The Effect And Mechanism Of High Concentration Of Glucose And Insulin In Reglulating Immune Maturation And Expression Of Scavenger Recptors In Dendritic Cells

PART1:High concentration of glucose and insulin induced immune maturation of human monocyte derived dendritic cellsObjective:Type2diabetes, a presently rapidly expanding disease, is a major risk factor for atherosclerosis. The precise mechanism of how diabetes induces atherosclerosis is still not known. Dendritic cells are potent antigen-presenting and immune modulating cells which might be implicated in the pathogenesis of atherosclerosis. Hyperglycemia and hyperinsulinemia have been reported as important risk factors for atherosclerotic diseases. The aim of this study is to explore the effect of high level concentration of glucose and insulin on the maturation of monocyte-derived dendritic cells (MoDCs).Methods:Human peripheral blood mononuclear cells (PBMCs) were separated by density gradient centrifugation and the monocytes (over98%) were purified from PBMCs by positive selection with anti-CD14magnetic beads. After cultured in RPMI-1640medium containing recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF,100ng/ml) and recombinant human interleukin-4(rhIL-4,50ng/ml) for5days, monocytes were derived into immature DCs. On culture day6, cells were treated with various concentrations of glucose (5.5mol/L、15mol/L、30mmon/L) or insulin(lnmol/L、10nmol/L、100nmol/L) for24hours. The immunophenotypic expressions (CD86,CD83and HLA-DR) were analyzed by FACS. Endocytosis function of the MoDCs was evaluated using FITC-Dextran. The cytokine secretions of culture supernatants (IL-6, IL-10, IL-12and TNF-a) were measured with ELISA.Results:Compaired with normal gluscoe(5.5mmol/L), high glucose(15mol/L、 30mmon/L) upregulated the immunophenotypic expressions (CD83,CD86and HLA-DR) in a dose-dependent manner(p<0.05). The endocytosis function of DCs were significantly inhibited in the group of high glucose(p<0.05). And high glucose significantly increased the cytokine secretions of IL-6,IL-12and TNF-a(p<0.05), but the cytokin secretion of IL-10was significantly descreased(p<0.05). Furthermore,we found that CD83,CD86and HLA-DR expression on DCs in group of lOnM and100nM insulin were significantly higher compared with group of1nM insulin (both p<0.05). The endocytosis function of DCs were significantly inhibited in the group of10nM、100nM insulin compared with the1nM group. Cytokine secretions of IL-6,IL-12and TNF-a in group of10nM100nM insulin were also significantly higher compared with1nM group (all p<0.05), but the secretions of IL-10was decreased in group of10nM、100nM insulin(p<0.05).Conclusion:High concentration of glucose and insulin induced immune maturation of DCs. These interactive roles of high glucose and high concentration of insulin with DCs may contribute to the immunopathogenesis of atherosclerosis. PART2:High concentration of glucose and insulin induce upregulation of scavenger-receptors in dendritic cellsObjective:Scavenger receptors which are expressed by monocyte-derived DCs, are major receptors for oxLDL. Stimulation of DCs by oxLDL through binding of scavenger-receptors leads to their activation and can be accompanied by enhanced cytokine production. We examined whether high concentration of glucose and insulin regulate scavenger receptors expression in DCs, especially focusing on CD36, SR-A and LOX-1.Methods:Human PBMCs were separated by density gradient centrifugation and the monocytes (over98%) were purified from PBMCs by positive selection with anti-CD14magnetic beads. After cultured in RPMI-1640medium containing100ng/ml rhGM-CSF and50ng/ml rhIL-4for5days, monocytes were derived into immature DCs. On culture day6, cells were treated with various concentrations of glucose(5.5mol/L、15mol/L、30mmon/L) and insulin(lnmol/L、10nmol/L、50nmol/L、100nmol/L) for24hours. The expression of the scavenger-receptors SR-A,CD36,LOX-1were determined by western blot analysis and real-time PCR. Furthermore, DCs were incubated with Dil-marked-oxLDL.The Dil-oxLDL incorporated fraction was investigated by confocal microscopy and quantified by flow cytometer analysis.Results:Incubation of DCs with high glucose(15mol/L、30mmon/L) enhanced, in a dose-dependent manner, gene and protein expression of SR-A,CD36,LOX-1(p<0.05). And high glucose can increase the oxLDL-uptake capacity of DCs. Blockage of the scavergen-receptors SR-A,CD36can reduce oxLDL uptake, but blockage of LOX-1can’t redue oxLDLuptake. Futhremor, we found that compared with1nmol/L inslulin, high concentration of insluin enhanced gene and protein expression of SR-A,CD36,LOX-1(p<0.05), and increased the oxLDL-uptake capacity of DCs which can be blocked by antibodies aginst CD36and SR-A.Conclusion:High concentration of glucose and insulin can increase the expression of the scavenger-receptors SR-A, CD36and LOX-1. High concentration of glucose and insulin can also increase the oxLDL-uptake capacity of DCs, which depends on SR-A and SR-A. PART3:Mechanism of high concentration of glucose and insulin induce upregulation of scavenger-receptors in dendritic cellsObjective:We aimed to investigate the mechanism high concentration of glucose and insulin induce upregulation of scavenger-receptors in dendritic cells.Methods:Human PBMCs were separated by density gradient centrifugation and the monocytcs (over98%) were purified from PBMCs by positive selection with anti-CD14magnetic beads. After cultured in RPMI-1640medium containing100ng/ml rhGM-CSF and50ng/mI rhIL-4for5days, monocytes were derived into immature DCs. In order to investigeate the mechanism of upregulation of scavenger receptors by high glucose, on culture day6, cells were treated DCs in the following five groups:①5.5mmol/L glucose②30mmol/Lglucose③30mmol/L glucose+SB203580(inhibitor of p38MAPK)④30mmol/Lglucose+NAC (inhibitor of ROS)⑤30mmol/L glucose+BAY11-7082(inhibitor of NF-κB). The expressions of three scavenger receptors SR-A、CD36and LOX-1were examined by Real-time PCR and Western-blot. Furthmore, we investiged expression of NF-κB p65in the first four groups by Western-blot. In order to investigeate the mechanism of upregulation of scavenger receptors by high concentration of insulin, on culture day6, cells were treated DCs in the following four groups:②1noml/L insulin③100nmol/L insulin③100mmol/L insulin+LY294002(inhibitor of PI3K)④100mmol/Linsulin+PD98059(inhibitor of MAPK). We also used Real-time PCR and Western-blot to determine the expressions of three scavenger receptors SR-A、CD36and LOX-1.Results:Induction of scavenger-receptors (SR-A,CD36,LOX-1) expression by high glucose(30mmol/L) was abolished by ROS inhibitor NAC (p<0..05). A similar effect was observed when the cells were pre-incubated with the p38MAPK inhibitor SB203580, or the NF-κB inhibitor BAY11-7082. NF-κB p65can be upregulated by high glucose, and this effect of high glucose can be partly blocked by NAC and SB203580. Furthmore, the MAPK inhibitor PD98059totally abrogated high cocentration of insulin-induced DCs SR-A,CD36,LOX-1expression. but LY294002didn’t have this effect.Conclusion:We proposed the notion that high glucose induces DCs to generate ROS and activate MAPK pathway, which leads to the subsequent activation of NF-κB, and finally up-regulates scavenger receptors expression. And insulin up-regulates cavenger receptors expression by MAPK pathway, not PI3K pathway.