Effect Of ?-glucan On Bone Marrow-derived Dendritic Cells Function And Its Mechanism

Backgroud:Due to the aging population and bad life style,the incidence of cancer is increasing year by year.Cancer has been a serious threat to human health and has became a hot topic and difficult problems in the field of medical research.?-glucan,a carbohydrate found ubiquitously in nature and as an important biological response modifier,has been widly used for the study of tumor immunotherapy.Previous studies have shown that ?-glucan can upregulate the secretion of proinflammatory cytokines that induce Thl and antigen-specific CD8+ cytotoxic T lymphocytes(CTLs)immune responses by activating immune cells such as dendritic cells(DCs),macrophages and neutrophils,resulting in enhanced anti-tumor efficacy in human body.Therefore,?-glucan can be served as an efficient adjuvant to enhance anti-tumor immune responses.DCs are now the most powerful antigen-presenting cells(APCs)discovered in immune system,specializing in activating naive T lymphocytes.Thus,DCs are the main initiator of the innate immune response,playing a critical role in anti-tumor effects.In recent years,DCs-based cancer immunotherapy has been gained more and more attention in the medical world,and has achieved good clinical efficacy in the treatment of prostate cancer,renal cell carcinoma,melanoma,malignant lymphoma and other tumors.In the present study,we intends to investigate the effect of ?-glucan on the function of DCs,and probe into its molecule mechanism,contributing to provide a new strategy for the development and optimization of DCs vaccine and offer a theoretical basis for further application of ?-glucan in the treatment of malignant tumors.Objective To determine the impact of ?-glucan on the maturation and migration of the bone marrow derived dendritic cells(BMDCs),and explore its potential mechanism of action.Methods BMDCs were generated from mouse bone marrow cells in vitro by culturing in a medium in the presence of GM-CSF and IL-4.Cultured BMDCs at day 7 were used for experiments.The expressions of cell surface markers including MHC class ?,CD80,CD86,CD40 were detected by flow cytometry(FCM).The cytokines in the supernatant were measured by ELISA,and the expressions of intracellular CC chemokine receptor 1(CCR1),CCR2,CCR5,CCR7 were determined by real-time quantitative PCR.Furthermore,the chemotactic response to CC chemokine ligand 19(CCL19)and CCL21,i.e.CCR7-ligands,was measured by TranswellTM migration assay.Moreover,the number of migrated cells in the draining lymph nodes was analyzed by FCM.The phosphorylation levels of MAPKs(p38 MAPK,ERK1/2,and JNK)were performed by Western blot analysis.In addition,ERK1/2 inhibitor U0126 and p38 MAPK inhibitor SB203580,which are thought to block the corresponding pathway,respectively,were used to determine whether these kinases are involved in BMDCs function induced by ?-glucan.Results1.Compared with the control group,the expression of co-stimulation molecules(MHC ? CD40,CD80,CD86)on BMDCs was up-regulated in the presence of?-glucan.Furthermore,P-glucan could prompt BMDCs to secret high levels of IL-6,TNF-?,IL-12 p40 and increase the production of CCR7 mRNA.After?-glucan treatment,BMDCs were more sensitive to CCL19/CCL21.The amount of BMDCs migrated from subcutaneous injection site into the draining lymph nodes was significantly increased in ?-glucan group.2.Western blot analysis showed that ?-glucan stimulation triggered dual phosphorylation of ERK1/2 and p38 MAPK in BMDCs.3.Treatment with both the ERK1/2 inhibitor U0126 and the p38 MAPK inhibitor SB20350 obviously impaired the augmentation of migratory ability of chemotaxis towards CCL19 or CCL21 and decreased the upregulation of CCR7 mRNA expression in BMDCs following ?-glucan treatment,while only the ERK1/2 inhibitor U0126 was able to dramatically suppress the expression of CD86 and CD80,significantly decrease the production of TNF-?,IL-6 and IL-12p40,and profoundly enhance the endocytosis of FITC-dextran.Conclusion1.?-glucan can promote the maturation of BMDCs,and enhances the migration ability of BMDCs in vitro and in vivo.2.?-glucan could significantly activate ERK1/2 and p38 MAPK signaling pathways in BMDCs.3.The ERK1/2 and P38 MAPK pathways was involved in ?-glucan-induced CCR7 mRNA expression and migration of BMDCs,while only the ERK1/2 signaling pathway is responsible for BMDCs maturation stimulated by ?-glucan.