The Function Of T-Lymphocyte Mediated Immunoreaction In The Pathogenesis Of Abdominal Aortic Aneurysms

Abdominal aortic aneurysms (AAA) is a kind of serious blood vessel disease that is featured by the progressive dilatation of abdominal aorta. This disease has the characteristics of concealment in forming and high mortality in the end. With the aging population and the rapid development of imaging diagnosis, the incidence of AAA increases year by year. It has been the focus of attention that how to effectively prevent and treat the forming and dilatation of AAA, but its early pathogenesis still remains unclear up to now, which seriously blocks the preventing and treating effects of AAA. Artery atherosclerosis, hypertension and smoking have been considered as the high risk factors resulting in AAA. Keeping far away from or controlling these high risk factors can prevent the development of AAA to a certain extent. We have much deeper recognition of the pathogenesis of AAA with the rapid development of modern science, such as genetics, molecular biology and immunity. Now AAA is considered as the result of combined action of such factors as environment, heredity and biochemistry, and so on. With the combined action of infection, arteriosclerosis, hypertension and smoking, the artery vessel's wall gets damaged and the body immunoreaction comes to be activated that results in the vessel's wall inflammatory cell infiltration, vascular smooth muscle cell (VSMC) apoptosis, the imbalance of extracelluar matrix (ECM) and the rupture of arterial middle elastic fiber, which finally leads to the forming, accelerated dilatation and break of the aneurysm. Therefore, the viewpoint has been consentaneously accepted that AAA most possibly represents the inflammatory response of immune media.As the chronic inflammation and immunoreaction in arterial local parts are the key factors of pathogenesis of AAA, the present researches give emphasis to the type of inflammatory cell infiltration and the immunoreaction mechanism mediated by them in the forming process of AAA. Firstly, research indicates that arterial vessel's wall has the micro-environment leading to immunoreaction. There is a type of vascular-associated lymphoid tissue (VALT) in normal arterial intima, similar to the mucosa-associated lymphoid tissue in respiratory and gastrointestinal tracts, in which distribute some cell sets organized by immunocompetent cells and antigen presenting cells, which scout and select the deleterious endogenous or exogenous antigen in blood vessel tissue. The great amount of VALT in the outer membrane of AAA provide suitable place for arterial local immunoreaction. At the same time, the type of inflammatory cell infiltration in the forming process of AAA has been made clear. Research indicates that there is a lot of inflammatory cell infiltration in local AAA, among which the infiltration of T-lymphocyte and macrophage cell are the most prominent ones. Further research indicates that there are a great amount of inflammatory factors produced by the proliferation of activated T-lymphocyte. Such research results arouse people's interests in the function of T-lymphocyte in the pathogenesis of AAA. With the progress in research, more and more evidence shows that the forming and dilatation is the process of adaptive cellular immunoreaction under the interaction of T-lymphocyte subset and macrophage cell with the combined action of several factors, but the accurate immunoreaction mechanism still remains unclear. To further study the T-lymphocyte mediated immunoreaction mechanism in the forming and developing process of AAA has very important significance for making clear the pathogenesis of AAA and so as to have targeted intervention. The peripherally matured T-lymphocyte can be classified to two subsets according to CD molecule:CD4+ and CD8+; to help T cell (Th), cytotoxic T cell (Tc) and suppressor T cell (Ts) according to the function. And CD4+Th cell can further be classified to the subgroups of ThO, Th1, Th2 and Th3 according to the cytokine secreted. Activated by antigen, the original T cell, i.e. ThO cell, can produce many kinds of cytokines. Then ThO cell differentiates to Th1 and Th2 under the influence of such factors as cytokine, antigen characteristic and other hormone. Th1 cell leans to secrete IL-2, IFN-γand TNF-α, which relates to Tc cell and the proliferation, differentiation and maturity of TDTH and mainly promotes the immune response of cellular media. While Th2 leans to differentiates IL-4 and IL-10, which relates to the proliferation and maturity of B cell and the forming of antibody, so as to strengthen the humoral immune of antibody media. In normal conditions, there is net adjustment in Th1/Th2 and the cellular function tends to dynamic balance. But in pathological conditions, there may be functional imbalance in Th1/Th2 cellular subgroup, which promotes the forming of body immune and inflammatory response.What is the inflammation status in the body and arterial local parts of AAA patients in the process of AAA? Is there functional imbalance of T-lymphocyte subgroup in the forming and developing process of AAA? Does T-lymphocyte mediate the immunoreaction and inflammatory reaction in early period of AAA, and how does it work? This experimental study, which starts at testing T-lymphocyte subset and subgroup in the peripheral blood of AAA patients in different periods, discusses the function of T-lymphocyte mediated immunoreaction in the forming and dilatation of AAA, which provides a certain experimental basis for clarifying the immune pathogenesis of AAA.This experiment divides into three parts. Firstly, we select 30 patients from three clinical groups:normal control group, small AAA patients group (the diameter of abdominal aorta<5.5cm) and large AAA patients group (the diameter of abdominal aorta>5.5cm). By enzyme-linked immunosorbent assay (ELISA), it tests the level of Thl/Th2 cytokine in the peripheral blood and T-lymphocyte culture supernate of the patients in all groups, in order to understand the inflammation activating status and the balancing situation between Th1/Th2 cellular subgroups in the AAA patients at different period. By testing cellular subset distribution of CD4+CD28-T and CD4+CD28+T in peripheral blood by FCM technology, it discusses the function of the change of T-lymphocyte subsets at different developing periods of AAA. To further understand the function of T-lymphocyte in the early process of AAA forming, an AAA animal model is established by enhancing perfusing elastase to the infrarenal abdominal aorta of the rats. The samples of the peripheral blood and abdominal aorta of the rats in every group are taken after 3 days,7 days and 14 days of the surgery, in order to dynamically observe the distribution and infiltration of the T-lymphocyte subsets in the rats' abdominal aorta in all the groups and analyze the influence of their change on the forming and development of AAA. Finally, the emphasis is set on observing the gene expression difference between arterial local IFN-γand IL-4 and its relations with the change of arterial matrix metalloproteinase during the forming process of the rats' AAA, so as to discuss the relations between T-lymphocyte mediated inflammation and immunoreaction and the change of matrix metalloproteinase, in order to further understand the immune pathogenesis of AAA. The study of the function of T-lymphocyte in the early period of AAA forming and development aims to provide an experimental basis for immune media theory of AAA forming and development and provides a theoretical platform for AAA prevention and treatment. The results are listed in the following sections.1. The change of T-lymphocyte activation and subsets in peripheral blood of AAA patients1.1 The comparison of the patients'clinical data of all groupsAll the 30 patients selected are separated into three groups, that is,10 cases in small AAA group,10 in large AAA group and 10 in normal control group. The comparison of clinical data indicates that:there is no significant difference between the age and gender ratio (P>0.05), and no significant difference between the high risk factors, such as smoking and hypertension in every group. All the studying objects have no such disease as infection, autoimmune disease, malignant tumor and hepatic and renal insufficiency.1.2 The change of Th1/Th2 subgroup-associated inflammatory factors in the patients' plasma of all groupsThe expression level of the serum IFN-γof the normal control group, small AAA group and large AAA group are 4.10±0.9,17.25±2.20pg/ml and 21.26±3.35, and expression level of the serum TNF-αare 33.3±6.63,55.05±5.20 and 59.62±7.21pg/ml. IFN-γand TNF-αhave significant difference among three groups (FIFN-γ=142.70 & FTNF-α=51.76, P<0.001). The expression level of the serum IL-4 of the normal control group, small AAA group and large AAA group are 5.27±1.72,6.82±1.72 and 5.26±1.32 pg/ml, and expression level of the serum IL-10 are 32.62±8.69, 33.37±9.37 and 30.67±10.46pg/ml. IL-4 and IL-10 have no significant difference among three groups (FIL-4=5.38 & FIL-10=0.27, P>0.05).1.3 The test of peripheral blood isolated T-lymphocyte culture supernate Thl/Th2 cytokine of all groupsThe expression level of the culture supernate IFN-γof the normal control group, small AAA group and large AAA group are 620.05±146.48,1740.05±291.58 and 1932.26±145.86 pg/ml, and expression level of the culture supernate TNF-αare 510.39±62.41,820.39±151.88 and 935.14±80.83pg/ml. IFN-γand TNF-αhave significant difference among three groups (FIFN-γ=117.93 & FTNF-α=43.24, P<0.05). The expression level of the culture supernate IL-4 of the normal control group, small AAA group and large AAA group are 54.69±9.82,67.69±7.74 and 60.8±6.66pg/ml, and expression level of the culture supernate IL-10 are 28.69±6.22,36.39 ±7.26 and 29.46±4.45pg/ml (FIL-4=6.323 & FIL-10=4.85, P>0.05).1.4 The relation between diameter of abdominal aorta and concentration of Th1/Th2 cytokine of AAA patients of all groupsThe diameter of abdominal aorta of the normal control group, small AAA group and large AAA group are 2.49±0.29,4.03±0.59,5.16±1.49cm, and the correlation coefficient with the concentration of IFN-γ, TNF-α, IL-4 and IL-10 in plasma are: RIFN-γ=0.847, P<0.01; RTNF-α=0.695, P<0.01; RIL-4=-0.071, P>0.05; RIL-10=-0.01, P>0.05. And the correlation coefficient with the concentration of IFN-γ, TNF-α, IL-4 and IL-10 in cell culture supernate are:RIFN-γ=0.718, P<0.01; RTNF-α=0.765, P<0.01; RIL-4=0.18, P>0.05; RIL-10=0.083, P>0.05.1.5 The amount of CD4+ and CD8+ T-lymphocyte in peripheral blood and ratio of CD4+CD28- and CD8+CD28- T-lymphocyte subgroup to total amount of CD4+ and CD8+ T-lymphocyteThe amount of CD4+ T-lymphocyte of the normal control group, small AAA group and large AAA group are 1211±654.1,906.3±250.62,717.4±200.76/ul. The amount of CD8+ T-lymphocyte are 333.1±235.48,301.3±153.13,333.5±140.09/ul. The ratio of CD4+/CD8+ are 3.01±1.91,3.65±1.89,2.70±1.68. CD4+ and CD8+ T-lymphocyte has no significant difference among three groups (FCD4+=0.693, FCD8+=0.369, P>0.05).The ratio of CD4+CD28- in CD4+ lymphocyte of the normal control group, small AAA group and large AAA group are 3.87±0.73%,12.45±5.36%,8.77±3.01%. The ratio of CD8+CD28- in CD8+ lymphocyte are 3.93±0.56,6.2±1.61,5.8±2.3. CD4+CD28-has significant difference among three groups (FCD4+CD28-= 14.49, P<0.05). CD8+CD28-has significant difference between large AAA group and normal control group (FCD8+CD28-=5.347, P<0.001), and it has no significant difference among other two groups (P<0.05). The correlation coefficient among the diameter of abdominal aorta and CD4+CD28-and CD8+CD28-of the normal control group, small AAA group and large AAA group are:RCD4+CD28-=0.532, P<0.05, RCD8+CD28-=0.418,P<0.05.2. The function of T-lymphocyte infiltration in the forming process of experimental AAA2.1 The establishment of AAA rat modelThe AAA animal model is established by improved method of enhancing perfusing elastase to the infrarenal abdominal aorta of the rats. The diameter of the rats' abdominal aorta of the groups before surgery (normal control,3 days after surgery,7 days after surgery and 14 days after surgery) are 1.11±0.13,1.12±0.15,1.13±0.05, 1.12±0.16. There is no significant difference among the groups (F=0.073, P>0.05). During the difference comparison of the rats' abdominal aorta dilatation rate of all groups when sampling, there is no significant difference between normal control group and 3 days after surgery group (F=141.25, P>0.05), and there is significant difference among other groups (F=141.25, P<0.05).2.2 The change in morphology and histology of the rats'abdominal aorta of all groupsThe general shape of the abdominal aorta of normal control group does not have obvious change. That of 3 days after surgery starts dilatation. That of 7 to 14 days after surgery has accelerated dilatation, and the AAA occurs 14 days after surgery, with obvious adhesion and hardening of local artery. The pathological section HE staining and EVG staining indicate that the intima of the rat's artery gets thickened, vascular smooth muscle cell gets apoptosis and the middle elastic fiber decreases or disappears 14 days after surgery.2.3 The function of T-lymphocyte infiltration in early period of AAA formingThere is no obvious inflammatory cell infiltration in artery of normal control group. There is a little T-lymphocyte (20.20±11.89,F=88.58,P=0.000), B-lymphocyte (10.40±3.92,F=29.99,P=0.000) and macrophage cell (7.70± 2.58,F=17.50,P=0.000) infiltration in the outer membrane of the artery of 3 days after surgery group. There is obvious T-lymphocyte(39.60±15.29), B-lymphocyte(14.0±5.63)and macrophage cell (8.20±3.05)infiltration both in middle and outer membrane of the artery of 7 days after surgery group. There is a great amount of T-lymphocyte(165.40±44.22), B-lymphocyte(52.10±22.76) and macrophage cell (28.90±15.98)infiltration in middle and outer membrane of the artery of 14 days after surgery group.2.4 The ratio of CD4+ and CD4+CD28- T-lymphocyte subgroup to total amount of CD3+ and CD4+ T-lymphocyteThe ratio of CD4+ in CD3+ lymphocyte of the normal control group,3 days,7 day,14 day after surgery group are22±8.88%,25.6±6.32%,26.7±7.04%,27.4±4.17%(F=0.30,P=0.309). The ratio of CD4+CD28- in CD4+ lymphocyte are2.03±0.73%,4.16±1.31%,8.13±2.12%,5.63±1.23%(F=31.768,P=0.000)3. The relation between Th1/Th2 cytokine and matrix metalloproteinase 9 gene expression and the forming of AAA3.1 The change of Th1/Th2 cytokine in the rats'plasma of all groupsThe concentration of IFN-γin the rats'plasma of all groups (normal control,3 days after surgery,7 days after surgery and 14 days after surgery) are 30.04±11.18ng/L,37.88±11.86ng/L,52.18±17.72ng/L,77.69±19.71ng/L. There is no significant difference between normal control group and 3 days after surgery group (F=18.11, P>0.05), and there is significant difference among other groups (F=18.11, P<0.05). The result indicates that the concentration of IFN-γincreases obviously with the increase of the diameter of abdominal aorta of all groups. The concentration of IL-4 in the rats' plasma of all groups (normal control,3 days after surgery,7 days after surgery and 14 days after surgery) are 24.15±10.74ng/L,27.45±9.02ng/L, 29.23±9.04ng/L,29.95±9.15ng/L (F=0.738, P>0.05).3.2 The test by fluorescence quantitative PCR of the IFN-γ,IL-4 and MMP-9mRNA expression in the rats' abdominal aorta of all groupsThe expression of IFN-γIFN-γmRNA in the rats' artery of all groups (normal control,3 days after surgery,7 days after surgery and 14 days after surgery) are 30.04±11.18,37.88±11.86,52.18±17.72,77.69±19.71. There is no significant difference among 7 days,3 days and 14 days after surgery groups (F=7.678, P>0.05), and there is significant difference among other groups (F=7.678, P<0.05). The expression of IL-4mRNA in the rats' artery of all groups (normal control,3 days after surgery,7 days after surgery and 14 days after surgery) are 3.58±1.68,4.52±1.92, 5.77±2.07,8.72±2.84. There is no significant difference among 3 days after surgery, normal control and 7 days after surgery groups (F=10.58, P>0.05), and there is significant difference among other groups (F=10.58, P<0.05). The expression of MMP-9mRNA in the rats'artery of all groups (normal control,3 days after surgery,7 days after surgery and 14 days after surgery) are 2.25±1.43,4.52±2.08,5.98±2.54, 8.03±3.10. There is no significant difference among 3 days after surgery, normal control and 7 days after surgery groups, and between 7 days and 14 days after surgery groups (F=10.82, P>0.05), and there is significant difference among other groups (F=10.82, P<0.05). The Spearrnan correlation analysis of the data shows that the expression level of IFN-γand MMP-9mRNA in the rats' blood vessel's wall of all groups does not have obvious correlation (R=0.329, P<0.05), while the expression level of IL-4 and MMP-9mRNA has positive correlation (R=0.74, P<0.05).Through the above three experiments, the conclusion can be made that:1. with the increase of Th1-associated inflammatory factor IFN-γand TNF-αin the peripheral blood of AAA patients, the concentration of IFN-γand TNF-αin T-lymphocyte culture supernate increases obviously, and the concentration of IFN-γand TNF-αin the peripheral blood of AAA patients has positive correlation with the diameter of abdominal aorta, which indicates that the AAA patients are in the inflammation activated status.2. the ratio of CD4+CD28-cell in the peripheral blood of AAA patients increases obviously, and it increases more obviously in that of small AAA patients, which indicates that the ratio of T-lymphocyte subsets has obvious change in the early period of AAA forming and development and the immunoreaction in the body is in the activated status.3. Elastase perfusion can lead to the forming of experimental AAA. A stable AAA animal model can be established by the improved experimental method. The inflammatory cell infiltration and the rupture and even disappearance of elastic fiber are the main mechanism of AAA. T-lymphocyte infiltration in the abdominal aortic wall is the most important inflammatory cell during the early period of AAA forming process.4. Thl cytokine IFN-γmay have relations with the early assembling and infiltration of abdominal aortic inflammatory cells. The assembling of Th2 cytokine IL-4 in local parts of abdominal aorta may have relations with the increase of the activity of matrix metalloproteinase during the forming process of AAA and the inducement of the reconstruction of the extracellular matrix (ECM).5.The method of achieving the effect of anti-inflammation and immune adjustment through regulating and controlling associated cytokine and antigen present has broad prospects in the prevention and treatment of the forming and development of AAA.