The Effects And Mechanism Of Angiotensin Ⅱ On Immune Function Of Human Monocyte-derived Dendritic Cells

【Background and Objectives】Currently Atherosclerosis(AS)is regarded as a chronic inflammatory response.It is a kind of proliferative reaction to inflammatory factors and a reaction of specific cells to exogenous risk factors or antigens.Dendritic cell is an antigen presenting cell widely distributed in various body tissues and may have the central role in AS immune reaction.Numerous studies showed renin-angiotensin system may take a role in the onset and development of arterial atherosclerosis.Basic studies have found angiotensinâ…¡(Angâ…¡)was involved in the arterial atherosclerosis process by stimulating the excretion of inflammatory mediators,inducing apoptosis,accelerating ox-LDL uptake,generating oxygen free radicals and influencing fibrinolysis via AT1 receptor.The excretion of inflammatory mediators is the most important cause in development of AS.Some researchers even called Angâ…¡a special cytokine.Previous studies found Angâ…¡could promote almost all cells associated with plague (endothelial cells,smooth muscle cells,macrophages)to excrete inflammatory mediators.It could stimulate the expression of adhesive molecules of endothelial cells, increase the adhesion of mononuclear cells to the endothelium,induce the expression of MMP-2 of endothelial cells.It could induce the mononuclear cells or macrophages to generate oxygen free radicals.It could induce smooth muscle cells to excrete IL-6, MCP-1 and enhance their migration.So far there are few researches on the effect of Angâ…¡on DCs.Renin-angiotensin system was found expressed in DCs and the expression of renin-angiotensin system genes changed during maturation of DCs.The differentiation of DCs is regulated by Angâ…¡.In the presence of the AT1 receptor antagonist losartan,DCs expressed lower levels of CD11c,CD40,Ia,and displayed a lower ability to endocyte horseradish peroxidase compared with controls.Currently there is no consensus on whether Angâ…¡,which is the most bioactive factor in the RAS system,could induce maturation of DCs,enhance the T cell activity and formation of plaque.If Angâ…¡can promote DCs maturation,what is the mechanism? The objective of this study is to evaluate the effect of Angâ…¡on the immune function of DCs and its mechanism by using in vitro cultured human monocyte-derived dendritic cells.【Methods】1.Cell dissociation and culture:Peripheral blood mononuclear cells were isolated from healthy volunteers by standard density gradient centrifugation on FicollHypaque. CD14⁺ peripheral blood mononuclear cells were purified by using CD14⁺ immunomagnetic micro beads(＞98%)and incubated in RPMI-1640 medium supplemented with GM-CSF(20ng/mL)and IL-4(10ng/mL)in 6-well tissue culture plates at 37℃and an atmosphere of 5%CO₂.The medium was replaced every 2 days.After 5 days,immature DCs were collected.Added 100ng/ml LPS for 24 hours,mature DCs were collected.2.Immunophenotype,such as HLA-DR,CD40,CD86 and CD83 of DCs was monitored by flow cytometry.3.Allogenic T cell activation by DCs was tested by mixed lymphocyte reaction (MLR).4.The endocytic activity of DCs was measured by flow cytometry using a FITCdextran uptake assay.5.The expressions of IL-12p40,IL-6 and TNF-αwere measured by ELISA.6.Chemokines in DCs were measured by semi-quantity RT-PCR.7.Western Blot was conducted to determine the change of protein expression of phosopho-ERK1/2 and p38 and TLR4.8.Nuclear NF-κB activation level in DCs was measured by EMSA. 【Results】1.Compared with control DCs,FACS analysis showed that the expression of CD40 in Angiotensinâ…¡-treated DCs increased significantly.There were no significant differences in the expression of CD86,CD83,HLA-DR.In the present of LPS, there were also no significant differences between two groups.2.There were no significant differences regarding the phagocytosis and ability of enhancing allogenic lymphocytes proliferation between Angiotensinâ…¡-treated DCs and control DCs.In the present of LPS,the ability of enhancing allogenic lymphocytes proliferation of DC was significantly enhanced by Angâ…¡while Valsartan could inhibit it.3.The production of IL-6,TNF-αin the supernatant was significantly higher in Angiotensinâ…¡-treated DCs and Valsartan could inhibit this.In the present of LPS, production of TNF-α,IL-12 was significant higher in Angiotensinâ…¡-treated DCs and Valsartan could inhibit this.4.The expression of MCP-1,RANTES,MIP-1αin DCs was enhanced after Angâ…¡intervention.Valsartan could inhibit the expression of MCP-1,MIP-1α.In the present of LPS,The expression of MCP-1,RANTES,MIP-1αand IP-10 were all significantly higher in Angiotensinâ…¡-treated DCs than control DCs and Valsartan could inhibit this.5.Angiotensinâ…¡stimulated phosphoylation of ERK1/2,p38 while the inhibitors of ERK1/2,p38 could inhibit the expression of MCP-1,RANTES,MIP-1αand secretion of IL-6,TNF-αinduced by Angiotensinâ…¡.6.Angiotensinâ…¡induced NF-κB activation in DC while Valsartan could inhibit this.7.The expression level of phosphor-ERK1/2 was higher in Angiotensinâ…¡-treated DCs than control DCs at 5 and 15 minutes induced by LPS.While Valsartan could inhibit it.No significant differences regarding phosphor-p38 between two groups.8.After 24 hours intervention by 0.0001-1μmol/L Angiotensinâ…¡,the expression of TLR4 protein and membrane surface protein was significantly increased and achieved peak in 0.1μmol/L.Expression increased from 12h and culminated on 24h.Angiotensinâ…¡upregulated the expression of TLR4 in a dose and timedependent manner.While Valsartan could inhibit it.The inhibitors of ERK1/2 and p38 could not inhibit the expression of TLR4 induced by Angiotensinâ…¡.【Conclusions】1.Angiotensinâ…¡could enhance the.expression of CD40,but it is not a typical inducer to promote DCs maturaion.2.Angiotensinâ…¡could induce DCs to secrete IL-6,TNF-αand express MCP-1, MIP-1αthrough AT1 receptor,activation of ERK1/2,p38 and NF-κB.3.Angiotensinâ…¡could induce DCs to express RANTES through AT2 receptor, activation of ERK1/2,p38 and NF-κB.4.Angiotensinâ…¡increases LPS-induced proliferation of allogenic T lymphocytes, production of cytokines and chemokines.Angiotensinâ…¡-treated DCs are hypersensitive to LPS.5.Angiotensinâ…¡further enhances the DCs immune function induced by LPS through AT1 receptor.6.Angiotensinâ…¡could enhance the activation of ERK1/2 induced by LPS,but no significant effect on activation of p38.7.Angiotensinâ…¡upregulates the expression of TLR4 through AT1,this maybe the mechanism of Angiotensinâ…¡on the immune functions in DCs.