Role Of Dendritic Cells In The Mechanism And Management Of Atherosclerosis

Part 1. Experimental researchSession 1 Effect of heat shock protein 60 on murine dendritic cell function in vitroObjective: To investigate the effect of heat shock protein 60 (HSP60), a significant inflammatory factor in atherosclerosis, on the maturation of murine dendritic cells (mDCs) in vitro. Methods: Myeloid mDCs were extracted in vitro from mouse marrow and incubated to mature with two distinct concentrations of mHSP60, observed the morphological changes; detected the variation of phenotype with flow cytometric analysis; the stimulating capacity determined in allogenic mixed lymphocyte reaction (MLR); ELISA was used to analyze the level of cytokines secreted by mDCs and stimulated lymphocyte in the medium of MLR. Results: Dendrites of mHSP60-DCs raised obviously. The expression of co-stimulating factor CD11c+, CD86, CD80 was up-regulated. The stimulating capacity of mHSP-DCs in MLR was higher. T lymphocytes co-cultured with mHSP-DCs in MLR secreted higher levels of pro-inflammation cytokines (IFN-γ, IL-12) but no significant difference of IL-4, and the stimulatory action was dose dependent and was evident at higher concentrations. The ratio of IFN-γ/ IL-4 was increased. Conclusion: mHSP60 progressed the maturation of mDC dose-dependently in vitro. Session 2 Recovery of endothelial dysfunction with tolerogenic dendritic cell loaded with heat shock protein60 in apolipoprotein E-/- miceAIM To examine whether tolerogenic dendritic cell(DC) loaded with heat shock protein60(HSP60) could restore normal endothelial function in hypercholesterolemic apolipoprotein (apo) E-/- mice. METHOD Bone marrow derived Dc of the mice were loaded with HSP60 and co-cultured with Rapamycin to generate tolerogenic DC. The tolerogenic DC ,DC loaded only with HSP60 and saline were injected into the ApoE-/- mice at 6 weeks of age for two times at a one–week interval.C57BL/6mice at the same age were taken as normal control two weeks after the last injection, aorta were harvested for ex vivo vascular ring tension test. immune parameter were also analyzed in vitro and in vivo. RESULTS Compared with the non loaded DC,Hsp60 pulsed DC expressed higher levels of CD86,and stimulated T lymphocytes to proliferation significantly; while the tolerogenic DC expressed lower levels of CD86,and inhibited T lymphocytes to proliferation. After immunization with different injection, Ach nduced relaxation was reduced significantly in DChsp group vs in saline group (P<0.01);treatment of mice with tolerogenic DC restored endothelium-dependent dilation in a dose-dependent manner (p < 0.01); The improvement in endothelial function was associated with a reduction in T cell response to HSP60. CONCLUSION Our results indicate a rapid improvement in endothelial dysfunction with HSP60 tolerogenic DC immunization, and suggest significant vasculoprotective effects with this immune therapy. Session 3 Effects of HSP60-pulsed tolerogenic DC on the progression of the atherosclerotic plaque in mouseAIM To evaluate whether tolerogenic dendritic cell(DC) loaded with heat shock protein60(HSP60) could inhibit the progression of aortic atherosclerotic plaque in hypercholesterolemic apolipoprotein (apo) E-/- mice. METHODS Bone marrow derived Dc of the mice were loaded with HSP60 and co-cultured with rapamycin to generate tolerogenic DC. The tolerogenic DC ,DC loaded only HSP60 and PBS were injected into the ApoE-null mice at 8 weeks of age for three times at a one–week interval. 8 weeks after the last injection, aorta were harvested for HE stained and anti-CD4+ T cell immunostained. Responses of pleenic cells to HSP60 were also evaluated.RESULTS Compared with DC, DCHSP60 expressed higher levels of CD86,and stimulated T lymphocytes to proliferation significantly; while the tolerogenic DC expressed lower levels of CD86,and inhibited T lymphocytes to proliferation. After immunization with different injection, the number of CD4+T cell in plaque were increased significantly in DCHSP60 group vs in PBS group (P<0.01),on the other hand ,they were reduced significantly in rap- DCHSP60 group vs in PBS group (P<0.01).Plaque areas in the tolerogenic DC groug were smaller than that in the PBS group (p < 0.01). Stimulated by HSP60, pleenic cells in tolerogenic DC group secreted more IL-10,while in DCHSP60 group more IFN-γ. CONCLUSION HSP60 specific tolerogenic DC immunization attenuated the progression of plaque, which indicate a new immune therapy for atherosclerosis. Part 2 Clinincal investigationSession 1 The function of dendritic cells in patients with unstable angina pectorisObjective To investigate the function of dendritic cells(DC) in patients with unstable angina pectoris(UAP). Methods PBMCs from 34 UAP patients and 21 healthy subjects were incubated and induced to mature DC in the completed medium containing GM-CSF and IL-4.The flow cytometric analysis was used to detect the expression of co-stimulating factor CD86(B7-2)on DC. The stimulating capacity of DC was determined in allogenic mixed lymphocyte reaction (MLR).ELISA was used to analyze the level of cytokines(IL-1β,IL-6,IL-10 and TNF-a) in the medium of MLR. Relationship of expression of CD86 to risk factors was also analyzed. Results When compared with normal group,CD86 on DC was much more expressed in UAP(P<0.01); the stimulating capacity of DC in MLR was higher ; T lymphocytes in MLR secreted higher levels of pro-inflammation cytokines(IL-1β,IL-6,and TNF-a) and lower level of anti-inflammation cytokine(IL-10),P<0.001.Blood LDL-C was positively related to the expression of CD86(r=0.63,P<0.01). Conclusion DCs in UAP are activated, which may play an important role in initiating immune reaction in the vulnerable plaques. Session 2 Dendritic cell–mediated heat-shock protein 60 specific T cytotoxicity in unstable angina pectorisObjective To investigate whether dendritic cells(DC) derived from patients with unstable angina pectoris(UAP) can induce heat-shock protein 60(HSP60) specific T cell immune response to endothelial cells. Methods : DCs generated from peripheral blood mononuclear cells from 34 patients with UAP and 24 patients with stable angina and 21 healthy subjects were investigated. CD86 expression on HSP60-pulsed and no-pulsed DCs ,and percentage of CD45RO+ T cells were detected by flow cytometry; MTT was used to assay the ability of DCs from each group to stimulate autologous T cell proliferation and cytotoxic T lymphocytes (CTL) mediated lysis of HSP60 loaded endothelial cells; ELISA was used to assay interferin-γrelease by T cells. Results Compared with DCs from stable angina and healthy subjects , CD86 was much more expressed in DCs from UAP and from DCS pulsed with HSP60;capacity of DCs from UAP and from DCs pulsed with HSP60 to stumulate autologous T cell proliferation was increased; DCs from UAP and pulsed with HSP60 could induce HSP60 specific CTLs which could efficiently kill endothelial cells loading HSP60; percentage of HSP60 specific CD45RO+ memory T cells was higher in ACS. Conclusion There exists DC- mediated HSP60 specific T cell cytotoxity in unstable angina pectoris . Session 3 Effects of Atorvastatin on the Function of Dendritic Cells in Patients with Unstable Angina PectorisObjective To investigate the function of dendritic cells(DC) in patients with unstable angina pectoris(UAP)and effects of atorvastatin on it. Methods 34 patients with UAP were divided into two groups treated respectively with regular pharmacotherapy and regular pharmacotherapy plus atorvastatin. PBMC from UAP patients(before and 2 weeks after the treatment) and 21 healthy subjects were incubated and induced to mature DC in the completed medium containing GM-CSF and IL-4.The flow cytometric analysis were used to detect the expression of co-stimulating factor CD86(B7-2)on DC. The stimulating capacity of DC was determined in allogenic mixed lymphocyte reaction (MLR).ELISA was used to analyze the level of cytokines(IL-1β,IL-6,IL-10 and TNF-a) in the medium of MLR. Relationship of expression of CD86 to risk factors and blood CRP level was also analyzed. Results When compared with normal group,CD86 on DC was much more expressed in UAP; the stimulating capacity of DC in MLR was higher ; T lymphocytes in MLR secreted higher levels of pro-inflammation cytokines(IL-1β,IL-6 and TNF-a) and lower level of anti-inflammation cytokine(IL-10).Blood LDL-C before treatment was positively related to the expression of CD86.Atorvastatin inhibited the function of DC and lowered blood level of CRP, CD86 correlated with CRP significantly. Conclusion 1. DCs in UAP are activated, which may play an important role in initiating immune reaction in the plaque;2.LDL-C may be one of the activators of DC;3.inhibitory effect of atorvastatin on inflammation in UAP may be due to its inhibition on DC.