Role of high glucose in maturation and immunologic function of dendritic cells

Objective To explore the effect and mechanism of high glucose environmenton mouse bone marrow-derived dendritic cells′ (DCs) differentiation, maturation, immune function, to further explore the role of DCs in inflammation of diabetic nephropathy. Methods Bone marrow mononuclear cells (MNCs) were isolated and cultured in the medium containing recombinant human granule macrophage colony stimulating factor (rhGM-CSF, 20 ng/ml) and recombinant human interleukin-4 (rhIL-4, 10 ng/ml). On 7th day, immature DCs were collected and cultured in RPMI1640 complete medium containing 5.5 mmol/L D-glucose, 30 mmol/L D-glucose, 30 mmol/L D-glucose+100 μg/ml NF-κB inhibitor pyrrolidinedithiocarbamate (PDTC) for 48 h, and then flow cytometry (FCM) was used to detect the phenotype of DCs. Cells were stimulated with DCs, allogeneic T lymphocytes were mixed in proportion and the reaction intensity of mixed lymphocytes was used to observe the effect on dendritic cell antigen presentation, lymphocyte proliferation, lymphocyte apoptosis. ELISA assay was adopted to detect cytokines concentration of cell culture supernatant. Western blot was adopted for detection of NF-κB activation. Student-t test was used to analyze the significance of the differences between the groups. Results Compared with the normal control group, 30 mmol/L D-glucose significantly increased the activation of NF-κB in DCs (IκBɑ:0.29±0.18 vs 0.69±0.79, t=-1.27; p65: 0.87±0.18 vs 0.35±0.13, t=2.19; all P<0.01) and the expression of eCD11c, MHC class Ⅱ molecules, CD80, CD86 and CD40 on DCs (t=8.97-10.45, all P<0.05). It promoted the T lymphocyte proliferation (1∶100:1.80±0.23 vs 1.57±0.20, t=2.46; 1∶25:1.74±0.17 vs 1.32±0.18, t=2.82; 1∶10:1.59±0.25 vs 1.28±0.19, t=3.06; all P<0.05), weakened the T lymphocyte apoptosis rate (12±2 vs 44±13, t=4.03, P<0.05). And it promoted DCs′ secretion of cytokines IL-12, IFN-γ and TNF-α (t=3.68-7.68, all P<0.05), while the inhibition of NF-κB agent PDTC could block the above effects to varying degrees (all P<0.05). Conclusion High glucose can significantly increase the activation of NF-κB thereby promote the maturation of DCs, which accelerates and amplifies the inflammatory response. This may be one of the important mechanisms of dendritic cells in diabetic nephropathy. Key words: Diabetic nephropathy; Dendritic cells; NF-kappa B; Immuno-inflammatory responses