Maturation of murine bone marrow-derived dendritic cells induced by Radix Glycyrrhizae polysaccharide

Abstract

Radix Glycyrrhizae polysaccharide (GP), the most important component of Radix Glycyrrhizae, has been reported to have many immunopharmacological activities. However, the mechanism by which GP affects dendritic cells (DCs) has not been elucidated. In this study, we investigated the effect of GP on murine bone marrow-derived DCs and the potential pathway through which GP exerts this effect. Mononuclear cells (MNCs) were isolated from murine bone marrow and induced to become DCs by culturing with GM-CSF and IL-4. Six days later, DCs were divided into three groups: control group, GP group and LPS group. After 48 h of treatment, phenotypic figures and antigen uptake ability were determined by FACS analysis. The proliferation of DC-stimulated allogenic CD3+ T cells was detected by WST-1. IL-12 p70 and IFN-γ, which are secreted by DCs and CD3+ T cells respectively, were quantified by ELISA. Additionally, IL-12 p40 mRNA expression was determined by real-time PCR. Alterations in TLR4-related signaling pathways were examined by performing an antibody neutralization experiment. Treatment of DCs with GP resulted in the enhanced expression of the cell surface molecules CD80, CD86 and MHC I-A/I-E. GP also increased the production of IL-12 p70 by DCs in a time-dependent manner. The endocytosis of FITC-dextran by DCs was suppressed by GP administration. Furthermore, GP-treated DCs enhanced both the proliferation and IFN-γ secretion of allogenic CD3+ T cells. Finally, the effects of GP on DCs were partially reduced by using inhibitors of TLR4, NF-κB, p38 MAPK or JNK. In conclusion, GP can induce the maturation of DCs, and does so, in part, by regulating a TLR4-related signaling pathway.

Figures

Figure 1

Effects of GP on phenotypic maturation of DCs. Hematopoietic progenitor cells from the bone marrow of C57BL/6 mice were cultured in the presence of GM-CSF and IL-4 for 6 days. Then, day 6 BMDCs were stimulated with GP (100 μg/mL) or LPS (100 ng/mL) for 48 h. Cells were then stained with two Abs, the first Ab was PE-conjugated CD11c, and the second Ab was FITC-CD80, FITC-CD86 or FITC-MHC I-A/I-E. Cells were analyzed by flow cytometry. The results are representative of three independent experiments. (A) Morphological characteristics of BMDCs (×200). The left picture is that bone morrow cells induced by GM-CSF and IL-4 for 1 day; the right picture is that bone morrow cells induced by GM-CSF and IL-4 for 6 days; (B) Flow cytometry results.

Figure 2

Effects of GP on the endocytotic capacity of DCs. Day 6 BMDCs were stimulated with GP (100 μg/mL) or LPS (100 ng/mL) for 48 h; and then incubated with FITC-dextran for 1 h at 37 °C. As a control, cells were also incubated at 4 °C. (A) 4 °C control; (B) 37 °C control; (C) GP; (D) LPS. The results are representative of three independent experiments.

Figure 3

GP-treated DCs strengthen allogenic T cell proliferation and IFN-γ secretion. Day 6 BMDCs were stimulated with LPS (100 ng/mL) or GP (100 µg/mL) for 48 h. Allogeneic CD3+ T cell proliferation was measured after 5 days of co-culture with DCs. DCs were cultured with CD3+ T cells at a ratio of 1:5, 1:10, 1:20 or 1:40. After co-culturing DCs with T cells at a ratio of 1:10 for 48 h, the culture supernatants were collected and IFN-γ was measured. (A) GP-treated DCs strengthen allogenic T cell proliferation; (B) GP-treated DCs increase IFN-γ secretion of allogeneic T cells. Compared with the control group, *p < 0.05, **p < 0.01. The results are representative of three independent experiments.

Figure 4

Effects of GP on IL-12 p70 production in DCs. For the IL-12 p70 assay, DCs were established in culture as described in materials and methods. GP was then added to the wells for 48 h, and the culture supernatants were harvested at various times for measurement of IL-12 p70 concentration using ELISA. IL-12 p40 mRNA expression was measured using real-time PCR. (A) Concentration of IL-12 p70 produced by GP-treated DCs; (B) IL-12 p40 mRNA expression in GP-treated DCs. Compared with the control group, **p < 0.01. The results are representative of three independent experiments.

Figure 5

Effects of TLR4-related signaling pathways on IL-12 p70 production in GP-treated DCs. Day 6 BMDCs were pre-treated with inhibitors of TLR4, NF-κB, p38 MAPK, ERK or JNK for 1 h and then incubated with GP for 24 h. Isotype control mAb was added into control group. The supernatant was then collected for IL-12 p70 detection by ELISA. (A) Effect of anti-TLR4 mAb on IL-12 p70 production of GP-treated DCs. Compared with the no anti-TLR4 mAb group, **p < 0.01; (B) Effect of TLR4 downstream signaling pathways on IL-12 p70 production of GP-treated DCs. Compared with the GP group, *p < 0.05, **p < 0.01. The results are representative of three independent experiments.