Deficient CD4~+CD25~+ Regulatory Cell Function In Patients With Abdominal Aortic Aneurysms

ObjectiveAbdominal aortic aneurysm(AAA) represents a common vascular condition associated with progressive aortic dilation and life-threatening risk of rupture.The western clinical epidemiology survey shows that the incidence of AAA has risen rapidly in recent years.The prevalence rates estimated at between 1.3%and 8.9%in men and between 1.0%and 2.2%in women.Rupture of these aneurysms causes about 45,000 deaths per year in the USA.Most AAAs have proven to be a complex and dynamic remodeling process rather than a simple degenerative process.Recent human tissue studies and animal models have shown that the immune response contributes importantly to aneurysm disease,raising the probability that autoimmunity may be responsible for the pathogenesis and development of AAA.In immune response,T lymphocytes can free body of the encroachment of exogenous material by discriminating the exogenous material and initiating a series of immune reactions. Recent study shows that these T lymphocytes are controlled by the second-group cells (regulatory T cell,Treg),which can guarantee that T lymphocytes can't attack the normal cells and tissues,and thus avoids immunologic injury.So Treg cells play an indispensable role in maintaining self tolerance and stabilization of the peripheric immune system.The impaired Fas-induced apoptosis of T lymphocytes in AAA patients identified in our recent study results in the fact that autoreactive cells have the capacity to survive and may proliferate in the circulatory system of AAA patients, which initiates a series of biochemical and biomechanical activities that direactly relate to the dilation of th eabdominal aorta.There are many factors affecting the development and activity of Treg cell.Recently discovered forkhead/ winged-helix family of transcription factor(forkhead box P3,Foxp3.Foxp3 for mouse,and FOXP3 for human) plays a specific role in the autoimmune stabilization because of its control to the phenotype and activity of Treg cells.A number of examples front mouse models (and a few from patients) have shown that quantitative or qualitative defects in CD4~+CD25~+ Treg cells contribute to disease development.Meanwhile,mutations, absence and decreasing expression of the human gene FOXP3 were found to be the causes of some autoimmune diseases.Thus,the aim of the present work was to evaluate expression of FOXP3 mRNA and protein as well as functional suppressive activity in CD4~+CD25~+T cells isolated from blood mononuclear cells from AAA patients and age-and gender-matched healthy controls(HC) and to prove that the immune response contributes importantly to AAA.Methods1.We analyzed peripheral blood from AAA patients and HC to determine the percentage of CD3~+,CD4~+,CD8~+,CD4~+CD25~+ T cells in the total CD4~+ T cell population by means of flow cytometry.2.We analyzed peripheral blood from AAA patients and HC to determine the percentage of CD4~+CD25~+FOXP3~+Treg in the total CD4~+ T cell population by means of flow cytometry.We compared the expression of FOXP3 in the PBMC with both mRNA and protein detection assays in AAA patients and HC by Western blots and quantitative Real time PCR.3.By comparing CD4~+CD25~+ Treg suppressive capacity between AAA patients and HC,we investigated the defective functions of CD4~+CD25~+ Treg in AAA patients and there existed a correlation between functional suppression and expression of FOXP3 message and FOXP3 protein.ResultsWe analyzed peripheral blood from 22 AAA patients and 32 HC to determine the percentage of CD4~+CD25~+ T cells in the total CD4~+ T cell population by means of flow cytometry.There was no significant difference in the frequency of the CD4~+CD25~+Tregs population between AAA patients and HC(mean±SD 5.69±0.99% vs.mean±SD 5.88±1.55%;P>0.01).After CD4~+CD25~+ Treg of peripheral blood from AAA patients and HC was isolated and purified by means of flow cytometry,we identified the purity of cell,which exceeded 95%.The frequency of CD4~+CD25~+FOXP3~+ T cells in AAA patients(median 2.45%;mean±standard deviation(SD) 2.45±0.57%) was significantly lower than that in HC(median 3.80%; mean±SD 3.69±0.82%;P<0.01).We compared the expression of FOXP3 in PBMC with both mRNA and protein detection assays in AAA patients and HC.A comparison of FOXP3 mRNA expression by quantitative Real time PCR revealed lower message levels in PBMC from each of the AAA patients than those of HC and a significant difference between the two groups(P<0.01).Similarly,comparison of FOXP3 protein expression in PBMC isolated from these AAA patients and HC by Western blots demonstrated a consistent reduction in FOXP3 protein and a highly significant difference between the two groups(P<0.01).To assess functional suppression from the paired AAA and HC,we stimulated CD4~+CD25~-indicator cells mixed with CD4~+CD25~+T cells at the ratio of 1:1.Freshly isolated CD4~+CD25~+Tregs from AAA patients showed the same low proliferative response to immobilized anti-CD3 as the HC.The data indicated that freshly isolated CD4~+CD25~+ Tregs from patients with AAA exhibited significantly less suppressive activity than those from HC(P<0.01).To determine whether the loss of regulatory function in AAA was explained by a decrease in the intrinsic function of CD4~+CD25~+Tregs or an increase in the resistance of CD4~+CD25~- effector T cells to inhibition.we conducted mixing experiments with cells from patients with AAA and HC.Tregs from AAA failed to suppress the proliferation of autologous CD4~+CD25~- effector T cells as well as CD4~+CD25~- effector T cell from HC,whereas CD4~+CD25~+Tregs from HC readily suppressed the proliferative response of CD4~+CD25~- effector T cell from AAA.These data clearly indicate that the primary regulatory defect is in the function of CD4~+CD25~+Tregs isolated from the circulation of patients with AAA,and not a resistance of lupus CD4~+CD25~- effector T cells to suppression.Linear regression analysis shows that there is a correlation between functional suppression and expression of FOXP3 message and FOXP3 protein. ConclusionsAlthough our finding reveals no alterations in the frequency of CD4~+CD25~+ T cells in AAA patients,our data demonstrate for the first time a functional defect in CD4~+CD25~+ Tregs and a significant reduction in FOXP3 expression in a cohort of AAA patients enrolled in our study.Our study also for the first time correlates quantitative differences in human FOXP3 expression and functional suppression in CD4~+CD25~+ Tregs in AAA patients.The data clearly indicate that the primary regulatory defect is in the function of CD4~+CD25~+Tregs isolated from the circulation of patients with AAA, and not a resistance of lupus CD4~+CD25~- effector T cells to suppression.Linear regression analysis shows that there is a correlation between functional suppression and expression of FOXP3 message and FOXP3 protein.