Correlation Of CD4~+CD28~-T Lymphocyte And HSP60 On The Mechanism Of Acute Coronary Syndrome

Acute coronary syndrome(ACS) is coronary heart disease(CHD) special spectrum,including unstable angina pectoris(UAP),ST-segment elevation acute myocardial infarction(STEMI),non-ST-segment elevation acute myocardial infarction(NSTEMI) and sudden cardiac death.Research shows that the ACS are almost invariably associated with irregular erosion or ruptured plaques,that is unstable plaques.Ruptures preferentially occur where the fibrous cap is thin and partly destroyed.At these sites,activated immune cells are abundant.They produce numerous inflammatory molecules and proteolytic enzymes that can weaken the cap and activate cells in the core,transforming the stable plaque into a vulnerable, unstable structure that can rupture,induce a thrombus,and elicit an acute coronary syndromeWhat causes a silent atherosclerotic lesion to rupture? Activated macrophages,T cells,and mast cells at sites of plaque rupture produce several types of molecules—inflammatory cytokines,proteases,coagulation factors,radicals,and vasoactive molecules—that can destabilize lesions.They inhibit the formation of stable fibrous caps,attack collagen in the cap,and initiate thrombus formation.All these reactions can conceivably induce the activation and rupture of plaque,thrombosis,and ischemia.Today,the concept that atherosclerosis is an inflammatory disease is no longer controversial,and instead of proving that point,we can focus on investigating how this inflammation is regulated.T cells are of particular interest,both because of their secretion of mediators that influence plaque development and because their activity depends on the triggering of specific antigens that are found within the disease site.T cells are regulated by soluble and membrane-bound molecules from many cell types and,in turn,they act on most other cells.This network of cell-to-cell interactions affects the development of many inflammatory and autoimmune diseases. patients with acute coronary syndrome may exhibit a peripheral burst of an unusual and aggressive CD4~+T cell subset that lacks the expression of the CD28 receptor (CD4~+CD28~-T cells).In these patients,CD4~+CD28~-T cells can represent up to 50%of the circulating CD4~+T cell compartment,and are also preferentially recruited into culprit lesions.These long-lived clonal T cells deficient in CD28 expression are commonly found in patients with inflammatory syndromes and persistent infections.Considering that CD28 loss is the most consistent immunological marker of aging,CD28~-T cells represent prematurely senescent cells resulting from persistent immune activation. These unusual lymphocytes have aberrant functions that contribute to disease-related immune abnormalities,and the degree of accumulation of CD28~-T cells predicts the severity of clinical manifestations.Senescent and pre-senescent T cells are memory cells characterized by the loss of CD28 expression,and have limited or no capacity for cell division due to telomere erosion and to increased transcription of mitotic inhibitors,such as p16 and p21.They are generally long-lived and functionally active. They have novel functions that might be attributed to their acquisition of NK-related receptors(NKRs) such as KIR,CD94,NKG2 and KLRG1.KLRG1 is an inhibitory receptor known to block TCR-driven proliferation.NKR+CD28-CD4+ and CD8+ T cells are distinct from the so-called natural killer(NK) T cells. CD4~+CD28~-T cells are expanded in the peripheral blood of patients with ACS and infiltrate unstable coronary plaques,where they undergo clonal expansion, probably triggered by specific antigens.These cells are capable of releasing large amounts of interferon IFN-γand they are the dominant population of IFN-γproducing cells in the peripheral blood of patients with UAE Because of the increased IFN-γproduction,one of their functions is the activation of monocytes and macrophages; indeed,monocytes from patients with UA display a molecular fingerprint of ongoing IFN-γstimulation.Therefore,CD4~+CD28~-T cells might be involved in the control of plaque-infiltrating macrophages.Endothelial cells are susceptible to this T-cell-mediated injury.Furthermore,in the presence of CRP at concentrations frequently found in patients at risk for coronary events,susceptibility of endothelial cells to T-cell-mediated cytotoxicity is increased.CD4~+CD28~-T cells also can effectively kill endothelial cells and smooth muscle cell in vivo.However,they acquire their cytolytic capabilities independent of the T cell receptor due to coexpression of APO2 ligand/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) capable of inducing apoptosis and stimulatory killer immunoglobulinlike receptors that recognize HLA classⅠmolecules and the adapter molecule DAP12 that leads to mitogen-activaed protein kinase cascade and ultimately induces degranulation of cytotoxic granules and the release of cytokines.These CD4~+CD28~-T cells also share features of central memory T cells because they can undergo clonal expansion within lesions and persist for years in the circulation,but they functionally resemble natural killer cells.Therefore,alongside other proinflammatory mechanisms, we hypothesized that the expansion of T lymphocytes with the functional profile of CD4~+CD28~-T cells might represent a key pathogenetic mechanism of ACS.CD4~+CD28~-T lymphocyte proliferation may reflect the existence of the atherosclerotic plaque microbial antigens or self-antigen.Epidemiological Investigation found coronary heart disease closely associated with Chlamydia pneumoniae,Helicobacter pylori,and other microorganisms antigen,which these organisms common carried HSP60.An autopsy coronary artery found HSP60 was strongly expressed in atherosclerotic plaque area,while in normal coronaries and in lesion-free coronary circumference,very weak reaction in medial smooth muscle cells was observed.In the lesion area,endothelial cells,intimal and medial smooth muscle cells,and intimal foam cells demonstrated HSP60 immuno-positivity. Furthermore,it found HSP60 expression in adventitial mononuclear cell infiltrates,in adventitial vasa vasorum.CD4~+CD28~-T cells is reported to be the result of their chronic exposure to a restricted spectrum of antigens,which may be present in the atherosclerotic plaque and/or the peripheral circulation.HSP60 presentation by target cells was a prerequisite for activation of CD4~+CD28~-T cells in at least a proportion of ACS patients.the above study proposed correlation of ACS and HSP60over-expression in atherosclerosis caused by microbial infection and stress factor,also of ACS and CD4~+CD28~-T lymphocyte proliferation resulted of/from enhanced inflammation. Possible mechanism is the CD4~+T cell persistently responsed against self-HSP60 antigen,has strongly aroused CD4~+T lymphocytes immune senescence and Reconstruction and changed its phenotype,resulting in down-regulation expression of CD28 and the de novo expression of KIRs and NKRs and acquired some newly function,which mediated damage result in the atherosclerotic plaque destabilization and caused ACS.However,in patients with ACS,correlation between HSP60 and frequency of CD4~+CD28~-T lymphocyte has not yet been reported.A study found that HSP60 can act on CD4~+T lymphocytes directly through TLR2 receptors,inhibit secretion of Th1 cytokines.animal experiments found that HSP60 can promte functional mature of DCs and DCs loaded HSP60 can promote atherosclerosis progress,but it is still not clear that wheather HSP60 can cause CD4~+T lymphocyte phenotype bias and promte expansion frequency of CD4~+CD28~-T lymphocyte induced by loaded HSP60 DCs.It has become evident that statins have immunomodulatory properties that alter the function of both T cells and antigen presenting cells.Statin can inhibit inflammatory response,enhance plaque stability, benefit to patients with ACS.It has been confirmed in clinic that correlation between statin use and reduction of the frequency of CD4~+CD28~-T lymphocytes in patients with UAP and an association between statin use and reduce concentrations of Hs-CRP. the benefits of statin therapy ACS at least in part,from the CD4~+CD28~-T lymphocyte decline,but that correlation has not been confirmed in vitro experiments.In view of the above problems,our study first observed correlation between levels of serum HSP60 and frequency percentage of peripheral blood CD4~+CD28~-T lymphocytes in patients with ACS.then,using cell culture and magnetic cell separation techniques in vitro,to observe frequency of CD4~+CD28~-T lymphocytes detected by flow cytometry and the secretion of IFN-γdetected by enzyme-linked immunosorbentassay(ELISA) of CD4~+T cells,wheather it result in CD4~+CD28~-T lymphocyte proliferation and secretion of IFN-γincrease when CD4~+T lymphocyte treated HSP60.In addition,we observe the effect of atorvastatin and intervention of mevalonic acid on interaction of DCs loaded HSP60 and CD4~+CD28~-T lymphocyte.We try to prove that HSP60 is one of self-antigens to promote the CD4~+CD28~-T lymphocyte proliferation,and to demonstrate HSP60 and CD4~+CD28~-T lymphocyte association with development of ACS.This study was divided into three parts:①Clinical Research:all subjects were divided into three groups:the normal control group,patients with SAP and patients with ACS.The serum CRP,HSP60,TNF-αand IFN-γ,were detected by ELISA,the percentage of CD4~+CD28~-T lymphocytes in CD4~+T lymphocytes were detected by FCM,emphasis on the expression of these indicators in ACS patients and the correlation between these indicators and the traditional cardiovascular risk factors.②Research in vitro:the peripheral blood mononuclear cells(PBMC) are obtained by cell separation technology,with whose orientation are induced to differentiate into mature DCs;the CD4~+T lymphocytes are obtained by magnetic activated cell sorting technology,which are co-cultured with mature DCs in vitro.Using FLM and ELISA to investigate at whether the HSP60 can promote the cells from PBMC of human source to the mature DCs and whether the HSP60-loaded DCs can reduced CD28 molecule expression and IFN-γsecretion of CD4~+T lymphocytes.③Drug Intervention Trial:the HSP60-loaded DCs are co-cultured with CD4~+T lymphocytes with the intervention of atorvastatin(Ato) and mevalonate acid(MVA),observing the variation of CD28 molecule expression and IFN-γsecretion of CD4~+T lymphocytes. The results are described as below:1.the relationship of peripheral blood CD4~+CD28~-T lymphocyte and serum HSP60 in patients of ACS.1.1 The expression of serum HSP60,CRP,TNF-α,IFN-γand the number of peripheral blood CD4~+CD28~-T lymphocytes were significantly higher(P=0.000) in ACS group than in SAP and normal group,while no significant difference of those were exsited between SAP and normal group(P＞0.05),except of higher serum IFN-γexpression in SAP group compared to normal(P=0.000).ACS had a relationship with the high expression of serum CRP,HSP60,TNF-α,IFN-γand the increased number of peripheral blood CD4~+CD28~-T lymphocytes.1.2 ACS correlated with peripheral blood CD4~+CD28~-T lymphocytes when the percentage of peripheral blood CD4~+CD28~-T cells exceed to the median 4.29%in the percentage of total samples(x~2=32.759,P=0.000).The number of peripheral blood CD4~+CD28~-T cells was found be valuable for the ACS diagnosis,as the result of ROC curve analysis described the area under the curve was 0.763 and the standard error was 0.031(P=0.000).ACS also correlated with serum HSP60 when the expression of serum HSP60 exceed to the median 317.7ng/L in the total samples(x~2=34.550,p= 0.000).The high expression of serum HSP60 was valuable for the ACS diagnosis too, as the result of ROC curve analysis described the area under the curve was 0.810 and the standard error was 0.028(P=0.000).1.3 Spearman Correlation Analysis found the number of peripheral blood CD4~+CD28~-T cells correlated unusually with the expression of serum HSP60,IFN-γand ages in ACS group(r=0.763,r=0.660),but not significant with serum HsCRP, TNF-α,gender,hypertension and smoking.After control of the age,IFN-γ,HsCRP, TNF-α,sex,hypertension,smoking and other factors,Partial Correlation Analysis showed the number of peripheral blood CD4~+CD28~-T cells correlated significantly with the expression of serum HSP60(r=0.527,P=0.000).These analysis maybe show the number of peripheral blood CD4~+CD28~-T cells correlated with The expression of serum HSP60 in the ACS group.2.HSP60 induced CD4~+CD28~-T lymphocytes proliferation.2.1 HSP60 effect on CD4~+T lymphocyte phenotype and secretion of cytokines Using cell co-culture,experiment is divided into four groups:①negative control group;②1mg/L HSP60;③10mg/L HSP60;④100mg/L HSP60 group(n=6,each group with different concentrations of HSP60 for antigen stimulation),observing each change after 26 hours and 72 hours.①Concentrations of IFN-γin the supernatant:The different doses of HSP60 groups had lower concentrations of IFN-γthan control group(P＜0.05),but the difference of IFN-γconcentrations in each HSP60 groups wasnt significant(P＞0.05).The differance of IFN-γconcentrations in four groups also wasnt significant in the time of 26h and 72h(P＞0.05).in the secretion of IFN-γin contrast differences.This mean HSP60 could directly stimulate CD4~+T lymphocytes and inhibite those secretion of IFN-γ,but seem to have no connection with the doses.②CD4~+CD28~-T lymphocytes number detection:The four groups showed no significant difference(P＞0.05),But the different numbers of CD4~+CD28~-T lymphocytes in four groups was significant in the time of 26h and 72h(P＜0.05).This mean the number of CD4~+CD28~-T lymphocyte would increase with the time,but HSP60 may not directly affect CD4~+CD28~-T lymphocyte proliferation.2.2 HSP60 impact on human PBMC DCs mature:Test divided into three groups(n=6):the control group,low-dose HSP60 treatment group and high-dose HSP60 treatment group.①The expression of CD80 and CD86 positive rate in HSP60 groups were significantly higher than that of the control group(P=0.000).The expression of CD80 and CD86 positive rate in two HSP60 groups also increased significantly(P=0.000). This mean HSP60 maybe promote human PBMC DCs expressing CD80 and CD86 costimulatory molecules,it seem that the function of antigen presenting to T lymphocytes enhance by the doses of HSP60.②The proliferation of T lymphocytes in HSP60 groups were significantly higher than that of the control group(P=0.000).The proliferation of T lymphocytes in two HSP60 groups also increased significantly(P=0.000).This mean HSP60 maybe promote human PBMC DCs proliferation,it seem to depend on the doses.2.3 The effect of HSP60,DCs and CD4~+T lymphocytes mixed culture on the expression of CD4~+CD28~-T lymphocyte and secretion of IFN-γ.Test divided into 4 groups:PBS group(PBS+CD4~+T,n=6);DCs group(DCs+ CD4~+T,n=6);HSP60-DCs low dose group(low HSP60-DCs+CD4~+T,n=6); HSP60-DCs high dose group(high HSP60-DCs+CD4~+T group,n=6).Each group had mixed culture with DCs and CD4~+ cells by 1:10 and 1:1. The differances of supernatant IFN-γconcentrations in all group were significant (P＜13.05).Three factors of DCs,HSP60 dose and CD4~+T cells could affect the IFN-γconcentrations.DCs could promote the CD4~+T lymphocyte secrete IFN-γ;DCs add HSP60 could be more obvious,and high doses of HSP60 was particularly notable role in the co-culture system.Detection of CD4~+CD28-T lymphocyte percentage:the CD4~+CD28T lymphocyte percentage in HSP60-DCs low-dose and high-dose groups were significantly increased comparison with PBS group and DCs group(P＜0.05), but the high-dose and low-dose HSP60-DCs groups showed no significant difference (P＞0.05);DCs group and PBS group also showed no significant difference(P＞0.05); the group of mixed culture with DCs and CD4~+ cells by 1:10 and 1:1 had no significant difference too(P＞0.05).This mean only with HSP60 the DCs could significantly induce CD4~+T lymphocyte lower expressing CD28 molecules and increase CD4~+CD28-T-lymphocytes percentage,but it seem not to depend on the doses of HSP60.Mature DCs in vitro but not loading HSP60 antigen couldn't decrease CD4~+T lymphocytes expression of CD28.In addition,the increased amount of DCs also couldn't decrease CD4~+T lymphocytes expression of CD28.3.Atorvastatin effect DCs loaded HSP60 on the CD4~+CD28-T lymphocytes proliferation.Test divided into three groups:①HSP60-DCs+CD4~+T group(control group,n= 6);②HSP60-DCs+CD4~+T +Ato group(Ato group,n=6);③HSP60-DCs+ CD4~+T +Ato+MVA group(MVA group,n=6);Each group had mixed culture with DCs and CD4~+T cells by 1:10.3.1 Ato and MVA impact on HSP60-DCs promoting the IFN-γsecretion of CD4~+T lymphocytes.the supernatant IFN-γlevels in Ato group was significantly lower than that of control group(P＜0.05),there was no significant difference between MVA group and control group(P＞0.05).but the IFN-γlevels in MVA Group was higher significantly than that of Ato group(P＜0.05);this mean Ato inhibiting HSP60-DCs to induce CD4~+T lymphocyte secretion of IFN-γ,but it can be reversed by MVA.3.2 Ato and MVA impact on HSP60-DCs promoting the CD28 expression of CD4~+T lymphocytes.The CD4~+CD28T lymphocytes percentage in Ato group was significantly lower than that of control group(P＜0.05),there was no significant difference between MVA group and control group(P＞0.05).but the CD4~+CD28-T lymphocytes percentage in MVA Group was higher significantly than that of Ato group(P＜0.05);this mean Ato inhibiting HSP60-DCs to induce CD4~+CD28-T lymphocyte proliferation,but it can be reversed by MVA.Through this three-part experiment,we can draw the following conclusions:①Clinical study confirmed ACS patients with significantly proliferating of CD4~+CD28-T lymphocyte and expressing of Th1 cytokine,this suggests CD4~+T lymphocytes bias maybe exist in ACS patients,its unique cytotoxic properties was closely related to atherosclerosis unstability:the significantly higher HSP60 in ACS patients may be the reasons of unstable plaque rupture,HSP60 as a self-antigen related to ACS development and CD4~+CD28-T lymphocytes proliferation;②HSP60 could reduce the DCs cell surface molecules CD80 and CD86 in vitro, and impact of DCs mature and function,it also reduced CD4~+T lymphocytes to express CD28 molecules,resulting in CD4~+CD28-T lymphocyte proliferation and IFN-γincreasing secretion;③clinical and in vitro studies have confirmed that atorvastatin can upregulate CD28 molecule expression of CD4~+T lymphocytesThe MVA metabolic pathway may be involved in the mechanism.The antigen presenting by DCs with HSP60 and CD4~+T lymphocyte phenotype wrer effected by MVA,so CD4~+T lymphocytes increased the expression of CD28 molecule and inhibited the the secreting of IFN-γ.MVA maybe have an anti-inflammatory and immunoregulatory role and has broad prospects for the therapy of ACS development.