| Objective: Different subsets of T lymphocytes have different functions in atherosclerosis advancement. T helper cells and T regulatory cells have been demonstrated to play opposite roles in rupture of atherosclerotic lesion. However, the roles of T helper 1 cells and novel subset of T regulatory cells, known as CD4+CD25+Foxp3+Tcells, remain largely unknown in coronary artery disease (CAD). In this study, we investigated the peripheral Th1 cells and CD4+CD25+Foxp3+Tcells of patients with CAD and controls to test the hypothesis that the imbalance between regulatory and pathogenic immunity substantially contributed both to plaque destabilization and systemic inflammation in ACS.Methods: 1) Study Population and PBMC isolation. 58 consecutive patients with CAD and 12 hospitalized healthy volunteers were included in the present study. The peripheral blood mononuclear cells (PBMC) were isolated by Ficoll density gradient centrifugation and washed twice with serum-free RPMI 1640 medium.2) Flow Cytometric Analysis for Th1 and Th2 cells. The mononuclear cells were stimulated and incubated for 4 h at 37°C/5% CO2. Stimulated samples were then stained with CD3-PerCP antibody at room temperature for 15 min. Mononuclear cells were permeabilized and stained with IFN-γ-FITC and IL-4-PE antibodies for 30 min at room temperature. Finally, samples were acquisited by FACS. Results were expressed as percentage of all CD3+ T-cell subtypes staining positive for the specific cytokine.3) ELISA detection of plasma IFN-γ,IL-4,IL-2 and IL-10. Heparinized blood samples were immediately centrifuged and plasma was frozen and kept at–80°C before being assayed. Plasma cytokine (IFN-γ, IL-2, IL-4and IL-10) concentrations were measured by ELISA procedure using the ELISA kit.4) Flow Cytometric Analysis for peripheral CD4+CD25+Foxp3+ regulatory T cells. The mononuclear cells were resuspended at 107 cells/ml in staining buffer and labeled by Human Treg Flow? Kit (FOXP3 Alexa Fluor? 488/CD4 PE-Cy5/CD25 PE). Samples were analysed by FACS. At a threshold set to gate on CD4+CD25+Tcells, 6,000 events were collected. Isotype controls enabled correct compensation and confirmed antibody specificity. Results were expressed as percentage of all CD4+ CD25+T-cell subtypes staining positive for Foxp3.5) CD4+CD25+T cells isolation and RT-PCT for Foxp3 mRNA. CD4+CD25+T cells were purified from PBMC by isolation with Dynal CD4+CD25+Treg isolation Kit. RT-PCR for Foxp3 and controlβ-actin mRNA was performed using SuperScript One-Step RT-PCR kit. One representative experiment out of five performed on different donors in each group is shown. Results: 1) Patient characteristics. There were no significant differences in age, gender, lipid, blood glucose, hypertension, or frequency of smoking in patients with SA, UA or MI.2) Frequencies of Th1 cells and Th2 cells in CD3+T cells subsets among the groups. The frequencies of Th1 cells were 5.91±0.92% in the SA group, 19.71±5.14% in the UA group, 29.40±5.81% in the MI group and 6.81±1.43% in the control group. The frequencies of Th1 cells in the UA and MI groups were significantly higher than that in the SA group (p<0.001). The MI group even had a higher level of IFN-γ/CD3+ lymphocytes frequency than the UA group (p<0.01). Difference in frequencies between the SA and control groups was not significant (p>0.05). Frequencies of peripheral IL-4-producing/CD3+T cells were 0.79±0.25% in the SA group, 0.69±0.22% in the UA group, 0.73±0.21% in the MI group and 0.79±0.19% in the control group. The differences in frequency among the 4 groups were not significant (p>0.05).3) Th1 cells and Th2 related cytokines among the groups. The level of IFN-γand IL-2 were elevated in patients with UA and MI compared to those with SA and controls (p<0.001). Difference in concentrations of IFN-γand IL-2 between the SA and control or the UA and AMI groups was not significant (p>0.05). The concentrations of IL-10 had significant difference among the groups (p<0.001). The concentrations of IL-4 were not detectable in any group.4) Frequencies of CD4+CD25+Foxp3+ regulatory T cells in CD4+CD25+T cells subsets among the groups. The frequencies of CD4+CD25+T cells were 6.33±1.39% in the SA group, 6.57±1.24% in the UA group, 6.93±1.16% in the MI group, and 6.80±1.15% in the control group. Patients with ACS, including UA and AMI, had no significant difference in peripheral CD4+CD25+T cells compared to patients with SA and control groups (p>0.05). Patients with CAD showed a significant lower level of Foxp3+T cells (71.75±8.05% in the SA group, 51.77±7.07% in the UA group, 40.43±5.73% in the MI group) than controls (84.78±6.15%) (p<0.001). Patients with UA and MI had an even lower level of Foxp3+T cells than SA patients (p<0.001).5) Expression of Foxp3 mRNA in CD4+CD25+T cell subsets. The level of FoxP3 mRNA was much lower in SA, UA and MI groups than that in control group. MI group even had more attenuated expression of Foxp3 mRNA than UA and SA groups.Conclusion: Our study provided the direct proof that Th1 cells expansion and CD4+CD25+Tregs reduction are consistent with the development of plaques instability. The imbalance between pathogenic Th1 cells and CD4+CD25+Tregs might contribute to atherosclerotic plaque destabilization in patients with CAD. These results suggested a new point for the modulation of atherosclerosis and may shed light on the stabilization of vulnerable plaque.
|
PDF Full Text Request