Losartan Suppresses Human Monocyte-Derived Dendritic Cells Maturation Via Downregulation Of Lectin-like Oxidized Low Density Lipoprotein Receptor-1

PART1:Immune Maturation of Human Monocyte-Derived Dendritic Cells Induced by Oxidized-Low Density Lipoprotein and Inhibited by LosartanObjective:To investigate the effects of oxidized low-density lipoprotein (OxLDL) and angiotensin II receptor1antagonist (ARB) losartan on the immune maturation of human monocyte-derived dendritic cells (DCs).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 OxLDL (10、50、100μg/ml) for24hours. Another group of human monocyte-derived DCs was pretreated with losartan (10μM) for24hours and subsequently stimulated with50μg/ml OxLDL for24hours. The immunophenotypic expressions (HLA-DR and CD80) were analyzed by FACS and the cytokine secretions of culture supernatants (IL-10、IL-12and IFN-γ) were measured with ELISA.Results:OxLDL upregulated the immunophenotypic expressions (HLA-DR and CD80) and the cytokine secretions of culture supernatants (IL-10、IL-12and IFN-γ) in a dose-dependent manner, which were inhibited by losartan pretreatment.Conclusion:Our study suggested that the anti-atherosclerosis effect of ARB losartan was partly mediated by inhibiting the OxLDL-induced immune maturation of DCs. PART2:Immune Maturation of Human Monocyte-Derived Dendritic Cells Induced by Angiotensin II and Inhibited by LosartanObjective:To investigate the effects of angiotensin Ⅱ (Ang Ⅱ) and losartan on the immune maturation of human monocyte-derived DCs.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 Ang Ⅱ (10、100nM) for24hours. Another group of human monocyte-derived DCs was pretreated with10μM losartan for24hours and subsequently stimulated with100nM AngⅡ for24hours. The immunophenotypic expressions (HLA-DR and CD80) were analyzed by FACS and the cytokine secretions of culture supernatants (IL-10、IL-12and IFN-γ) were measured with ELISA.Results:Ang II upregulated the immunophenotypic expressions (HLA-DR and CD80) and the cytokine secretions of culture supernatants (IL-10、IL-12and IFN-γ) in a dose-dependent manner, which were inhibited by losartan pretreatment.Conclusion:Our study suggested that the anti-atherosclerosis effect of ARB losartan was partly mediated by inhibiting the Ang Ⅱ-induced immune maturation of DCs. PART3:Mechanism of Suppression of the Immune Maturation of Human Monocyte-Derived Dendritic Cells by LosartanObjective:To investigate the mechanism through which losartan suppressed the immune maturation of human monocyte-derived DCs.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 OxLDL (10、50、100μg/ml) and AngⅡ (10.100nM) for24hours. The other two groups of DCs were pretreated with10μM losartan for24hours and subsequently stimulated with50μg/ml OxLDL or100nM Ang Ⅱ for24hours. The Ang Ⅱ secretion of culture supernatants in the OxLDL-treated groups was measured with ELISA. The expressions of three scavenger receptors SR-A、CD36and LOX-1were examined by Real-time PCR and Western-blot.Results:Human monocyte-derived DCs produce Ang Ⅱ, which was promoted by OxLDL in a dose-dependent manner. Little scavenger receptor expression was shown in the PBS control group. OxLDL upregulated the expressions of three scavenger receptors SR-A、CD36and LOX-1in a dose-dependent manner. Losartan reduced OxLDL-induced LOX-1expression, but not SR-A and CD36expressions. Ang Ⅱ could also upregulate the LOX-1expression dose-dependently, which was reduced by losartan. The expressions of SR-A and CD36were similar between the Ang Ⅱ-and losartan-treated groups.Conclusion:Ang II could upregulate the LOX-1expression of human monocyte-derived DCs through ATI activation, which was suppressed by losartan. OxLDL could promote the autocrine of Ang II and upregulate the expressions of three scavenger receptors SR-A、CD36and LOX-1; Losartan could suppress the OxLDL-and Ang Ⅱ-induced immune maturation of human monocyte-derived DCs partly through downregulation of the LOX-1expression.