate (1 µg ml–1; Sigma) and ionomycin (0.5 µg ml–1; Sigma) in the presence of Brefeldin A (0.05 µg ml–1; Sigma) for blocking Golgi activity during the last 5 h. Intracellular cytokine staining was performed according to the manufacturer’s instruction. Briefly, cells were fixed, permeabilized and washed with Cytofix/CytopermTM and Perm/WashTM solutions (BD PharMingen). Then, cells were stained with IFN-
–FITC and IL-4–PE antibodies (BD PharMingen). Cells were analyzed using flow cytometer.
Statistical analysis
Comparisons between treatments and control were made using non-parametric Student’s t-test (two tailed). The difference was statistically significant when P < 0.05.
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Results |
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Stimulation of human PBMC proliferation by polysaccharides
To study the general effect of GL and barley on immune cells, we compared their effect on human PBMCs in terms of cell proliferation. In Fig. 1(A and B), we found that both GL-M and GL-PS could induce cell proliferation in a dose- and time-dependent manner. GL-PS was more potent than GL-M as 10-fold less GL-PS (1 versus 10 µg ml–1) was needed to induce similar effect as GL-M. As previously done (26), GL-S induced similar growth pattern as the extraction vehicle control (Fig. 1C and D). A cytotoxic effect was observed when using a high dose of 1 mg ml–1. Both GL-SG and MSK-BG did not significantly stimulate PBMC proliferation though modest increase was noted in some independent experiments (Fig. 1D and E). When compared with the effect of our positive control LPS on PBMCs, we found that both the increases in cell proliferation from GL-M and GL-PS in all 3 days were comparable to those from 10 µg ml–1 LPS (Fig. 1G).
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Phenotypic maturation of polysaccharide-treated DCs
We then studied the effect of different polysaccharides on DC maturation. LPS was used as positive control. From the trypan blue exclusion assay, there was no significant difference in the cell survival between the polysaccharide-treated and negative control medium-treated DCs (data not shown). As shown in Fig. 2, GL mycelium, either in the form of GL-M orGL-PS, up-regulated the surface and co-stimulatory molecules HLA-DR, CD40, CD80 and CD86. The maturity of cells was comparable to that induced by LPS (positive control). GL-S-, GL-SG- and MSK-BG-treated DCs remained immature without significant increase in maturation marker expression.
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Endocytosis of polysaccharide-treated DCs
To evaluate DC maturation other than using phenotype switch, the functional assessment for treated DCs is important (12, 19). In terms of endocytosis, it is known that immature DCs can engulf antigen but upon maturation, DCs subsequently lose this endocytotic and processing ability. In our studies, GL-M- and GL-PS-treated DCs had reduced endocytic activity for FITC–dextran, suggesting that they were functionally mature (Fig. 3). The GL-S-, GL-SG- and MSK-BG-treated DCs, however, retained their antigen-engulfing potential, suggesting that the DCs remained immature. To rule out the possibility of non-specific binding, parallel experiments were performed at 4°C for inhibition.
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IL-12 and IL-10 production by polysaccharide-treated DCs
As shown in Fig. 4, we could detect significant amount of both IL-12p70 and IL-10 in the culture supernatant of GL-PS-treated DCs. GL-PS was as potent as LPS (positive control) in stimulating the production of IL-12p70 and IL-10. Interestingly, the GL-M-treated DCs induced significant amount of IL-10 but not IL-12 (Fig. 4B). The IL-10 concentrations in GL-S-, GL-SG- and MSK-BG-treated DC cultures were as low as the untreated DCs (negative control). Similar pattern was observed for IL-12p70 production. Therefore, the spore and MSK-BG-treated DCs were relatively inactive with minimal cytokine production.
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Allogeneic mixed lymphocyte reactions with polysaccharide-treated DCs
One of the important functions for mature DCs is their ability to induce T cell proliferation after priming. In allogeneic mixed lymphocyte reaction, DCs were co-cultured with CD3+ T cells in the ratio of 1:100. As shown in Fig. 5, GL-PS significantly increased the T cell proliferation. Although GL-M could also induce T cell proliferation in some experiments, the changes did not reach statistical significance. Interestingly, GL-S-treated DCs suppressed T cell proliferation. The relative cell proliferation of T cells decreased significantly to 68.7 ± 14.7%. For GL-SG and MSK-BG, they did not induce any T cell response as the negative control.
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T cell polarization by polysaccharide-treated DCs
To determine whether T cells would be polarized by polysaccharide-treated DCs, we performed intracellular cytokine staining for IFN-
and IL-4. As shown in Fig. 6, all T cells showed some background IFN- and IL-4 productions. All GL-treated DCs did not induce any significant change in IFN-+ T cells (Fig. 6A). For barley-treated DCs (MSK-BG), a significant increase in IFN-+ T cell was induced as LPS. However, no significant change in terms of IL-4+ cells was observed in all treatments (Fig. 6B). We have also measured the level of IFN- and IL-4 in the supernatants with ELISA. Similarly, no significant increase in IFN- and IL-4 was detected (data not shown).
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CD4+CD25+ expression and IL-10 and TGF-ß production by T cells
CD25, the
chain of IL-2 receptor, has been used as one of the activation markers for T cells. It will be expressed when T cells are optimally stimulated by DCs or cytokines (28). Since GL-S-, GL-SG- and MSK-BG-treated DCs were phenotypically and functionally immature, we determined the activation status of co-cultured T cells by checking the CD4 and CD25 expressions. As shown in Table 1, we detected <10% of CD4+CD25+ T cells in the co-culture with GL-S-, GL-SG- and MSK-BG-treated DCs. There was no significant increase when compared with the negative control. In the culture supernatants, we examined the production of immunosuppressive cytokine IL-10 and TGF-ß1. We detected a significant amount of IL-10 in GL-S- and GL-SG-treated DC:T co-cultures (Fig. 7A), but not TGF-ß1 (Fig. 7B).
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Discussion |
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This is the first report to describe how natural immunopotent polysaccharides of different sources affect the human DC maturation and their effects on T cell subsets. We demonstrated that mushroom polysaccharides rather than MSK-BG can induce human DC maturation. Even within the same GL species, polysaccharides derived from either mycelium or spore have different potency of action on both PBMCs and DCs. Aware of the differences, the appropriate and logical use of specific maturation stimulus for specific immune purpose can be designed. Our study provides scientific evidences and rationale on using some of the natural-derived polysaccharides in various clinical settings, especially for DC-based immunotherapy in the future.
Similar observations on the effect of GL mycelium in inducing DC maturation and cytokine production have been reported recently (25). Both Lin and our team found that GL mycelium polysaccharides could induce DC maturation with IL-12 and IL-10 production. However, the two studies differ in two aspects: (i) In our study, we also included GL spore polysaccharides, spore crude extracts and MSK-BG for comparison of their immune effects on DCs. We then demonstrated how the variation in sources might affect the potency of DC maturation. (ii) By comparing the DC-based immunostimulating effects by either crude or purified polysaccharides from GL mycelium and spore, we confirmed that the purity of polysaccharides affect the immune responses. The GL-M was not as potent as the purified GL-PS in inducing immune cell proliferation and DC maturation (Figs 1 and 2) and stimulation of IL-12p70 production (Fig. 4). However, we did not detect high level of IL-10 as in the other study. The induction of high IL-12p70 but low IL-10 observed in our study may be beneficial for anti-cancer purpose. It is because increase in IL-12p70 may help to restore or enhance immunity in cancer patients; whereas decrease in IL-10, one of the key cytokines involved in the induction of immunotolerance, might help in maintaining the cancer immune surveillance.
The crude extract GL-M was extracted by repeated hot water extraction methods under Good Manufacturing Practice (GMP) guidelines. The standard GMP guidelines are set out by the FDA to ensure drug development is carried out in safe and quality processes, to avoid contamination and ensure repeatability. By using liquid chromatography–mass spectrometry and UV–vis spectroscopic techniques, we examined and found that the extracts produced consisted of other polysaccharide signals although they all passed the pharmacological tests including uniformity of weight, disintegration time, water loss on drying, solubility test, pH measurement and uniformity of polysaccharide to protein ratio. In contrast, the purified polysaccharides such as GL-PS were prepared by hot water extraction followed by the repeated ethanol precipitations, reserve dialysis and Sevag protein depletion. This purification controlled the polysaccharides to be extracted at the molecular weight of 584 900 with the yield of 35–36%. The difference in how the polysaccharides inside them were controlled could possibly explain the differential responses resulted from PBMCs and DCs.
Both crude and purified polysaccharides from GL spore (GL-S and GL-SG, respectively) were weak mitogens in terms of inducing PBMCs proliferation and DC maturation. It has been proposed that GL spores contain relatively little amount of polysaccharides so that it would be less potent than its mycelium counterpart (6, 29). In our study, we used equal amount of purified GL spore polysaccharides (GL-SG). But it was still less potent than that of the GL mycelium, on an equivalent weight to weight comparison basis. Thus, we concluded that GL spore polysaccharides were intrinsically less immunogenic in terms of their effect on human DCs. We also noted that there was a significant suppression of T cell proliferation from the GL-S-treated DC:T mixed lymphocyte reaction (Fig. 5). It was associated with a significant increase of IL-10 production from the GL-S-treated DC:T co-culture supernatant after the T cell engagement (Fig. 7B). The immature phenotype, function and cytokine profile of GL-S-treated DCs matched that of the regulatory DC phenotype that has been reported recently (19). We speculated that GL spore may be useful for the prevention of graft-versus-host in transplantation situation due to its potential immunotolerant property. This hypothesis remains to be proven.
Recent studies demonstrated that the therapeutic use of MSK-BG was effective only when it is in combination with antibodies in vivo (9). We noted that MSK-BG-treated DCs in vitro remained immature after stimulation. T cells primed with them, however, showed increase in cytokines secretion including IFN- and IL-4 (Fig. 6). These observations can help us to optimize the use of MSK-BG as an adjunct for antibody-based targeted therapy among cancer patients in the future.
Although we were not able to precisely correlate the exact polysaccharide structure with their immunomodulating activity, we postulate that 1 6 branching is important for the initial recognition by DCs and subsequent maturation. The MSK-BG that we used in this study has no effect on human DCs and was indeed a linear (1 3), (1 4)-ß-glucan. In contrast, GL polysaccharides contain branched (1 3), (1 6)-ß-glucans and are more immunogenic. The other factors leading to differential responses may be the specific recognition of polysaccharides. In previous papers (25, 30, 31), it has been shown that GL polysaccharides can activate TLR4, a key receptor for activation of innate immune response, on either murine macrophages or human DCs. And the cytokine productions from the TLR activation could change when collaboration of receptors happened (32). Based on the differences in the polysaccharides, it is likely that other receptors are involved in the signaling pathways leading to DC maturation and cytokine productions.
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Funding |
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Committee on Research and Conference Grant (#10203870, #10204396 & #10205033); Paediatric Departmental Research Grant (RFPOIT-#2003-3); Edward Sai Kim Hotung Paediatric Education & Research Fund (#20000663); Shun Tak District Min Yuen Tong Fund (STMYT-#2004-1); Pau K.W. Charitable Grant (#25003587).
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Acknowledgements |
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The authors warrant that there is no conflict of interests, including conflicts of a financial nature involved with any pharmaceutical company. We would like to thank Cheung V. N. K. for providing the barley glucan to us.
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Abbreviations |
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| BrdU, bromodeoxyuridine |
| CLR, C-type lectin receptor |
| DC, dendritic cell |
| FBS, fetal bovine serum |
| GL, Ganoderma lucidum |
| GL-M, crude mycelium polysaccharide |
| GL-PS, pure mycelium polysaccharide |
| GL-S, crude spore polysaccharide |
| GL-SG, pure spore polysaccharides |
| IFN-, interferon-gamma |
| IL, interleukin |
| LPS, lipopolysaccharides |
| MSK-BG, barley polysaccharide |
| PBMC, peripheral blood mononuclear cell |
| TGF-ß, tumor growth factor-ß |
| TH, T helper |
| TLR, toll-like receptor |
| Treg, regulatory T cell |
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Notes |
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Transmitting editor: J. Allison
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References |
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