Macrophages generate reactive oxygen species in response to minimally oxidized low-density lipoprotein: toll-like receptor 4- and spleen tyrosine kinase-dependent activation of NADPH oxidase 2.
Macrophages generate reactive oxygen species in response to minimally oxidized low-density lipoprotein: toll-like receptor 4- and spleen tyrosine kinase-dependent activation of NADPH oxidase 2.
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DOI：
10.1161/circresaha.108.181040
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发表时间：
2009-01-30
影响因子：
20.1
通讯作者：
Miller YI
中科院分区：
文献类型：
--
作者：
Bae YS;Lee JH;Choi SH;Kim S;Almazan F;Witztum JL;Miller YI
低密度脂蛋白（LDL）的氧化修饰在动脉粥样硬化的发展中起着重要作用。在这项研究中，我们证明，最低限度氧化的低密度脂蛋白（mmLDL）刺激细胞内活性氧（ROS）通过NADPH氧化酶2（gp 91 phox/Nox 2），这反过来又诱导产生RANTES和平滑肌细胞的迁移在巨噬细胞的产生。来自gp 91 phox/Nox 2 −/−小鼠的腹膜巨噬细胞或其中通过siRNA敲低Nox 2的J774巨噬细胞不能响应于mmLDL产生ROS。由于mmLDL诱导的细胞骨架变化依赖于TLR 4，我们分析了野生型、TLR 4 −/−或MyD 88 −/−小鼠腹腔巨噬细胞中ROS的产生，发现mmLDL介导的ROS以TLR 4依赖性但MyD 88非依赖性方式产生。此外，我们发现ROS的产生需要脾酪氨酸激酶（Syk）的募集和激活，mmLDL还诱导PLCγ1磷酸化和PKC膜转位。重要的是，在表达Syk特异性shRNA的J774细胞中，PLCγ1磷酸化减少。Nox 2通过调节促炎细胞因子IL-1β、IL-6和RANTES的表达来调节巨噬细胞的mmLDL活化。我们发现，纯化的RANTES能够刺激小鼠主动脉平滑肌细胞（MASMC）的迁移，并添加针对RANTES的中和抗体消除了由mmLDL刺激的巨噬细胞刺激的MASMC的迁移。这些结果表明，mmLDL通过依次激活TLR 4、Syk、PLCγ1、PKC和gp 91 phox/Nox 2诱导ROS的产生，从而刺激促炎细胞因子的表达。这些数据有助于解释内源性配体（如mmLDL）可诱导巨噬细胞TLR 4依赖性致动脉粥样硬化激活的机制。
Oxidative modification of low-density lipoprotein (LDL) plays a causative role in the development of atherosclerosis. In this study, we demonstrate that minimally oxidized LDL (mmLDL) stimulates intracellular reactive oxygen species (ROS) generation in macrophages through NADPH oxidase 2 (gp91phox/Nox2), which in turn induces production of RANTES and migration of smooth muscle cells. Peritoneal macrophages from gp91phox/Nox2−/− mice or J774 macrophages in which Nox2 was knocked down by siRNA failed to generate ROS in response to mmLDL. Because mmLDL-induced cytoskeletal changes were dependent on TLR4, we analyzed ROS generation in peritoneal macrophages from wild type, TLR4−/−, or MyD88−/− mice and found that mmLDL-mediated ROS was generated in a TLR4-dependent, but MyD88-independent manner. Furthermore, we found that ROS generation required the recruitment and activation of spleen tyrosine kinase (Syk) and that mmLDL also induced PLCγ1 phosphorylation and PKC membrane translocation. Importantly, the PLCγ1 phosphorylation was reduced in J774 cells expressing Syk-specific shRNA. Nox2 modulated mmLDL activation of macrophages by regulating the expression of proinflammatory cytokines IL-1β, IL-6 and RANTES. We showed that purified RANTES was able to stimulate migration of mouse aortic smooth muscle cells (MASMC) and addition of neutralizing antibody against RANTES abolished the migration of MASMC stimulated by mmLDL-stimulated macrophages. These results suggest that mmLDL induces generation of ROS through sequential activation of TLR4, Syk, PLCγ1, PKC, and gp91phox/Nox2 and thereby stimulates expression of proinflammatory cytokines. These data help explain mechanisms by which endogenous ligands, such as mmLDL, can induce TLR4-dependent, proatherogenic activation of macrophages.