Study On The Relationship And Genesis Between The Deadexis Of Dendritic Cells And CRP In The Development Of Atherosclerotic Plaques

Coronary artery disease(CAD) has been recognized as the leading cause of morbidity and mortality in developed as well as developing countries.The recruitment of immune cells,macrophages,and T-cells plays a key role in the initiation and progression of atherosclerosis.Later on,activation of these inflammatory cells may elicit plaque rupture resulting in acute cardiovascular events.It has been suggested that dendritic cells(DCs) mediate T-cell activation in atherosclerosis.DCs are potent antigen presenting cells required for initiation of innate and adaptive immune responses.Indeed some proatherogenic factors such as oxidized low density lipoproteins and nicotine were reported to induce DC activation resulting in T-cell stimulatory capacities.Recently we and others demonstrated that numbers of DC were lower in the circulation of patients with coronary artery disease in comparison to healthy subjects.Serum CRP levels slightly above normal have been put forward as indicator of mild inflammation associated with cardiovascular disease(CVD) and an independent predictor of CVD.CRP is the prototypical acute phase protein in humans,rising rapidly in response to inflammation.As a well-known ligand of phosphorylcholine residues,CRP binds avidly to oxidized low density lipoproteins and may induce foam cell formation in atherosclerosis.Indeed in atherosclerotic lesions colocalization of CRP with apolipoprotein B was demonstrated and foam cells underearnth the endothelium were shown to stain positive for CRP.There is accumulating evidence that CRP is not only a risk marker of CVD,but actively contributes to atherosclerosis. It has been reported that CRP induces endothelial cell activation and dysfunction. Other in vitro studies show effects on vascular smooth muscle cells,monocytes,and macrophages suggesting that CRP may promote atherosclerotic plaque formation, plaque maturation,plaque destabilization,and eventually plaque rupture,as reviewed by Verma et al.Very recent in vitro studies showed that CRP attenuates the very early steps of DC differentiation,mainly through interaction with FcγyRII.As all we known,DCs and CRP play an important role in the proatherogenic of atheroscelrosis.However,the relationship between those factors and the communication with the function of DCs were not showed.Therefore,we want to know the function and mechanisms of the DCs in the difference types of CHD. Forethermore,we study the effects on the DCs with incubated with difference of CRP. It is made up of two parts:Objective:Inflammation and immune reactions are predictive atherosclerosis and coronary artery disease(CAD).Recent findings suggest dendritic cells might play an important role in the development of atherosclerosis.Therefore,DCs in humanity Coronary heart disease remains unstudied.Methods:62 patients diagnosed as CHD initially were divided into four groups.18 patients in the control group with negative result in coronary angiography(CAG);15 patients in AMI group,15 patients in UAP(CTNI+)groups,15 patients in UAP(CTNI-)group and 17 patient in SAP group.Ten of SAP,UAP(CTNI-)and UAP(CTNI+)examined by IVUS and divided into 7 patients in atherosclerosis plaque ruptured,12 and 11 patients in the plaque in unstabilization and stabilization.The subgroup of distibtions of mDCs and pDC in peripheral blood were detected by four flow cytometric technique and identify blood denditic cell antigen:CD1C and CD14,CD303.IgG1-PE and IgG2a-PE were used as isotope-matched controls.Enzyme-linked immunosorbent assay(ELISA)was employed to the analysis of IL-6 and CRP in the peripheral blood of cases.Results: clinical trails:1).Comparison of the clinical general data such as age and gender showed no significant difference among different groups(p＞0.05).And there are no significant difference the main CHD risk factors such as smoking,hypertension, diabetes mellitus,total cholesterol,triglyceride,HDL cholesterol,and LDL cholesterol among those groups(p＞0.05).2)A:the four color FCS was showed that there is a significant difference of the number and function among in the periphery blood DCs,mDCs,mDC1 and pDCs in the UAP(CTNI-)groups(p＜0.05).In the SAP groups showed no changes of the subjects,which in the periphery blood DCs, mDCs,mDC1s and pDCs(p＞0.05).Furthermore,the significant decrease in the ratio and the number of DCs,mDCs,mDC1s,pDCs and mDC2s in the AMI.The ratio of DCs,mDCs and mDCls were declined(p＜0.05)and the numbers of its were no changed(p＞0.05).B.IVUS was used in coronary ateries in 10 patients with SAP,10 patient with UAP(CTNI-)and 10 patients with UAP(CTNI+)to analyze the coronary lesions.IVUS found that patients in SAP groups mainly had 8 of stable plaques and 2 unstable plaques,while patients in UAP(CTNI-)groups mainly had 7 of unstable,2 stable and 1 rupture groups.Nonetheless,6 of rupture,3 unstable and 1 stable plaques were found in UAP(CTNI+)group.Those groups according to IVUS could be reflection the degrees and development of coronary atherosclerosis arteries.The four colour FCS results show that in unstable plaque patient,the ratio and number of DCs, mDCs and mDC1 were significantly higher than stable plaque(p＜0.05).However, the ratio of mDC2s and pDCs were no change when comparion of the other groups(p＞0.05).The ratio and number of DCs,mDCs,mDCls and pDCs were significantly desease in rupture plaque groups(p＜0.05).Forethermore,the mDC2s were no changed(p＞0.05,respective).Cell trails:1)after success cultured the PMBC in vitro,we detected the functional changes of DCs,the proliferative response in T cells and the mixed lymphocyte reaction(MLR)and the cytokine were coculrured with difference factors respective. The CD86 expression was higher than SAP group in UAP(CTNI-),UAP(CTNI+)and AMI,(p＜0.05).However,there is no significantly changed in SAP and CTL(p＞0.05).2)T-cell proliferation in UAP(CTNI-),UAP(CTNI+)and AMI incubated with 2×10⁵/ml DCs evoked a strong proliferation,which compared with CTL and SAP(p＞0.05,respectly).3)The supernatant of in MLR secret the IL-12 and IFN-a were higher than UAP(CTNI-),SAP and AMI.4)There are relatively regression between the expression of CD86 and activate the T-cell proliferation,when compared with plasma hs-CRP,IL-6,(p＜0.01).Partâ…¡:The CRP Stimulates Immune Maturation of Human Dendritic Cell: Pothential Implication For Progression of Atherosclerotic LesionsObjective:CRP plays a key pathogenic role in the development of atherosclerosis. Recent findings suggested that dendritic cells(DCs)may play a crucial role in T cell activation in it.We studied the effects of difference concentration on the maturation and immune function of monocyte-derived dendritic cell(MDC)and its underlying mechanisms.Methods:Human peripheral blood mononuclear cells(PBMC)were isolated by Ficoll Paque PLUS.RPMI 1640 medium supplemented with 5%human AB serum,10 mM pyruvic acid,10 mM nonessential amino acids,100lg/mL kanamycin, 800U/mL recombinant human GM-CSF and500 U/mL IL-4 for 5 days.At days 2 and 4,half the volume of the medium was replaced by fresh medium supplemented with 800 U/mL GM-CSF and 500 U/mL IL-4.For DC maturation,cells were treated with 5μg/mL LPS at day 5 and subsequently incubated for 2 days in 3 mL RPMI 1640 medium supplemented with 800 U/mL GM-CSF and 500 U/mL IL-4.During DC differentiation,various concentrations(0.6,2,6,20 and 60μg/mL)of CRP were added at 24h and replenished every other day.Cells were harvested at 24h for analysis of cell-surface marker expression as well as other functional assays.Normal DC cultures(without addition of CRP)were used as untreated controls.Cells were subsequently stained with 2 mL/10⁵ cells of the DC surface markers CD40,CD80,CD83,CD83.Cells were then washed with 3mL PBS and fixed with 2% paraformaldehyde for FUAP(CTNI+)analysis.FACs data were analyzed using CELLQuest software.Cells were then cultured for 4 days in AIM-V medium and then ^[3H]-thymidine was added,followed by incubation or another 12h.Labeled cells were then harvested onto glassfiber filters with a Filtermate Harvester,and the T-cell proliferation rate was determined by the amount of ^[3H]-thymidine incorporation, which was measured by TopCount-NXT.After 24-hour pulsation with 60μg/mL CRP,DCs were washed and plated in fresh IMDM supplemented with 5%human serum at 10⁵cells per well in 48-well plates with 9-10⁵ autologous PBL labeled with CFSE.With each cell division CFSE fluorescence intensity of the cells is reduced.After 8 days of coculture,cells were harvested,labeled with mouse anti-human CD3-PE and propidium iodide;CFSE fluorescence of propidium iodide-negative and CD3-positive T-cells was determined by fluorescence-activated-cell sorter(FACS). The supernatants were collected for measurement of IL-10,IL-12,IFN-λand IL-2 by ELISA.Results:After 24-hour incubation with CRP(60μg/mL),DCs showed an activated phenotype consisting of multiple enlarged clusters of elongated cell morphology compared with DCs incubated with control group.CRP induced DC activation as shown by the2 to 4 fold upregulation of the median fluorescence intensity(MFI)of CD40,CD80,CD83,and CCR7 when compared with control.Upregulation of CD80 and CD40 by DCs was evident from 24 hours and increased further after 48-hour incubation with 60μg/mL.CD80 and CD40 expression gradually increased with rising CRP concentrations,starting at 2μg/mL and 6μg/mL CRP,respectively.The maximum expression was reached at 60(CD80)μg/mL and CRP(CD40)and was equivalent to that seen with 0.1μg/mL LPS.The DC activation by CRP was further evaluated by analysis of their ability to stimulate autologous T-cell proliferation.DCs incubated with control buffer or CRP for 24 hours were thoroughly washed and then added to T-cells for 8 days.Control DCs hardly induced T-cell proliferation.In contrast,prepulsation of DCs with CRP evoked a strong proliferative T-cell response, as indicated by the reduced CFSE fluorescence.Furthermore,T-cell activation was confirmed by the significant increase of IFN-A,in the supernatant,compared with T-cells incubated with control DCs. Conclusions These findings suggest that CRP in plaques or circulating in CVD patients can influence DCs function during atherogenesis.