Role for Lipids in the Maintenance of Chemokine and T-Ce

脂质在维持趋化因子和 T-Ce 中的作用

• 批准号: 6674127
• 负责人: DENNIS D. TAUB
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6674127
• 关键词: T lymphocyte; aging; biological signal transduction; chemokine; cholesterol; conformation; cytokine receptors; human immunodeficiency virus; human tissue; membrane lipids; receptor binding; virus infection mechanism

Chemokine receptors (CRs) have drawn much attention since their description as human immunodeficiency virus (HIV) co-receptors by several groups in 1996. Prior to that time, HIV tropism was defined as either macrophage (M)- or T cell (T)-tropic, which corresponded to non-syncytia- or syncytia-inducing viruses, respectively. Today, the classification of HIV tropism is defined by chemokine receptor usage of either CCR5, CXCR4, or both receptors. Chemokine receptors are a family of seven transmembrane spanning G protein-coupled receptors that are differentially expressed by a number of immune and non-immune cell populations. Certain CRs have been shown to be palmitoylated and targeted to cholesterol-and sphingolipid-rich membrane microdomains termed lipid rafts. Lipid rafts is a broad term for the collection of membrane microdomains enriched in cholesterol, sphingolipids, glycosylphosphatidylinositol (GPI)-anchored proteins, and acylated signaling molecules. Lipid rafts are believed to be important signaling platforms enriched in many signaling proteins, including but not limited to src kinases, Ga subunit, H-Ras, LAT, and NOS. Signal transduction through the T and B cell receptors as well as the IgE receptor involves the recruitment of signaling assemblies to lipid rafts. CCR5 and CXCR4 have been shown to be present in lipid rafts, colocalizing at the leading edge of migrating cells. CCR5 has also recently been shown to be palmitoylated, which is one of the important modifications in lipid raft targeting of proteins. However, the role of cholesterol and these lipid rafts on T cell chemokine binding and signaling through CCR5 and CXCR4 remains unknown. We found that cholesterol extraction by beta-cyclodextrin (BCD) significantly reduced the binding and signaling of CXCR4 and MIP-1b using CXCR4- or CCR5-expressing T cells, respectively. Reloading treated cells with cholesterol but not 4-cholesten-3-one, an oxidized form of cholesterol, restored chemokine binding to BCD-treated cells. Antibodies specific for distinct CXCR4 or CCR5 epitopes lost their ability to bind to the cell surface after cholesterol extraction. Moreover, cells stained with fluorescently-labeled MIP-1b extensively co-localized with the GM1 lipid raft marker while using anti-CCR5 antibodies, the majority of CCR5 on these cells co-localized with CD59 and only partially with GM1 suggesting that active ligand binding facilitates receptor association with lipid rafts or that raft association promotes a higher affinity conformation of CCR5. Together, these data demonstrate that cholesterol and lipid rafts are important for the maintenance of the chemokine receptor conformation and are necessary for both the binding and function of this chemokine receptor. This cholesterol and lipid raft requirement for ligand binding may play a significant physiological role in controlling immune cell signaling and migration. Oxidized forms of cholesterol, known as oxysterols, are abundant in various food products and can be found naturally in membranes and mitochondria of a variety of cell types. Oxysterols have been reported to modulate a number of cellular functions and signals, including the inhibition of HMG Co-A reductase activity and sterol synthesis, increasing calcium influx, decreasing cholesterol efflux, the induction of cellular apoptosis, and the initiation of atherosclerosis. The incorporation of oxysterols, such as 22-hydroxycholesterol (22-OHC) and 25-OHC, into cell membranes has previously been shown to exert immunosuppressive effects on lymphocytes. Our results revealed that 22-OHC and 25-OHC, but not cholesterol, significantly reduced the binding of both SDF-1a and MIP-1? to T cells and PBMCs. These oxysterols also inhibited the intracellular calcium mobilization and chemotaxis in response to SDF-1a and MIP-1b treatment. The inhibitory effects of oxysterols were rapid and approached maximum levels within 1 hour of incubation. In contrast to the ligand binding studies, anti-CXCR4 and CCR5 antibody binding to T cells was not inhibited by oxysterol treatment. In addition, while the presence of oxysterols in cell membranes significantly inhibits chemokine receptor function, this effect does not involve alterations to receptor conformation, receptor internalization, or direct blocking of chemokine binding. We hypothesize that the increased presence of circulating and/or membrane-associated oxysterols in athlerosclerotic patients and in various chronic disease states may play a role in the diminished immune responses observed in elderly and frail subjects. Similarly, enzymatic cholesterol oxidation using cholesterol oxidase (CO) alone or in combination with sphingomyelinase (SMase) results in similar inhibitory effects as those observed with OHC treatment. CO is an enzyme that converts cholesterol (3b-hydroxy-5-cholestene) into 4-cholesten-3-one and is produced by various pathogens including Streptomyces, Pseudomonas, Mycobacterium, Brevibacterium, Nocardia, Rhodococcus and Corynebacterium. We have found that that CO treatment inhibits chemokine binding to CXCR4 and CCR5 on T cells, resulting in the significant inhibition of chemokine-mediated intracellular calcium mobilization and chemotaxis. Thus, we propose a novel mechanism for pathogen modulation of chemokine function in the immune cell response to infection and inflammation.

自从 1996 年多个研究小组将趋化因子受体 (CR) 描述为人类免疫缺陷病毒 (HIV) 共受体以来，趋化因子受体 (CR) 引起了广泛关注。在此之前，HIV 向性被定义为巨噬细胞 (M) 向性或 T 细胞 (T) 向性，分别对应于非合胞体诱导病毒或合胞体诱导病毒。如今，HIV 向性的分类是根据 CCR5、CXCR4 或两种受体的趋化因子受体的使用来定义的。趋化因子受体是由七种跨膜 G 蛋白偶联受体组成的家族，它们在许多免疫和非免疫细胞群中差异表达。某些 CR 已被证明是棕榈酰化的，并靶向富含胆固醇和鞘脂的膜微域（称​​为脂筏）。脂筏是一个广泛的术语，是指富含胆固醇、鞘脂、糖基磷脂酰肌醇 (GPI) 锚定蛋白和酰化信号分子的膜微结构域的集合。脂筏被认为是重要的信号传导平台，富含许多信号传导蛋白，包括但不限于src激酶、Ga亚基、H-Ras、LAT和NOS。通过 T 细胞和 B 细胞受体以及 IgE 受体的信号转导涉及将信号组件募集至脂筏。 CCR5 和 CXCR4 已被证明存在于脂筏中，共定位于迁移细胞的前缘。 CCR5 最近也被证明是棕榈酰化的，这是蛋白质脂筏靶向的重要修饰之一。然而，胆固醇和这些脂筏对 T 细胞趋化因子结合以及通过 CCR5 和 CXCR4 信号传导的作用仍然未知。我们发现，使用表达 CXCR4 或 CCR5 的 T 细胞，通过 β-环糊精 (BCD) 提取胆固醇分别显着降低了 CXCR4 和 MIP-1b 的结合和信号传导。用胆固醇而不是胆固醇的氧化形式 4-cholesten-3-one 重新加载处理的细胞，可以恢复趋化因子与 BCD 处理的细胞的结合。胆固醇提取后，针对不同 CXCR4 或 CCR5 表位的特异性抗体失去了与细胞表面结合的能力。此外，使用抗 CCR5 抗体时，用荧光标记的 MIP-1b 染色的细胞与 GM1 脂筏标记物广泛共定位，这些细胞上的大部分 CCR5 与 CD59 共定位，仅部分与 GM1 共定位，表明活性配体结合促进受体与脂筏的结合，或者筏结合促进了脂筏的更高亲和力构象。 CCR5。总之，这些数据表明胆固醇和脂筏对于维持趋化因子受体构象很重要，并且对于该趋化因子受体的结合和功能都是必需的。配体结合所需的胆固醇和脂筏可能在控制免疫细胞信号传导和迁移中发挥重要的生理作用。 氧化形式的胆固醇（称为氧甾醇）在各种食品中含量丰富，并且天然存在于各种细胞类型的膜和线粒体中。据报道，氧甾醇可调节许多细胞功能和信号，包括抑制 HMG Co-A 还原酶活性和甾醇合成、增加钙内流、减少胆固醇外流、诱导细胞凋亡和引发动脉粥样硬化。先前已证明，将氧甾醇（例如 22-羟基胆固醇 (22-OHC) 和 25-OHC）掺入细胞膜中可对淋巴细胞产生免疫抑制作用。我们的结果显示，22-OHC 和 25-OHC（而非胆固醇）显着降低了 SDF-1a 和 MIP-1 的结合？ T 细胞和 PBMC。这些氧甾醇还抑制 SDF-1a 和 MIP-1b 治疗引起的细胞内钙动员和趋化性。氧甾醇的抑制作用迅速，并在孵育 1 小时内达到最大水平。与配体结合研究相反，抗CXCR4和CCR5抗体与T细胞的结合不受氧甾醇处理的抑制。此外，虽然细胞膜中氧甾醇的存在显着抑制趋化因子受体功能，但这种效应不涉及受体构象的改变、受体内化或直接阻断趋化因子结合。我们假设，动脉粥样硬化患者和各种慢性疾病状态下循环和/或膜相关氧甾醇的增加可能在老年和体弱受试者中观察到的免疫反应减弱中发挥作用。 同样，单独使用胆固醇氧化酶 (CO) 或与鞘磷脂酶 (SMase) 组合进行酶促胆固醇氧化会产生与 OHC 治疗中观察到的类似的抑制效果。 CO 是一种将胆固醇（3b-羟基-5-胆烯）转化为 4-胆烯-3-酮的酶，由多种病原体产生，包括链霉菌属、假单胞菌属、分枝杆菌属、短杆菌属、诺卡氏菌属、红球菌属和棒状杆菌属。我们发现CO处理抑制趋化因子与T细胞上的CXCR4和CCR5结合，从而显着抑制趋化因子介导的细胞内钙动员和趋化性。因此，我们提出了一种病原体调节免疫细胞对感染和炎症反应中趋化因子功能的新机制。