Expresson Of PD-1 On Peripheral Blood T Cells Of Patients With Coronary Heart Disease And Regulation Of Simvastatin

Coronary Heart Disease (CHD) is the main killer of human health. Heart disease epidemiology data indicates that with the changes of lifestyle, CHD in China has gradually increasing morbidity and mortality. The World Health Organization (WHO) estimates:around 2020, China will face the epidemic peak of CHD. Atherosclerosis (AS) is the pathologic basis of cardiac-cerebro vascular disease.500 years ago, atherosclerotic lesions had been found in the arteries of Egyptian mummies. It has been more than 100 years of history for human to understand AS and study its pathogenesis. There are different theories to explain the pathogenesis of AS, including endothelial injury, lipid infiltration, thrombosis and arterial smooth muscle cell proliferation. AS was categorized to inflammatory diseases by Russell Ross in 1999, Ross proposed AS is the arterial wall endothelial cells and smooth muscle cells after injury by an inflammatory fibroid tissue reaction. Recent advances in basic science have established a fundamental role for inflammation in mediating all stages of this disease from initiation through progression, ultimately, the thrombotic complications of AS. Now, there is growing evidence that AS is a chronic inflammatory disease, autoimmunity in AS may play an important role. T lymphocytes (T cells) are important immune cells involved in immune response specificity. Current evidence supports a central role for T lymphocytes in all phases of the AS process.AS is a complex disease characterized by smooth muscle cell proliferation, cholesterol deposition, infiltration of macrophages and lymphocytes and foam cells formation. It is confirmed that at all stages of atherosclerosis there are different degrees of lymphocyte infiltration, in which T cells were predominant. Compared with stable plaques, T cells were significantly increased in unstable plaques. Evidence from many human and rodent studies has established that T lymphocytes enhance inflammation in atherosclerotic plaques and contribute to lesion progression and remodeling. T cells interact with other components in plaque, and promote atherosclerosis; T cells involved in atherosclerosis-related autoantibodies formation such as ox-LDL antibodies, Hsp antibodies; Natural killer T cell dissoved macrophages and released of a large number of substances to promote atherosclerosis; T cells contribute to atherosclerotic plaque instability.The precondition for playing its pathophysiological role is T cell activation. The T lymphocyte activation requires two signals. The first signal is the combine of TCR-CD3 on T cell surface with MHC-Ⅱantigen peptide on antigen presenting cells (APC) Giving the specificity of immune response, but not T cell proliferation and secretion of cytokines. The second signal is co-stimulation signal, the T cell costimulatory molecule on the surface of APC induced by ligand combnding, starts, maintains and regulates the activation cascade, determines the T cells to proliferate, respond to state and even into apoptosis. Important costimulatory molecule and its ligand are:B7/CD28, VCAM-1/VLA-4, ICAM-1, LFA-3/CD2, CD40/CD40L, CD70/CD27, etc.CD28/CTLA-4 (cytotoxic T lymphocyte-associated antigen 4)-B7-1/B7-2 and CD40-CD40L signaling pathways has been shown to play an important role in regulating T cell activation and immune tolerance. In recent years, some new B7/CD28 superfamily were successively found, they seem to have had unique importance on regulation of activated T cell response. Like CTLA-4/B7, PD-1/PD-L (Programmed death-1/Programmed death-1 ligand) interaction also provides inhibitory signals of lymphocyte activation. And the complex network composed by positive and negative costimulation signals plays a critical role in immune activation and tolerance.PD-1 molecule is the immunoglobulin superfamily inhibitory receptor, is a type 1 transmembrane protein containing an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM), which is induced upon the activation of CD4 and CD8 Tcells as well as B, NKT and myeloid cells. PD-L1 (B7-H1) and PD-L2 (B7-DC) are the two ligands of PD-1, PD-L1 is expressed mainly in dendritic cells, monocytes/macrophages, B cells, activated T cells, as well as endothelial cells, tumor cells, IFN-α,β,γcan induce its expression. PD-L2 is expressed on dendritic cells, monocytes, IL-4. GM-CSF in vitro strongly induced the expression of PD-L2 on dendritic cells. PD-1 delivers a negative costimulatory signal to effector T cells by engagement with its ligands, inhibits T cell proliferation and cytokine production, inhibits immune response at initial and effect stages to prevent excessive immune injury and autoimmune diseases, maintains immune homeostasis.PD-1 negative costimulatory signal deficiency will lead to a variety of autoimmune diseases. Animal experiments showed that PD-1 signal deficient could develop lupus-like glomerulonephritis and arthritis in mice, and leading to autoimmune diabetes with rapid progress in NOD mice. Increasing in PD-1 negative costimulatory signals decreased T cells infiltration and inflammatory reaction in AS rabbit plaque; relieved experimental autoimmune encephalitis and infiltration of macrophages, CD4+, CD8+T cells in spinal cord. In recent years, researchers have found that the expression of PD-1 and its ligands were abnormal in many clinical diseases:viral infections (such as HBV, HIV), parasitic infections, transplantation immunology, autoimmune disease (such as SLE, systemic sclerosis), cancer and other disease, showing that PD-1 signaling played a protective role in the maintenance of peripheral tolerance and immune homeostasis.Hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) are potent cholesterol-lowering drugs. In addition to their cholesterol-lowering properties, statins exert a number of so-called'pleiotropic', vasculoprotective actions that include improvement of endothelial function, increased nitric oxide (NO) bioavailability, antioxidant properties, stabilisation of atherosclerotic plaques, regulation of progenitor cells, inhibition of inflammatory responses and immunomodulatory actions. Anti-inflammatory and immunomodulatory effects of statins were demonstrated in various of diseases such as rheumatoid arthritis, multiple sclerosis.AS is a chronic inflammatory disease, and has the characteristics of autoimmune, in which the mechanism of PD-1 is unclear. In this study we had investigate the negative costimulatory molecule PD-1 expression on PBTC in CHD patients, a preliminary analysis of its pathogenesis in AS was conducted. We had used of specific blocking antibodies to speculated the mechanism of PD-1 in AS. At last, we observed the effect of Simvatatin (Sim) on the expression of PD-1, which might provide a new evidence of Sim in immunological therapy of AS. PartⅠ:PD-1 expression on PBTC in CHD patientsObjective:To investigate the expression of PD-1 on PBTC in CHD patients.Methods:55 patients with CHD were enrolled into our research at the Department of Cardiology of our hospital.30 patients without CHD confirmed by coronary angiography were served as controls.The PBTCs of CHD patients and controls were isolated by Ficoll density gradient medium and nylon wool column. The expression of PD-1 was detected by direct immunofluorescence, flow cytometry and half-quantitative RT-PCR.Results:PD-1 expressed on PBTC of CHD patients and controls.The expression level of PD-1 in stable angina(SA) group and acute coronary syndrome(ACS) group was found to be lower than that of the normal control group (P<0.001, P<0.001, respectively). But there was no statistically significant difference between SA group and ACS group (P>0.05).Conclusions:The expression of PD-1 on PBTC from patients with AS was significantly down-regulated, which might play some role in the pathogenesis of CHD.Part 2 Immunological regulation of PD-1 on PBTC of CHD patients cultured in vitroObjective:To detect the Immunological regulation of PD-1 on T lymphocyte of CHD patients cultured in vitro. Methods:PBTC separation and purification as former.7 CHD patients and controls were selected from the population involved in our study. PBTC were fresh purified from patients and controls, and mixed with purified mDC from a third healthy individual at ratio of 20:1. Blocking antibodies were added to some cultures at a concentration of 10μg/ml, and they were instead by equal volume of PBS in the PBTCs alone group, or isotype antibodies in the control group. Then cultures were divided into 4 groups:PBTC group, PBTC+mDC group, PBTC+mDC+PD-1-Ig group, and PBTC+mDC+Ig group. Cells were cultured in 24 well plates for 72 hours in CO2 incubators at a temperature of 37℃.3H-thymidine (3H-TdR) incorporation was measured to evaluate DNA synthesis of T lymphocytes. The supernatant of each group was collected for cytokine detection.Results:1. The effect of PD-1 on proliferation of PBTC:The difference between groups in both controls and CHD patients was significant (P<0.001).3H-TdR incorporation increased more in PBTC+mDC+PD-1Ig groups than PBTC+mDC groups (P <0.001). Changes in PBTC proliferation caused by PD-1 blocking were less in CHD patients2. The effect of PD-1 on production of cytokines by PBTC:Differences between groups on production of IFN-γand IL-4 were significant (P<0.001). LSD test showed that, IFN-γand IL-4 production was significantly induced in PBTC+mDC group than in the PBTC group (P<0.001). Adding PD-1-Ig into PBTC+mDC group, IFN-γand IL-4 production was increased more significantly (all P<0.001). Changes in cytokine production caused by PD-1 blocking were less in CHD patients.Conclusions:PD-1 plays a critical role in inhibition of PBTC proliferation and production of cytokine, both in cantrols and CHD patients. But its capacity of inhibition seemed weakened in CHD patients.Part 3:Regulation of Sim on the expression of PD-1 on PBTC from CHD patientsObjective:To observe the effects of Sim on the expression of PD-1 on PBTC from CHD patients, find out new mechanisms of statins on the immune regulation on AS.Methods:15ml fresh peripheral blood of 12 patients with CHD was drawn for PBMC purification. PBMC from each patient were divided into three groups:control group (equal volume of PBS were added) and two Sim groups (final concentrations of 10-6 mol/L,10-5 mol/L).24 hours later, cells were collected, washed and adjust the concentration of 5×105/ml. PD-1 expression on T cells was detected by flow cytometry.Results:PD-1expression on T cells was significantly up-regulated by Sim with a concentration of 10-5 mol/L in patients with CHD (P<0.001).Conclusions:Statins have capacity of up-regulation of PD-1 expression on peripheral blood T cells of patients with CHD, Which may be involved in its immunomodulatory action in CHD.