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Originally published online as doi:10.1189/jlb.0804481 on May 13, 2005

Published online before print May 13, 2005
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(Journal of Leukocyte Biology. 2005;78:533-543.)
© 2005 by Society for Leukocyte Biology

Polysaccharide purified from Ganoderma lucidum induced activation and maturation of human monocyte-derived dendritic cells by the NF-{kappa}B and p38 mitogen-activated protein kinase pathways

Yu-Li Lin*, Yu-Chih Liang{dagger}, Shiuh-Sheng Lee{ddagger} and Bor-Luen Chiang*,1

* Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, Republic of China;
{dagger} Graduate Institute of Biomedical Technology, College of Medicine, Taipei Medical University, Taiwan, Republic of China; and
{ddagger} Department of Biochemistry, National Yang-Ming University, Taipei, Taiwan, Republic of China

1 Correspondence: Department of Pediatrics, National Taiwan University Hospital, No. 7, Chungshan South Road, Taipei, Taiwan, R.O.C. E-mail: gicmbor{at}ha.mc.ntu.edu.tw


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ganoderma lucidum, a fungus native to China, has been widely used to promote health and longevity in the Chinese. The polysaccharide component with a branched (1->6)-ß-D-glucan moiety of G. lucidum (PS-G) has been reported to exert anti-tumor activity and activation of natural killer cells. In this study, we investigated the effects of PS-G on human monocyte-derived dendritic cells (DC). Treatment of DC with PS-G resulted in the enhanced cell-surface expression of CD80, CD86, CD83, CD40, CD54, and human leukocyte antigen (HLA)-DR, as well as the enhanced production of interleukin (IL)-12p70, p40, and IL-10 and also IL-12p35, p40, and IL-10 mRNA expression, and the capacity for endocytosis was suppressed in DC. In addition, treatment of DC with PS-G resulted in enhanced T cell-stimulatory capacity and increased T cell secretion of interferon-{gamma} and IL-10. Neutralization with antibodies against Toll-like receptor (TLR)-4 inhibited the PS-G-induced production of IL-12 p40 and IL-10, suggesting a vital role for TLR-4 in signaling DC upon incubation with PS-G. Further study showed that PS-G was able to augment inhibitor of {kappa}B (I{kappa}B) kinase and nuclear factor (NF)-{kappa}B activity and also I{kappa}B{alpha} and p38 mitogen-activated protein kinase (MAPK) phosphorylation. Further, inhibition of NF-{kappa}B by helenalin and p38 MAPK by SB98059 prevented the effects of PS-G in the expression of CD80, CD86, CD83, CD40, CD54, and HLA-DR and production of IL-12p70, p40, and IL-10 in various degrees. Taken together, our data demonstrate that PS-G can effectively promote the activation and maturation of immature DC, suggesting that PS-G may possess a potential in regulating immune responses.

Key Words: PS-G • signal transduction • T cells • IL-10 • IL-12 • IFN-{gamma}


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ganoderma lucidum, a native fungus from China, has been widely used in China and other Asian countries. G. lucidum has been reported to be effective in modulating immune functions and inhibiting tumor growth and in the treatment of chronic hepatopathy, hypertension, and hyperglycemia [1 ]. The polysaccharide from G. lucidum (PS-G) is a branched (1->6)-ß-D-glucan moiety. Studies have demonstrated the antineoplastic action of G. lucidum and attributed it to the activated host immune response [2 , 3 ]. PS-G has been reported to enhance the cytotoxic activity of natural killer (NK) cells and to increase tumor necrosis factor {alpha} (TNF-{alpha}) and interferon-{gamma} (IFN-{gamma}) release from macrophages and lymphocytes, respectively [4 , 5 ]. The polysaccharide component from G. lucidum also has been reported to elicit antiapoptotic effects on neutrophils, and this action primarily depends on the activation of Akt-regulated signaling pathways [6 ].

Dendritic cells (DC) are the most professional antigen-presenting cells (APCs), whose primary function is to capture, process, and present antigens to unprimed T cells [7 ]. Immature DC reside in nonlymphoid tissues, where they can capture and process antigens. Thereafter, DC migrate to the T cell areas of lymphoid organs, where they lose the antigen-processing activity and mature to become potent immunostimulatory cells [8 ]. The induction of DC maturation is critical for the induction of antigen-specific T lymphocyte responses and may be essential for the development of human vaccines relying on T cell immunity. Fully mature DC show a high surface expression of major histocompatibility complex (MHC) class II and costimulatory molecules (CD40, CD80, and CD86) but a decreased capacity to internalize antigens [9 ]. Up-regulation of CD83, a specific marker for DC maturation, also occurs [10 ]. Various stimuli, such as proinflammatory cytokines [e.g., TNF-{alpha} and interleukin (IL)-1], CD40 ligation, bacterial products [e.g., lipopolysaccharide (LPS) and unmethylated DNA CpG motif], and contact sensitizers, can induce DC maturation in vivo and in vitro [11 , 12 ]. Several reports have already indicated that the nuclear transcription factor (NF)-{kappa}B also plays an important role in DC maturation [13 ]. Another intracellular component involved in DC maturation, the three major mitogen-activated protein kinase (MAPK) signaling pathways in mammals, including p38 MAPK, extracellular signal-regulated kinases (ERK), and c-Jun N-terminal kinases (JNK), are activated in DC on maturation induced by LPS or TNF-{alpha} [14 ].

The exact effects of PS-G on human DC are yet to be defined. In the present study, we first examined the molecular mechanisms of PS-G on the activation and maturation of human monocyte-derived DC.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Escherichia coli LPS (L8274, E. coli) and lipoteichoic acid (LTA; L2515, from Staphylococcus aureus) were purchased from Sigma Chemical Co. (St. Louis, MO). Isotopes were obtained from Amersham Corp. (Arlington Heights, IL). Neutralization antibodies (without sodium azide) against Toll-like receptor (TLR)-2 and TLR-4 were purchased from eBiosciences (San Diego, CA), and helenalin, SB203580, PD98059, and JNK inhibitor II were purchased from Calbiochem (Germany). Treatment of immature DC with these inhibitors (helenalin, SB203580, PD98059, and JNK inhibitor II) before stimulation was performed for 60 min. These inhibitors were dissolved in dimethyl sulfoxide (DMSO), where a 0.1% (v/v) concentration of DMSO was used as a negative control whenever indicated.

PS-G purification from G. lucidum
As in our previous study [2 ], fruiting bodies of G. lucidum were washed, disintegrated, and extracted with boiling water for 8–12 h. Hot water extract of G. lucidum was fractionated into a polysaccharide fraction (alcohol-insoluble) and nonpolysaccharide fraction (alcohol-soluble). The crude polysaccharide obtained was then passed through a gel-filtration Sephadex G 50 column (Pharmacia, Uppsala, Sweden) and was further purified by anion exchange chromatography with a column of diethylaminoethyl cellulose [1 ]. The PS-G was a protein-bound polysaccharide consisting of ~95% polysaccharide and 5% peptides. To rule out possible endotoxin LPS contamination of PS-G samples, we determined LPS content by the chromogenic Limulus amebocyte lysate assay. We found that there was no detectable level of endotoxin (<0.10 endotoxin units/ml) in the PS-G samples.

Human DC generation
DC were generated from peripheral blood mononuclear cells (PBMC), as described previously [15 , 16 ], with some modification. Briefly, PBMC were obtained from healthy donors by centrifugation with Ficoll-Hypaque (Pharmacia), and the light-density fraction from the 42.5–50% interface was recovered. CD14+ cells were purified by positive selection using anti-CD14+ microbeads in conjunction with the MiniMACS system by following the manufacturer’s instructions (Miltenyi Biotec, Auburn, CA). The DC14+ cells were cultured at 1 x 106 cells per 1 ml RPMI 1640 containing 10% fetal calf serum in 24-well plates (Costar, Cambridge, MA) with granulocyte macrophage-colony stimulating factor (GM-CSF; 800 U/ml) and IL-4 (500 U/ml). Fresh medium containing GM-CSF and IL-4 was added every 2–3 days. Human monocyte-derived DC were used routinely at day 6 of culture.

Determination of cytokine levels
The IL-12 p70, IL-12 p40, IL-10, and IFN-{gamma} in the culture supernatant from DC or T cell were assayed with an enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN), as per the manufacturer’s instructions.

Reverse transcriptase-polymerase chain reaction (RT-PCR)
Total RNA was isolated from DC using TRIzol reagent (Life Technologies, Gaithersburg, MD) following the manufacturer’s instructions. Total RNA was converted to cDNA with Moloney-murine leukemia virus RT (Life Technologies) at 42°C for 1 h. The amplification of IL-12 p35, IL-12 p40, and IL-10 cDNA was performed by incubating equivalents of cDNA with Super Taq DNA polymerase. The IL-12 p35 primers used were the forward primer 5'-GAGTCCCGGGAAAGTCCTGCC-3' and the reverse primer 5'-TCTGGCCTTCTGGAGCATGTT-3'. The IL-12 p40 primers used were the forward primer 5'-GGGGTGACGTGCGGAGCTGCT-3' and the reverse primer 5'-TCTTGCCCTGGACCTGAACGC-3'. The IL-10 primers used were the forward primer 5'-TTTCTCTTGGAGCTTATTAAAG-3' and the reverse primer 5'-AAGACTTTCTTTCAAATGAAGG-3' (Invitrogen, Carlsbad, CA). The cDNA sequence of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was also amplified as a control using the following primers: 5'-CTCATGACCACAGTCCATGC-3' and 5'-CCCTGTTGCTGTAGCCAAAT-3'. These primers produced a 450-base pair product. A thermal cycle of 30 s at 94°C, 30 s at 52°C, and 1 min at 72°C was used for 35 cycles for IL-12 p35 and IL-12 p40. A thermal cycle of 30 s at 94°C, 30 s at 55°C, and 1 min at 72°C was used for 35 cycles for IL-10.

Intracellular staining of IL-10
Intracellular cytokine staining was performed by using the BD Cytofix/Cytoperm kit (BD Biosciences, San Diego, CA). Briefly, 1 x 105 CD3+ T cells and DC were incubated at a ratio of 5:1 at 37°C for 2 days. After adding the transport inhibitor monesine, the culture was incubated at 37°C for 2.5 h. After washing with staining buffer, cells were labeled with CD3-fluorescein isothiocyanate (FITC) and permeabilized. Intracellular staining was performed with phycoerythrin-labeled IL-10 antibodies or isotype control.

Flow cytometric analysis
DC were harvested and washed with cold buffer [phosphate-buffered saline (PBS) containing 2% fetal calf serum (FCS) and 0.1% sodium azide]. Cells were then incubated in cold buffer. Subsequent stainings with monoclonal antibodies (mAb) or isotype-matched controls were performed for 30 min on ice. Stained cells were then washed twice and resuspended in cold buffer and analyzed with a FACSort cell analyzer (Becton Dickinson, San Jose, CA). More than 1 x 104 cells were analyzed for each sample, and the results were processed by using Cellquest software (Becton Dickinson).

FITC-labeled dextran uptake
Cultured DC were washed twice and resuspended in 1 ml RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 U/ml streptomycin, and 25 mM HEPES. The cells were then incubated with FITC-labeled dextran (0.2 mg/ml) at 4°C or 37°C for 1 h. Finally, the cells were washed thrice with cold buffer and analyzed with a FACSort cell analyzer, as described above.

Allogeneic mixed leukocyte reaction (MLR)
PBMC were obtained as described above, and CD3+ T cells were purified from PBMC using magnetic beads (Miltenyi Biotec). Theallogeneic CD3+ T cells obtained were distributed at 1 x 105 cells per well and incubated for 5 days in the presence of graded numbers of irradiated DC (3000 rad, 137Cs source). Tritiated thymidine (1 µCi/well, New England Nuclear, Boston, MA) incorporation for 6 h was determined with a liquid counter.

Neutralization experiments
Human DC were preincubated for 1 h with 20 µg/ml antibody solution of TLR-2 and TLR-4. LPS, LTA, and PS-G were then added for 15 h. The cell culture supernatants were collected and were analyzed for IL-12 p70, IL-12 p40, and IL-10 by ELISA.

Inhibitor of {kappa}B (I{kappa}B) kinase (IKK) activity assay
The kinase activity assay was performed as described by Spiecker et al. [17 ] with some modifications. Whole cell extract was lysed with Gold lysis buffer [10% glycerol, 1% Triton X-100, 1 mM sodium orthovanadate, 1 mM EGTA, 5 mM EDTA, 10 mM NaF, 1 mM sodium pyrophosphate, 20 mM Tris-HCl, pH 7.9, 100 µM ß-glycerophosphate, 137 mM NaCl, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml aprotinin, and 10 µg/ml leupeptin] for 30 min at 4°C. The cell lysate was clarified by centrifugation at 12,000 g for 10 min at 4°C. Equal amounts of total cellular protein (100 µg) were immunoprecipitated with IKK1- and IKK2-specific antibody (Santa Cruz Biotechnology, CA) and protein A/G-PLUS agarose for 12 h at 4°C. Kinase assay was carried out in 45 µl kinase buffer [40 mM Tris-NaOH, pH 7.5, 500 mM NaCl, 0.1% Nonidet P-40 (NP-40), 6 mM EDTA, 6 mM EGTA, 10 mM ß-glycerophosphate, 10 mM NaF, 10 mM p-nitrophenyl phosphate, 300 µM sodium orthovanadate, 1 mM benzamidine, 2 µM PMSF, 10 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 mM dithiothreitol (DTT)] containing 5 µM cold adenosine 5'-triphosphate (ATP), 10 µCi [{gamma}-32P] ATP (5000Ci/mmol, Amersham), and 1 µg glutathione S-transferase (GST)-I{kappa}B{alpha} fusion protein (Santa Cruz Biotechnology) as substrate and incubated for 20 min at 25°C. Each sample was mixed with 8 µl 5x Laemmli’s loading buffer to stop the reaction, heated for 10 min at 100°C, and subjected to 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The gels were dried, visualized by autoradiography, and quantified by densitometry (IS-1000, Digital Imaging System).

Western blotting
Total cellular extract was prepared using Gold lysis buffer. Total protein (50 µg) was separated on 10% SDS-polyacrylamide minigels and transferred to Immobilon polyvinylidene difluoride membrane (Millipore, Bedford, MA). The membrane was incubated overnight at 4°C with 10% bovine serum albumin in PBS to block nonspecific immunoglobulins (Igs) and then incubated with anti-I{kappa}B-P polyclonal and anti-{alpha}-tubulin mAb (Santa Cruz Biotechnology) and anti-p38-P, anti-p42/44-P, anti-p46/54-P, and anti-total p38 polyclonal antibody (Cell Signaling Technology, Beverly, MA).

Preparation of nuclear extracts and electrophoretic mobility shift assay (EMSA)
Nuclear and cytoplasmic extracts were prepared as described previously [18 ]. At the end of the culture, the cells were suspended in hypotonic buffer A (10 mM HEPES, pH 7.6, 10 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF) for 10 min on ice and vortexed for 10 s. Nuclei were pelleted by centrifugation at 12,000 g for 20 s. The supernatants containing cytosolic proteins were collected. Pellets containing nuclei were resuspended in buffer C (20 mM HEPES, pH 7.6, 25% glycerol, 0.4 M NaCl, 1 mM EDTA, 1 mM DTT, 0.5 mM PMSF) for 30 min on ice. The supernatants containing nuclear proteins were collected by centrifugation at 12,000 g for 20 min and stored at –70°C. For EMSA, each 5 µg nuclear extract was mixed with the labeled, double-stranded NF-{kappa}B oligonucleotide, 5'-AGTTGAGGGGACTTTCCCAGGC-3', and incubated at room temperature for 20 min. The incubation mixture included 1 µg poly (dI-dC) in a binding buffer (25 mM HEPES, pH 7.9, 0.5 mM EDTA, 0.5 mM DTT, 1% NP-40, 5% glycerol, and 50 mM NaCl). The DNA-protein complex was electrophoresed on 4.5% nondenaturing polyacrylamide gels in 0.5x Tris-boric acid EDTA buffer (0.0445 M Tris, 0.0445 M borate, 0.001 M EDTA). A double-stranded, mutated oligonucleotide, 5'-AGTTGAGGCGACTTTCCCAGGC-3', was used to examine the specificity of the binding of NF-{kappa}B to DNA. The specificity of binding was also examined by comparison with the unlabeled oligonucleotide.

Statistical analysis
The Student’s t-test was used to analyze the results, and a P value of less than 0.05 was considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
PS-G induces maturation of human monoctye-derived DC
LPS has been described as an inducer of DC activation and maturation [19 ]. Therefore, we use LPS as a positive control in this study. To determine whether PS-G also can modulate the development of human DC in vitro, we compared the phenocyte of human DC treated with or without PS-G for 24 h. Our data demonstrated that PS-G increased the presentation of CD80, CD86, CD83, CD40, CD54, and MHC class II molecules on the cell membrane of human DC (Fig. 1 ).



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  		Figure 1. The effect of PS-G and LPS on DC phenotype. Human DC were treated with PS-G (10 µg/ml), LPS (100 ng/ml), or medium alone for 24 h, and surface markers were analyzed by flow cytometry (dotted line, isotype control; solid line, specific mAb). The values shown in the flow cytometry profiles are the mean fluorescence intensity (MFI) indexes. HLA, Human leukocyte antigen.

 
PS-G induces IL-12 p70, IL-12 p40, and IL-10 production in human DC
To determine whether PS-G can affect the cytokine production in human DC, we compared cytokine concentrations in the supernatants of DC cultured with different doses of PS-G, which enhanced the production of IL-12 p70, IL-12 p40, and IL-10 (Fig. 2A ). When human DC were treated with 10 µg/ml PS-G for 3, 6, 18, 24, and 48 h, we found that PS-G significantly enhanced the production of IL-12 p70, IL-12 p40, and IL-10 at 18, 24, and 48 h (Fig. 2B) . It was clear that the stimulatory effect of PS-G on IL-12 p70, IL-12 p40, and IL-10 production was dose- and time-dependent in manner. To determine whether PS-G could affect IL-12 p35, IL-12 p40, and IL-10 mRNA expression, human DC were activated with PS-G at indicated periods of time and assayed for IL-12 p35, IL-12 p40, and IL-10 mRNA expression by RT-PCR. We found that significantly higher levels of IL-12 p35, IL-12 p40, and IL-10 mRNA were expressed at 6 h, 6 h, and 18 h, respectively, in human DC, especially highly expressed IL-12 p40 mRNA (Fig. 2C) . In unstimulated DC, there was no detectable IL-12 p35, IL-12 p40, and IL-10 mRNA.



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  		Figure 2. PS-G induces IL-12 p70, IL-12 p35, and IL-10 production in human monocyte-derived DC. (A) Human DC were cultured for 24 h in the presence of 100 ng/ml LPS or various concentrations of PS-G. At the end of the incubation time, the culture medium was collected for cytokine assay by ELISA. (B) Human DC were incubated with PS-G (10 µg/ml) for the indicated period of time. At the end of the incubation time, IL-12 p70, IL-12 p35, and IL-10 production was subsequently analyzed by ELISA. Each data represent the mean ± SE for three determinations. Statistical analysis concerns unstimulated versus stimulated DC. *, P < 0.05. ND, Not determined. (C) RT-PCR analysis of mRNA expression of IL-12 p35, IL-12 p40, and IL-10. DC was incubated in the presence of PS-G (10 µg/ml) for 3, 6, 18, and 24 h. This experiment was repeated three times with similar results. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Lane M, marker.

 
PS-G down-regulation of endocytotic activity of human DC
Immature DC capture and process antigens via their high endocytic capacity, and they lose their endocytic/processing activities of antigens and mature into potent immunostimulatory APCs during differentiation [7 ]. The uptake of FITC-dextran is known to be maximal in the immature monocyte-derived DC and occurs by a combination of macropinocytosis and binding to the mannose receptor. Previous studies have shown that the endocytic capacity of DC is suppressed by LPS during their maturation process. Thus, we tested whether PS-G affected the uptake of FITC-labeled dextran by human DC. In our study, we demonstrated a reduction in FITC-dextran uptake when human DC were matured with PS-G (Fig. 3 ).



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  		Figure 3. PS-G on the endocytotic capacities of human DC. At day 6, immature DC were stimulated with medium alone, LPS (100 ng/ml), or PS-G (10 µg/ml) for 24 h, and cells were then incubated with FITC-dextran for 1 h at 4°C (dotted lines) or 37°C (solid lines). This experiment was repeated three times with similar results.

 
Enhancement of T cell activation by PS-G-treated human DC
Mature DC have the capacity to induce proliferation in allogenic T cells at a much higher level than immature DC [8 ]. In human DC, we found that PS-G up-regulated cell-surface markers, increased IL-12 production, and induced the activation of NF-{kappa}B. To test whether this maturation is sufficient to promote activation of naive T cells, DC were treated with LPS or PS-G. These cells were then used to activate allogenic, naive T cells. The results presented in Figure 4A show that PS-G-treated DC enhanced T cell activation, as evidenced by the secretion of IFN-{gamma} in the culture supernatant (Fig. 4B) . The IFN-{gamma} production induced under these experimental conditions was far higher than that seen following LPS treatment of DC, especially at low DC/T cells ratios. It is interesting that we demonstrated that PS-G-treated DC enhanced T cell secretion of IL-10, except for LPS-treated DC, which were not able to enhance T cell secretion of IL-10 in the supernatant (Fig. 4C) . To investigate if IL-10 is produced by the activated T cells in this experiment, we performed intracellular cytokine staining for IL-10 on CD3+ T cells (>95% purity by flow), which were cocultured with autologous DC. Intracellular cytokine staining results showed that PS-G-treated DC enhanced the T cell production of IL-10, such that the percentage gated of double-positive CD3 and IL-10 is 6.04%. In DC alone and LPS-treated DC, the percentages gated of double-positive CD3+ and IL-10 are 1.74% and 1.51%, respectively. Therefore, intracellular IL-10 staining of T cells in the PS-G-treated DC group is significantly higher than in DC alone or in the LPS-treated DC group. In the LPS group or the PS-G-treated group, DC could not induce T cell secretion of IL-4 cytokine (data not shown).



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  		Figure 4. PS-G enhances T cells response. (A) Immature DC were stimulated with LPS (100 ng/ml) or PS-G (10 µg/ml) for 24 h. Allogeneic T cell proliferation was measured after 5 days of coculture with DC. These data are means ± SEM of triplicates and representative of three independent experiments. Supernatants were analyzed for (B) IFN-{gamma} and (C) IL-10, produced by activated T cells after 2 days of culture. cpm, Counts per minute.

 
PS-G induces IL-12 p40 and IL-10 synthesis through TLR-4
TLRs have been shown to be involved in the antifungal defense mechanism in Drosophila and the antibacterial defense in humans. To determine the involvement of these receptors in the interaction of DC with PS-G, neutralization experiments were performed. Cell-surface TLR-2 and TLR-4 receptors were blocked by neutralizing concentrations of their respective antibodes before DC were treated with LPS, LTA, or PS-G. We showed positive and negative controls for the neutralization of TLR-4 (by using LPS) and TLR-2 (by using LTA) in Figure 5A and 5B . In Figure 5C , we demonstrated that the addition of an anti-TLR-4 mAb to human DC blocked PS-G (100 ng/ml, 1 µg/ml, and 10 µg/ml)-induced IL-12 p40 production ~70%, 31%, and 47%, respectively, and IL-10 production, ~55%, 39%, and 20%, respectively, but the addition of an anti-TLR-2 mAb failed to inhibit PS-G-induced IL-12 p40 and IL-10 production.



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  		Figure 5. Neutralization with TLR-4 mAb inhibits the synthesis of IL-12 p40 and IL-10 in PS-G-treated, human DC, which were preincubated with 20 µg/ml anti-TLR-2, TLR-4, and IgG1 antibodies separately for 1 h. DC were then challenged with LPS (A), LTA (B), or PS-G (C) for 15 h. The cell culture supernatants were collected for IL-12 p40 and IL-10 analysis. Data are represented as mean ± SE. Significant difference between DC treated with antibodies and no antibodies (noAb) is indicated by P < 0.05 (*).

 
PS-G induces IKK activity and phosphorylation of I{kappa}B{alpha} in human DC
As the activation of IKK activity is necessary for I{kappa}B{alpha} phosphorylation, the effect of PS-G on IKK activity was likewise studied. Human DC were treated with PS-G (10 µg/ml) for the indicated periods of time. To measure IKK1 activity directly in human DC, IKK1 and IKK2 proteins were immunoprecipitated from cell extracts, and the kinase activity in the immunocomplex was assayed using recombinant GST-I{kappa}B{alpha} (1–317) as a substrate. Figure 6A illustrates the relative effect on IKK activity. After stimulation with PS-G, the GST-I{kappa}B{alpha} fusion protein was strongly phosphorylated at 30 min, indicating the stimulation of IKK activity in human DC.



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  		Figure 6. PS-G induced IKK activity and I{kappa}B{alpha} phosphorylation in human DC. (A) Human DC were treated with PS-G (10 µg/ml) for the indicated time periods and collected the total cell lysates for IKK activity assay. Immunoprecipitated IKK was incubated with [{gamma}-32P] ATP and GST-I{kappa}B fusion protein, as substrates performed the kinase activity assay as described in Materials and Methods, and P-GST-I{kappa}B{alpha} is shown. (B) Human monocyte-derived DC were treated with LPS (100 ng/ml) for 45 min or PS-G (10 µg/ml) for the indicated time periods. Cytosolic fractions were prepared and analyzed for the phosphorylation level of I{kappa}B by Western blotting. The lower panel shows the blot probed for {alpha}-tubulin to demonstrate equal loading of samples. This experiment was repeated three times with similar results.

 
NF-{kappa}B is one molecular family whose activation is associated with DC maturation. NF-{kappa}B normally binds to I{kappa}B{alpha}, which impedes NF-{kappa}B nuclear translocation from the cytoplasm to the nucleus. Once cells are exposed to inflammatory stimuli, including LPS and TNF-{alpha}, I{kappa}B{alpha} is phosphorylated, leading to I{kappa}B{alpha} degradation and nuclear translocation of NF-{kappa}B. We thus examined whether PS-G had any effect on I{kappa}B{alpha} phosphorylation. The cytoplasmic levels of I{kappa}B{alpha}-P protein were examined by Western blot analysis. LPS and PS-G induced the phosphorylation of I{kappa}B{alpha}. After 60 min from the activation of human DC with PS-G, the cytosolic I{kappa}B{alpha} protein was significantly phosphorylated (Fig. 6B) .

PS-G induces NF-{kappa}B activation
DC maturation derived by LPS has been clearly associated with NF-{kappa}B activation. To determine whether PS-G uses a similar activation pathway, we monitored its ability to activate NF-{kappa}B translocation into the nucleus. DC were cultured in the presence of PS-G for 2 h, and nuclear extracts were analyzed for NF-{kappa}B binding by the EMSA. As shown in Figure 7 , PS-G was able to induce NF-{kappa}B translocation and activation. Identical results were obtained after treatment of DC with LPS. The binding of NF-{kappa}B was specific and could be blocked by unlabeled, competing NF-{kappa}B oligonucleotide.



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  		Figure 7. PS-G induces NF-{kappa}B activation. Human monocyte-derived DC were treated with LPS (100 ng/ml) or PS-G (10 µg/ml) for 2 h or remained unstimulated, and nuclear fractions were prepared and analyzed for NF-{kappa}B binding activity by EMSA. To assess the specificity of the binding, 100-fold excess of cold NF-{kappa}B probe or mutant probe was added to the LPS condition. This experiment was repeated three times with similar results.

 
PS-G induces phosphorylation of members of the three MAPK families in human DC
MAPK is a serine and threonine protein kinase whose activities are up-regulated through tyrosine and threonine residue phosphorylation by its upstream regulators [20 , 21 ]. This experiment focused on p38 MAPK, p42/44 ERK, and p46/54 JNK to further characterize the MAPK activation pathways involved in PS-G signaling. Human DC were stimulated with PS-G or none at all, and the level of MAPK phosphorylations was assessed by Western blotting with respective antityrosine-phosphorylated MAPK mAb. Total p38 mAb was used for internal control. Results presented in Figure 8 show that PS-G induced the phosphorylation of all MAPK tested, especially in inducing a higher p38 phosphorylation at 30 min. The total amount of p38 was unchanged following stimulation.



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  		Figure 8. PS-G induces the phosphorylation of p38 MAPK, p42/44 ERK, and p46/52 JNK kinase. Human monocyte-derived DC were treated with PS-G (10 µg/ml) inhibitors for the indicated time periods and then collected the cell lysate, and the level of MAPK phosphorylations was assessed by Western blotting with respective antityrosine-phosphorylated MAPK mAb, and total p38 mAb was for internal control.

 
Inhibition of NF-{kappa}B and MAPK prevents the maturation changes induced by PS-G
PS-G-treated DC produced many cytokines, including IL-12 p70, IL-12 p40, and IL-10, during maturation (Fig. 2A and 2B) . We investigated whether the PS-G-induced secretions of IL-12 p70, IL-12 p40, and IL-10 were affected by the inhibitors of NF-{kappa}B, p38 MAPK, p42/44 ERK, and p46/54 JNK. Immature human DC were pretreated with helenalin (a specific blocker of NF-{kappa}B), SB203580 (a specific blocker of p38 MAPK), PD98059 (an inhibitor of the ERK pathway), or JNK inhibitor II (an inhibitor of the JNK pathway) for 1 h and subsequently stimulated with PS-G for 24 h. The production of IL-12 p70, IL-12 p40, and IL-10 was quantified by means of ELISA. PS-G induced significant production of IL-12 p70, IL-12 p40, and IL-10, and these cytokine productions were abrogated significantly by helenalin and SB203580 (Fig. 9 ). In contrast, PD98059 and JNK inhibitor II down-regulated IL-12 p70 and IL-10 production but only had little effect on the inhibition of IL-12 p40 production induced by PS-G.



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  		Figure 9. The effect of inhibiting the NF-{kappa}B, p38 MAPK, ERK1/2, or JNK pathways on the PS-G-induced up-regulation of IL-12 p70, IL-12 p40, and IL-10 production in human monocyte-derived DC. Human DC were pretreated with 0.1% DMSO, 10 µM helenalin (a specific blocker of NF-{kappa}B), 20 µM SB203580 (a specific blocker of p38 MAPK), 50 µM PD98059 (an inhibitor of the ERK1/2 pathway), or 20 µM JNK inhibitor II (an inhibitor of the JNK pathway) for 1 h and then incubated with 10 µg/ml PS-G for 24 h. At the end of the incubation time, the supernatant was collected for IL-12 p70, IL-12 p 35, and IL-10 production by ELISA. Significant difference between DC treated with PS-G alone and pretreated with inhibitors is indicated by P < 0.05 (*). ND, Not determined.

 
To further examine the involvement of NF-{kappa}B, p38 MAPK, ERK, and JNK in the PS-G-induced expression of costimulatory and antigen-presenting molecules, NF-{kappa}B inhibitor, helenalin, p38 MAPK inhibitor, SB203580, ERK pathway inhibitor, PD98059, and JNK pathway inhibitor, JNK inhibitor II, in the expression of costimulatory and adhesion molecules as well as HLA-DR, were investigated. Blocking the NF-{kappa}B pathway with helenalin significantly inhibited the PS-G and induced the up-regulation of CD80, CD86, CD83, CD40, CD54, and HLA-DR (Fig. 10 ). In contrast, blocking the p38 MAPK and JNK pathway with SB203580 and JNK inhibitor II, respectively, had little effect on CD80, CD86, CD83, CD40, CD54, and HLA-DR expression. PD98059, a specific inhibitor of ERK, had no effect on these costimulatory molecules and MHC class II expression. These results show that certain features of human monocyte-derived DC maturation are regulated by signaling via NF-{kappa}B and p38 MAPK and imply that different aspects of the maturation process induced by PS-G may be regulated by distinct signal transduction pathways.



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  		Figure 10. The effect of inhibiting the NF-{kappa}B, p38 MAPK, ERK1/2, or JNK pathways on the PS-G-induced up-regulation of CD80, CD86, CD83, CD40, CD54, and HLA-DR in human monocyte-derived DC. Day 6 immature DC were pretreated with 0.1% DMSO, 10 µM helenalin (a specific blocker of NF-{kappa}B), 20 µM SB203580 (a specific blocker of p38 MAPK), 50 µM PD98059 (an inhibitor of the ERK1/2 pathway), or 20 µM JNK inhibitor II (an inhibitor of the JNK pathway) for 1 h before the addition of PS-G (10 µg/ml) for 24 h. The cell-surface expression of CD80, CD86, CD83, CD40, CD54, and HLA-DR was then measured using the flow cytometry (dotted line, isotype control; solid line, specific mAb). The values shown in the flow cytometry profiles are the MFI indexes. These results are representative of three independent experiments with similar results.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The biological process of DC maturation represents a crucial step in the initiation of adaptive immune responses [22 ]. This process is regulated by various extracellular stimuli, including cytokines, bacterial products, and membrane-bound ligands [10 , 23 ]. DC maturation is accompanied by changes in their morphological, phenotypic, and functional properties [24 ]. Recently, several researchers have demonstrated the immunomodulatory effects of polysaccharides purified from G. lucidum on T lymphocytes [25 ]. Cao and Lin [26 ] showed that G. lucidum polysaccharides could promote the maturation and function of murine DC. These results are similar to our finding in human DC. However, little is known about the molecular mechanisms responsible for the regulation of DC in their activation and maturation states by PS-G. In this study, we were the first to demonstrate the PS-G-induced morphological, phenotypical, and functional changes in human monocyte-derived DC. PS-G promoted the maturation of DC, and mature DC demonstrated characteristic morphology with enlarged size and numerous cytoplasmic processes, which gave rise to a stellate appearance (data not shown). Maturation of DC was characterized by a decreased antigen-processing capacity, an increased cell-surface expression of MHC class II molecule and costimulatory molecules CD80, CD86, and CD40, and the secretion of IL-12, which primed a strong stimulation of T lymphocyte growth and differentiation. The CD83 marker for mature human DC was also increased.

In immune responses, IL-12 plays a central role as a link between the innate and adaptive immune systems [27 ]. Thus, IL-12 induces and promotes NK and T cells to generate IFN-{gamma} and lytic activity. In addition, IL-12 polarizes the immune system toward a primary T helper cell type 1 (Th1) response. In this study, we found out that LPS and PS-G can induce IL-10 and IL-12 production in human DC. IL-10 is a pleiotropic cytokine produced by DC, T cells, and macrophages and anti-inflammatory and immunosuppressive properties [24 ]. We suggested that when IL-12 p40 is overexpressed in PS-G-treated human DC, IL-10 could act as a feedback regulatory role, although LPS has the similar effect in human DC, but the cytotoxicity of LPS is higher than PS-G. Therefore, PS-G is a safe immune modulator for human DC. IFN-{gamma} and IL-10 cytokines were induced in MLR by PS-G-treated human DC. In contrast to PS-G, only IFN-{gamma} cytokine was induced in MLR by LPS-treated, human DC. Therefore, LPS was described as a Th1 inducer. Our experimental data show that under some conditions, PS-G can induce a Th1 differentiation or promote the differentiation of naïve T cells for the secretion of IL-10. However, although DC are widely regarded as the most potent APCs, recent evidence also indicates that DC play an important role in inducing immune tolerance [28 ] and regulating Th1/Th2 immunity balance [29 ]. Furthermore, these immature DC could also be alternatively activated and induced to exert suppressive effects [30 ].

TLRs have been identified in humans as an important component of innate immunity against microbial pathogens. LPS is recognized by TLR-4 in DC. The effects of TNF-{alpha} are mediated by two distinct cell-surface receptors, TNF-R1 and TNR-R2, where TNF-R1 has been implicated in TNF-{alpha}-induced phenotypic and functional changes in DC [10 ]. Recent reports show that LPS and TNF-{alpha}, two potent DC maturation factors, induced the NF-{kappa}B activation and phosphorylation of p38, ERK1/2, and p46/54 JNK in monocyte-derived DC [31 ]. Our results demonstrated that PS-G activated NF-{kappa}B and all three MAPK pathways during maturation. Neutralization experiments with antibodies blocking TLR-2 and TLR-4 further demonstrated that TLR-4 played a critical role in the signal transduction cascade induced by PS-G. The blocking effect in high concentration of PS-G (10 µg/ml)-treated DC is not better than in low concentration (200 ng/ml). However, the percentage of inhibition by anti-TLR-4 antibody was similar between LPS and PS-G stimulation. This result is different from the zymosan, as zymosan particles are recognized simultaneously by dectin-1 and TLR-2 [32 33 34 ]. Recent reports show that NF-{kappa}B is responsible for LPS-induced DC maturation in an in vitro murine model [35 ] and that cytokine-induced maturation of human DC results in increased NF-{kappa}B nuclear translocation [36 ]. Many proinflammatory cytokines displayed NF-{kappa}B-responsive elements in their promoters, conferring a major role on immune responses [37 ]. Moreover, the p38 MAPK pathway has been shown to contribute to NF-{kappa}B-mediated transactivation [38 , 39 ]. Little is known about the signal transduction pathways involved in the maturation of human monocyte-derived DC by PS-G. We demonstrated that the NF-{kappa}B, p38 MAPK, ERK1/2, and p46/54 JNK pathways are activated when immature human DC are exposed to PS-G, suggesting a role of these pathways in the maturation process. The promoters of human (h)IL-12 p35 and hIL-12 p40 gene contain {kappa}B-binding sites [40 ]. It likely that NF-{kappa}B is also involved in the IL-12 p35 and IL-12 p40 expression. The lack of {kappa}B-binding sites in the hIL-10 promoter makes it unlikely that NF-{kappa}B is involved in IL-10 regulation [41 ]. Recently, it has been suggested that p38 MAPK is involved in the regulation of IL-10 production [42 ].

Early phosphorylation of p38 MAPK, ERK1/2, and p46/54 JNK was investigated in PS-G-treated, human monocyte-derived DC. Our results corroborate recent reports using the murine models, as well as human DC in vitro models showing activation of all three MAPK pathways during maturation [35 , 43 ]. The availability of specific inhibitory drugs for the NF-{kappa}B, p38 MAPK, ERK, and JNK pathways prompted us to investigate the respective roles of the NF-{kappa}B and these MAPK in DC maturation. In cytokines analysis, pretreatment of helenalin and SB203580 significantly inhibited the IL-12 p70, IL-12 p40, and IL-10 productions in PS-G-treated, human DC. In contrast, PD98059 and JNK inhibitor II were shown to inhibit IL-12 p70 and IL-10 production, although we only observed a little inhibitory effect of these compounds in the up-regulation of IL-12 p40 in the process of DC maturation triggered by PS-G. Concerning costimulatory molecules and MHC class II expression, helenalin-pretreated human DC were able to completely suppress these molecules’ expression induced by PS-G. The inhibition of p38 MAPK and p46/54 JNK by SB203580 and JNK inhibitor II, respectively, before PS-G stimulation had a weak effect on the CD80, CD86, CD83, CD40, CD54, and MHC class II expression induced during DC maturation. PD98059 had no effect on the costimulatory molecules and MHC class II expression in the process of DC maturation triggered by PS-G. Moreover, the inhibitory effects of these inhibitors were not a result of nonspecific toxicity, as these inhibitors did not modify the viability of DC (data not shown). Collectively, these results show that the NF-{kappa}B and p38 MAPK pathways play critical roles in the initiation of DC maturation. The human CD86 promoter has been cloned recently, and two canonical NF-{kappa}B binding sites have been revealed [44 ]. One of them is essential for the Th-induced CD86 gene transcription. Moreover, NF-{kappa}B activation has been shown previously to drive CD 80 transcription [45 ]. A recent report describes the generation of MAPK kinase 3-deficient mice to study the role of the p38 MAPK pathway in vivo [46 ]. Using this animal model, the authors showed that p38 MAPK is required for the production of IL-12 in macrophages and DC. It appears that different signal transduction pathways regulate the different aspects of DC maturation.

In conclusion, we demonstrated that PS-G can effectively and rapidly induce the significant activation and maturation of human DC by the NF-{kappa}B and p38 MAPK pathways. Therefore, PS-G is a good and potential part of the treatment regimen to regulate host immune responses.


   ACKNOWLEDGEMENTS
 
This study was supported by the National Science Council NSC 92-2314-B-002-201. The authors extend their gratitude to Ya-Hui Chuang for the discussions and comments about this work and manuscript.

Received August 31, 2004; revised April 7, 2005; accepted April 13, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Miyazaki, T., Nishijima, M. (1981) Studies on fungal polysaccharides, XXVII. Structural examination of a water-soluble, anti-tumor polysaccharide of Ganoderma lucidum Chem. Pharm. Bull. (Tokyo) 29,3611-3616[Medline]
  2. Wang, S. Y., Hsu, M. L., Hsu, H. C., Tzeng, C. H., Lee, S. S., Shiao, M. S., Ho, C. K. (1997) The anti-tumor effect of Ganoderma lucidium is mediated by cytokines released from activated macrophages and T lymphocytes Int. J. Cancer 70,699-705[CrossRef][Medline]
  3. Furusawa, E., Chou, S. C., Furusawa, S., Hirazami, A., Dang, Y. (1992) Anti-tumor activity of Ganoderma lucidum, an edible mushroom, on intraperitoneally implanted Lewis lung carcinoma in synergenic mice Phytother. Res. 6,300-304[CrossRef]
  4. Lee, S. S., Wei, Y. H., Chen, C. F., Wang, S. Y., Chen, K. Y. (1995) Antitumor effects of Ganoderma lucidum J. Chin. Med. 6,1-12
  5. Won, S. J., Lee, S. S., Ke, Y. H., Lin, M. T. (1989) Enhancement of spenic NK cytotoxic activity by extracts of Ganoderma lucidum mycelium in mice J. Biomed. Lab. Sci. 2,201-213
  6. Hsu, M. J., Lee, S. S., Lin, W. W. (2002) Polysaccharide purified from Ganoderma lucidum inhibits spontaneous and Fas-mediated apoptosis in human neutrophils through activation of the phosphatidylinositol 3 kinase/Akt signaling pathway J. Leukoc. Biol. 72,207-216[Abstract/Free Full Text]
  7. Banchereau, J., Steinman, R. M. (1998) Dendritic cells and the control of immunity Nature 392,245-252[CrossRef][Medline]
  8. Cella, M., Sallusto, F., Lanzavecchia, A. (1997) Origin, maturation and antigen presenting function of dendritic cells Curr. Opin. Immunol. 9,10-16[CrossRef][Medline]
  9. Cella, M., Pinet Engering, A., Pieters, J., Lanzavecchia, A. (1997) Inflammatory stimuli induce accumulation of MHC class II complexes on dendritic cells Nature 388,782-787[CrossRef][Medline]
  10. Sallusto, F., Cella, M., Danieli, C., Lanzavecchia, A. (1995) Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial products J. Exp. Med. 182,389-400[Abstract/Free Full Text]
  11. Caux, C., Massacrier, C., Vanbervliet, B., Dubois, B., van Kooten, C., Durand, I., Banchereau, J. (1994) Activation of human dendritic cells through CD40 cross-linking J. Exp. Med. 180,1263-1272[Abstract/Free Full Text]
  12. Jakob, T., Walker, P. S., Krieg, A. M., Udey, M. C., Vogel, J. C. (1998) Activation of cutaneous dendritic cells by CpG-containing oligodeoxynucleotides: a role for dendritic cells in the augmentation of Th1 responses by immunostimulatory DNA J. Immunol. 161,3042-3049[Abstract/Free Full Text]
  13. Yoshimura, S., Bondeson, J., Foxwell, B. M., Brennan, F. M., Feldmann, M. (2001) Effective antigen presentation by dendritic cells is NF-{kappa}B dependent: coordinate regulation of MHC, co-stimulatory molecules and cytokines Int. Immunol. 13,675-683[Abstract/Free Full Text]
  14. Ardeshna, K. M., Pizzey, A. R., Devereux, S., Khwaja, A. (2000) The PI3 kinase, p38 SAP kinase, and NF-{kappa}B signal transduction pathways are involved in the survival and maturation of lipopolysaccharide-stimulated human monocyte-derived dendritic cells Blood 96,1039-1046[Abstract/Free Full Text]
  15. Sallusto, F., Lanzavecchia, A. (1994) Efficent presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulation factor plus interleukin 4 and downregulated by tumor necrosis factor {alpha} J. Exp. Med. 179,1109-1118[Abstract/Free Full Text]
  16. Zhou, L. J., Tedder, T. F. (1996) CD14+ blood monocytes can differentiate into functionally mature CD83+ dendritic cells Proc. Natl. Acad. Sci. USA 93,2588-2592[Abstract/Free Full Text]
  17. Spiecker, M., Darius, H., Kaboth, K., Hubner, F., Liao, J. K. (1998) Differential regulation of endothelial cell adhesion molecule expression by nitric oxide donors and antioxidants J. Leukoc. Biol. 63,732-739[Abstract]
  18. Lin, Y. L., Lin, J. K. (1997) (–)-Epigallocatechin-3-gallate blocks the induction of nitric oxide synthase by down-regulating lipopolysaccharide-induced activity of transcription factor-{kappa}B Mol. Pharmacol. 52,465-472[Abstract/Free Full Text]
  19. De Smedt, T., Pajak, B., Muraille, E., Lespagnard, L., Heinen, E., De Baetselier, P., Urbain, J., Leo, O., Moser, M. (1996) Regulation of dendritic cell numbers and maturation by lipopolysaccharide in vivo J. Exp. Med. 184,1413-1424[Abstract/Free Full Text]
  20. Chan, E. D., Winston, B. W., Jarpe, M. B., Wynes, M. W., Riches, D. W. H. (1997) Preferential activation of the p46 isoform of JNK/SAPK in mouse macrophage by TNF-{alpha} Proc. Natl. Acad. Sci. USA 94,13169-13174[Abstract/Free Full Text]
  21. Reinhard, C., Shamoon, B., Shyamala, V., Williams, L. T. (1997) Tumor necrosis factor {alpha}-induced activation of c-jun N-terminal kinase is mediated by TRAF-2 EMBO J. 16,1080-1092[CrossRef][Medline]
  22. Roake, J. A., Rao, A. S., Morris, P. J., Larsen, C. P., Hankins, D. F., Austyn, J. M. (1995) Dendritic cell loss from nonlymphoid tissues after systemic administration of lipopolysaccharide, tumor necrosis factor, and interleukin 1 J. Exp. Med. 181,2237-2247[Abstract/Free Full Text]
  23. O’Sullivan, B. J., Thomas, R. (2002) CD40 ligation conditions dendritic cell antigen-presenting function through sustained activation of NF-{kappa}B J. Immunol. 168,5491-5498[Abstract/Free Full Text]
  24. Moore, K. W., de Waal Malefyt, R., Coffman, R. L., O’Garra, A. (2001) Interleukin-10 and interleukin-10 receptor Annu. Rev. Immunol. 19,683-765[CrossRef][Medline]
  25. Gao, Y., Zhou, S., Jiang, W., Huang, M., Dai, X. (2003) Effects of ganopoly (a Ganoderma lucidum polysaccharide extract) on the immune functions in advanced-stage cancer patients Immunol. Invest. 32,201-215[CrossRef][Medline]
  26. Cao, L-Z., Lin, Z-B. (2002) Regulation on maturation and function of dendritic cells by Ganoderma lucidum polysaccharides Immunol. Lett. 83,163-169[CrossRef][Medline]
  27. Trinchieri, G. (1998) Interleukin-12: a cytokine at the interface of inflammation and immunity Adv. Immunol. 70,83-243[Medline]
  28. Adler, A. J., Marsh, D. W., Yochum, G. S., Guzzo, J. L., Nigam, A., Nelson, W. G., Pardoll, D. M. (1998) CD4+ T cell tolerance to parenchymal self-antigens requires presentation by bone marrow-derived antigen-presenting cells J. Exp. Med. 187,1555-1564[Abstract/Free Full Text]
  29. Kalinski, P., Hilkens, C. M., Wierenga, E. A., Kapsenberg, M. L. (1999) T-cell priming by type-1 and type-2 polarixed dendritic cells: the concept of a third signal Immunol. Today 20,561-567[CrossRef][Medline]
  30. Goerdt, S., Orfanos, C. E. (1999) Other functions, other genes, alternative activation of antigen-presenting cells Immunity 10,137-142[CrossRef][Medline]
  31. Arrighi, J-F., Rebsamen, M., Rousset, F., Kindler, V., Hauser, C. (2001) A critical role for p38 mitogen-activated protein kinase in the maturation of human blood-derived dendritic cells induced by lipopolysaccharide, TNF-{alpha}, and contact sensitizers J. Immunol. 166,3837-3845[Abstract/Free Full Text]
  32. Brown, G. D., Taylor, P. R., Reid, D. M., Willment, J. A., Williams, D. L., Martinez-Pomares, L., Wong, S. Y. C., Gordon, S. (2002) Dectin-1 is a major ß-glucan receptor on macrophages J. Exp. Med. 196,407-412[Abstract/Free Full Text]
  33. Gantner, B. N., Simmons, R. M., Canavera, S. J., Akira, S., Underhill, M. (2003) Collaborative induction of inflammatory responses by dectin-1 and Toll-like receptor 2 J. Exp. Med. 197,1107-1117[Abstract/Free Full Text]
  34. Brown, G. D., Herre, J., Williams, D. L., Willment, J. A., Marshall, A. S. J., Gordon, S. (2003) Dectin-1 mediates the biological effects of ß-glucans J. Exp. Med. 197,1119-1124[Abstract/Free Full Text]
  35. Rescigno, M., Martino, M., Sutherland, C. L., Gold, M. R., Ricciardi-Castagnoli, P. (1998) Dendritic cell survival and maturation are regulated by different signaling pathways J. Exp. Med. 188,2175-2180[Abstract/Free Full Text]
  36. Neumann, M., Fries, H-W., Scheicher, C., Keikavoussi, P., Kolb-Maürer, A., Bröcker, E. B., Serfling, E., Kämpgen, E. (2000) Differential expression of Rel/NF-{kappa}B and octamer factors is a hallmark of the generation and maturation of dendritic cells Blood 95,277-285[Abstract/Free Full Text]
  37. Ghosh, S., May, M. J., Kopp, E. B. (1998) NF-{kappa}B and Rel proteins: evolutionarily conserved mediators of immune responses Annu. Rev. Immunol. 16,225-260[CrossRef][Medline]
  38. Vanden Berghe, W., Plaisance, S., Boone, E., De Bosscher, K., Schmitz, M. L., Fiers, W., Haegeman, G. (1998) p38 and extracellular signal-regulated kinase mitogen-activated protein kinase pathways are required for nuclear factor-{kappa}B p65 transactivation mediated by tumor necrosis factor J. Biol. Chem. 273,3285-3290[Abstract/Free Full Text]
  39. Carter, A. B., Knudtson, K. L., Monick, M. M., Hunninghake, G. W. (1999) The p38 mitogen-activated protein kinase is required for NF-{kappa}B-dependent gene expression: the role of TATA-binding protein (TBP) J. Biol. Chem. 274,30858-30863[Abstract/Free Full Text]
  40. Yoshimoto, T., Kojima, K., Funakoshi, T., Endo, Y., Fujita, T., Nariuchi, H. (1996) Molecular cloning and characterization of murine IL-12 genes J. Immunol. 156,1082-1088[Abstract]
  41. Kube, D., Platzer, C., von Knethen, A., Straub, H., Bohlen, H., Hafner, M., Tesch, H. (1995) Isolation of the human interleukin 10 promoter. Characterization of the promoter activity in Burkitt’s lymphoma cell lines Cytokine 7,1-7[CrossRef][Medline]
  42. Foey, A. D., Parry, S. L., Williams, L. M., Feldmann, M., Foxwell, B. M., Brennan, F. M. (1998) Regulation of monocyte IL-10 synthesis by endogenous IL-1 and TNF-{alpha}: role of the p38 and p42/44 mitogen-activated protein kinases J. Immunol. 160,920-928[Abstract/Free Full Text]
  43. Sato, K., Nagayama, H., Tadokoro, K., Juji, T., Takahashi, T. A. (1999) Extracellular signal-regulated kinase, stress-activated protein kinase/c-Jun N-terminal kinase, and p38mapk are involved in IL-10-mediated selective repression of TNF-{alpha}-induced activation and maturation of human peripheral blood monocyte-derived dendritic cells J. Immunol. 162,3865-3872[Abstract/Free Full Text]
  44. Li, J., Liu, Z., Jiang, S., Cortesini, R., Lederman, S., Suciu-Foca, N. (1999) T suppressor lymphocytes inhibit NF-{kappa}B-mediated transcription of CD86 gene in APC J. Immunol. 163,6386-6392[Abstract/Free Full Text]
  45. Zhao, J., Freeman, G. J., Gray, G. S., Nadler, L. M., Glimcher, L. H. (1996) A cell type-specific enhancer in the human B7.1 gene regulated by NF-{kappa}B J. Exp. Med. 183,777-789[Abstract/Free Full Text]
  46. Lu, H. T., Yang, D. D., Wysk, M., Gatti, E., Mellman, I., Davis, R. J., Flavell, R. A. (1999) Defective IL-12 production in mitogen-activated protein (MAP) kinase kinase 3 (MKK3)-deficient mice EMBO J. 18,1845-1857[CrossRef][Medline]



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