Expression Of Programmed Death Ligand 2 In Peripheral Blood Monocytes In Patients With Coronary Heart Disease And Regulation Of Simvastatin

Backgroud:Cardiovascular disease, epecially coronary heart diseases(CHD), is the leading cause of death in most developed country. The main pathological basis of CHD is Atherosclerosis (AS), present researchers believe that AS is an inflammatory immune process after vascular injury. Data from the Framingham Heart Study, demonstrated some independent risk factors of CHD and predictors for cardiovascular outcomes, such as dyslipidemia (high low-density lipoprotein, reduced high-density lipoprotein),diabetes mellitus,elevated blood pressure, smoking, age and gender.The response-to-injury theory suggests that all of the risk factors of coronary heart disease injuried arteries eventually, while the formation of atheromatous plaque is the response to endothelium injury. Various of cells and inflammatory cytokines participate in the formation of AS.Monocytes passing through artery endodermis into intima,was considered to be the earliest stage of AS. Monocytes adhere to endothelial cells with the help of integrin, selectin, selectin ligand. Monocytes chemotactic protein-1 (MCP-1) attract monocytes to entry into the artery endodermis. After the interaction between monocytes and endothelial cells, a lot of cytokines such as interleukin 6 (IL-6), platelet-derived growth factor (PDGF),granulocytes-macropha-ges colony stimulating factor (M-CSF) are produced by them. These cytokines may help expand the inflammatory response by stimulating the replication and secretion of monocyte-derived macrophages and the entry of new monocytes into lesions.Those activated macrophages have enhanced secretion property leading to increased level of MCP-1, IL-6, vascular cell adhesion molecule-1 (VCAM-1), intercellular cell adhesion molecule-1 (ICAM-1), tumor necrosis factor (TNF) and interferon (IFN), and such a positive-feedback result in the aggregation and adhesion to injury endothelium of monocyte-macrophages. These macrophages transformed into foam cells and, thus, led to the AS. Atherosclerotic plaques contain significant numbers of inflammatory immune cells including monocyte-macrophages, T cells and mast cells.They may activate cells to secrete cytokines, promote thrombosis molecules, protease, vascular active substances and so on, that may affect the the inflammatory process and vascular function in injury lesion. So inflammatory reaction produces a cascade effect, which eventually leads to the atherosclerotic plaque and thrombosis. Many researchs have proved that atherosclerosis was characterized by infiltration of monocytes into the arterial wall and the participantion of significant numbers of T lymphocytes at all development stages of AS,in both humans and mouse models.The pathological mechanism of inflammatory immune of AS is unclear yet. Pathways of the B7/CD28 family acting as ligand/receptor provide second signals that can modulate the activation,inhibition and fine-tuning of T-cell responses.These pathways regulate the balance between the stimulatory and inhibitory signals needed for defense against microbes and for self-tolerance. For example, the interaction of B7-1 with CD28 can attenuate, while interaction of B7-1 with CTLA-4 may inhibit T-cell responses. There are abundance of inhibitory pathways within the B7/CD28 family that can attenuate T-cell responses and promote T-cell tolerance. Programmed death-1 (PD-1,CD279)/Programmed death ligand (PD-L) which are immune inhibitory receptor/ligands discovered recently belong to the B7/CD28 family of costimulatory molecules.They are considered to be significant regulator of microbial infection and immune tolerance because of their unique effects in autoimmune response. PD-1/PD-L pathway may not only affect their autoimmune tolerance in many ways, but also affect the interactions between antigen presenting cells and T cells by positive or negative regulation. PD-1 is a member of the immunoglobulin superfamily. It is composed of an extracellular IgV domain, a transmembrane domain, and an intracellular signaling domain with tyrosine-based signaling motifs. PD-1 is constitutively expressed on the activated CD4+T cells, CD8+T cells, NK-T cells, B cells and monocytes. PD-L2(CD273) is one of ligand of PD-1, with expression on Dendritic cells and monocytes. The conventional view is that:after the interaction of PD-L2 with PD-1, the recruitment of phosphorylation of immunoreceptor tyrosine-based inhibitory motifs and tyrosine-based switch motifs in the cytoplasmic tail, and the SH2 domain protein, result in negative signals which may inhibit T lymphocyte proliferation and the secretion of cytokines. Some evidences proved that PD-1/PD-L may strengthen T cell activation, and even some scholars showed that PD-1/PD-L signal may provide both negative and positive signals. The balance of PD-1/PD-L passway is the key to maintain immunity. PD-1/ PD-L may inhibit T cell response, thus inhibit inflammation, and result less inflammatory injury in inflammatory disease. Further investigations maybe needed to do to study the role of the PD-L2 in AS.Inflammation and immune play an important role in the process of AS, the appropriate anti-inflammatory, immune regulator may influence the formation, progress and prognosis of AS. Statins, inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, are the first-line therapy in the treatment of cholesterol-induced cardiovascular disease, which may reduce complications of AS. It reduces cholesterol by the reduction synthesis of liver,and the strengthen expression of low-density lipoprotein (LDL) cholesterol receptor results in increase removement of plasma LDL. In addition to their cholesterol-lowering properties, statins exert a number of so-called pleiotropic, vasculoprotective actions, such as anti-inflammatory, anti-oxidation, regulate immune, stabilize plaques, inhibit platelet aggregation and thrombosis, improve endothelial function, inhibit proliferation of vascular smooth muscle cell, contra-neoplasms and so on. Anti-inflammatory and immunomodulatory effects of statins were demonstrated in various of diseases such as rheumatoid arthritis, multiple sclerosis and experimental autoimmune encephalomyelitis (EAE) in mouse models. The immunomodulatory effect of statins in AS and correlation between PD-L2 and statins deserves further study.Objectives:1.In order to find the relationship among programmed cell death ligand 2 (PD-L2/CD273), interleukin-6 (IL-6), monocyte chemoattractant protein-1(MCP-1) and high sensitivity C-reactive protein (hs-CRP) and explore the role of PD-L2 in the formation of atherosclerosis, we detect the expression of PD-L2 in peripheral blood monocytes in patients with different types of coroary heart diseae. 2. To study the effects of simvastatin with different concentration on the expression of PD-L2 interfered by interferon-gamma (IFN-y) in peripheral blood monocytes in vitro, and to provide some theoretical basis for the anti-atherosclerotic immunotherapy of simvastatin for CHD.Methods:Part I:According to the diagnostic criteria of CHD in guidelines of AHA/ACC of 2007, participants were diagnosed by coronary artery angiography, exclused by the criteria of autoimmune disease, serious diseases with liver and kidney, actue or chronic infections, drug administration of statins, immunosuppressants or steroid drug in the past 3 months. Peripheral blood of 116 participants were continuously collected, including 30 patients with acute myocardial infarction (AMI, n=30),30 patients with unstable angina pectoris (UAP, n=30),30 patients with stable angina pectoris (SAP, n=30), and 26 cases as control group (n=26). The expression of protein and mRNA of PD-L2 of monocytes were detected by flow cytometry and reverse transcriptase Real time Quantitative Polymerase Chain Reaction (RQ-PCR). Serum IL-6 and MCP-1 concentration were detected by enzyme-linked assay (ELISA), serum hs-CRP was detected by immunoturbidimetry. PartⅡ:Peripheral blood monocytes were separated and purified from 15 healthy volunteers, the monocytes of the same volunteer were randomly divided into six groups, they were control group, IFN-γintervention group (SIM 0 group), IFN-γwith 1×10-6mmol/L simvastatin group (SIM 1 group), IFN-γwith 5×10-6mmol/L simvastatin group (SIM 5 group), IFN-y with 10×10-6mmol/L simvastatin group (SIM 10 group), IFN-γwith 50×10-6 mmol/L simvastatin group (SIM 50 group), the monocytes of all the groups were cultured for 48 hours, after that, the expression of PD-L2 in monocytes were detected by flow cytometry.Statistical Methods:SPSS 13.0 statistical software was used to analyze data. Data was presented as Mean±S.D. Homogeneity of variance test is applied before ANOVA, One-Way ANOVA was used in comparision between groups, LSD method was used in multiple comparisons between groups while homogeneity of variance was assumed, Dunnett's T3 was used while homogeneity of variance was not assumed. Pearson correlation and stepwise regression was used in PD-L2 and IL-6, MCP-1, hs-CRP correlation analysis. In partⅡ, Paired T test was used in the comparison between control group and only the IFN-γintervention group of the same volunteer, while the analysis of Two-Way ANOVA was used in other comparisons, LSD method was used in multiple comparisons between groups while homogeneity of variance was assumed, Dunnett's T3 was used while homogeneity of variance was not assumed. P value< 0.05 was considered to be significant.Results:1. Compared with the control group, the expression of protein and mRNA of PD-L2 in peripheral blood monocytes in patients with SAP, UAP and AMI were significantly higher, the difference was statistically significant (P<0.001, respectively).2. Compared with the control group, the concentration of serum IL-6,MCP-1 and hs-CRP in group of UAP and AMI increased significantly (P<0.001, respectively).3. The level of serum hs-CRP was higher in CHD patients, but there was no statistically significant difference between SAP group and contol group (P=0.683).4. There was a significant positive correlation among the expression of PD-L2 and the concentration of IL-6, MCP-1, hs-CRP in patients with CHD (r=0.563, r=0.736, r=0.778,P<0.001, respectively). After eliminating the influences of age, gender, smoking, systolic blood pressure, diastolic blood pressure, total cholesterol and triglycerides, low density lipoprotein-C, fasting blood sugar, body mass index, the correlations of hs-CRP, MCP-1 and PD-L2 were still significant(P<0.001).5. Compared with the control group, the expression of PD-L2 in IFN-γintervention group was significantly higher (P<0.001).6. Compared with IFN-γintervention group (SIM 0 group), the expression of PD-L2 decreased gradually in SIM 1 group, SIM 5 group, SIM 10 and SIM 50 group, the difference has statistical significance (P<0.05).7. The expression of PD-L2 between SIM 0 group and SIM 1 group has no statistical significance(P=0.088).Conclusions:1. In patients with CHD, when inflammatory response is enhanced, the expression of PD-L2 on peripheral monocytes increases. 2. Inflammatory cytokines, including serum IL-6, MCP-1 and hs-CRP increase significantly in patients with CHD, particularly in UAP group and AMI group.3. In vitro, pro-inflammatory cytokine IFN-y can promote the expression of PD-L2 on monocytes, while different concentration of simvastatin can inhibit this action, it provides new theory for the effect of anti-imflammation and immunomodulation of simvastatin in AS.4. It needs further study to explore the influence of the function of proliferation and secretion of T lymphocyte after the interaction of PD-L2 and PD-1, and to investigate the mechanism of immunomodulation and signal transduction of PD-1/PD-L of AS in animal models.