Assistance Publique des Hôpitaux de Paris (AP-HP), Service d'Anesthésie-Réanimation, Hôpitaux Universitaires Paris-Sud, Centre Hospitalier Universitaire de Bicêtre, Université Paris XI, Paris, France.
Shock. 2013 Jan;39(1):83-8. doi: 10.1097/SHK.0b013e318278ae36.
Little is known about the endothelial mechanisms involved in the anti-inflammatory effects of interleukin 10 (IL-10). The goal of this study was to evaluate the effects of IL-10 on endothelial oxidative stress and endothelial inflammation induced by tumor necrosis factor α (TNF-α). Production of reactive oxygen species (ROS) in perfused human umbilical vein endothelial cells (HUVECs) was studied by fluorescent microscopy using dichlorodihydrofluorescein diacetate. Tumor necrosis factor α (1 ng/mL) was added to the perfusion medium in the absence and presence of IL-10 (1 ng/mL). The role of phosphatidylinositol 3-kinase (PI3-kinase) was assessed using wortmannin and LY 2940002 (inhibitors of PI3-kinase). Specific inhibition of p110 α and p110 γ/δ PI3-kinase subunits was studied using A66 and TG100-115. As well, levels of ceramide and intercellular adhesion molecule 1 (ICAM-1) expression were measured. Finally, the effect of IL-10 on TNF-α-induced leukocyte/endothelium interaction was examined using an ex vivo perfused vessel model. Interleukin 10 significantly reduced dichlorodihydrofluorescein diacetate fluorescence induced by TNF-α in HUVECs (12.5% ± 3.2% vs. 111.7% ± 21.6% at 60 min). Pretreatment by LY2940002 or wortmannin restored ROS production induced by TNF-α in the presence of IL-10. In HUVECs treated by TNF-α + IL-10, inhibition of p110 α PI3-kinase subunit significantly increased ROS production, whereas p110 γ/δ inhibition did not have a significant effect. Pretreatment with IL-10 significantly decreased TNF-α-induced increased levels of ceramide (TNF-α vs. TNF-α + IL-10: 6,278 ± 1,013 vs. 1,440 ± 130 pmol/mg prot), as well as ICAM-1 expression and leukocyte adhesion (TNF-α vs. TNF-α + IL-10: 26.8 ± 2.6 vs. 6.7 ± 0.4 adherent leukocytes/field at 15 min). Interleukin 10 decreases the level of inflammation induced by TNF-α in endothelial cells by reducing the TNF-α-induced ROS production, ICAM-1 expression, and leukocyte adhesion to the endothelium. The antioxidant effect of IL-10 is mediated through PI3-kinase and is paralleled by a decrease in ceramide synthesis induced by TNF-α.
关于白细胞介素 10（IL-10）抗炎作用涉及的内皮细胞机制知之甚少。本研究旨在评估 IL-10 对肿瘤坏死因子 α（TNF-α）诱导的内皮细胞氧化应激和炎症的影响。通过荧光显微镜使用二氯二氢荧光素二乙酸酯研究灌注人脐静脉内皮细胞（HUVEC）中的活性氧（ROS）的产生。在不存在和存在 IL-10（1ng/mL）的情况下，将 TNF-α（1ng/mL）添加到灌注培养基中。使用 wortmannin 和 LY 2940002（PI3-激酶的抑制剂）评估磷脂酰肌醇 3-激酶（PI3-kinase）的作用。通过 A66 和 TG100-115 研究 p110α 和 p110γ/δPI3-激酶亚基的特异性抑制。还测量了神经酰胺和细胞间黏附分子 1（ICAM-1）表达的水平。最后，使用体外灌注血管模型研究了 IL-10 对 TNF-α诱导的白细胞/内皮细胞相互作用的影响。IL-10 显著降低了 TNF-α诱导的 HUVEC 中二氯二氢荧光素二乙酸酯荧光（60 分钟时 12.5%±3.2%与 111.7%±21.6%）。LY2940002 或 wortmannin 的预处理恢复了存在 IL-10 时 TNF-α诱导的 ROS 产生。在 TNF-α+IL-10 处理的 HUVEC 中，p110αPI3-激酶亚基的抑制显著增加了 ROS 的产生，而 p110γ/δ抑制则没有显著作用。IL-10 的预处理显著降低了 TNF-α诱导的神经酰胺水平升高（TNF-α与 TNF-α+IL-10：6,278±1,013 与 1,440±130pmol/mgprot），以及 ICAM-1 表达和白细胞黏附（TNF-α与 TNF-α+IL-10：26.8±2.6 与 6.7±0.4 个黏附白细胞/场在 15 分钟时）。IL-10 通过减少 TNF-α诱导的 ROS 产生、ICAM-1 表达和白细胞与内皮细胞的黏附来降低内皮细胞中由 TNF-α诱导的炎症水平。IL-10 的抗氧化作用是通过 PI3-kinase 介导的，并伴随着 TNF-α诱导的神经酰胺合成减少。
