HUMAN BLOOD MONOCYTE RECEPTOR EXPRESSION AND MODULATION

单核/巨噬细胞受其Fc-γ受体的刺激，诱导信号转导，导致吞噬作用。在Fc-Gamma受体中，Fc-Gamma-RI是独一无二的，因为它有一个高亲和力结合配体的胞外区(EC)和一个不含酪氨酸的61个氨基酸的胞浆区(CY)。我们已经证实，在上皮细胞和小鼠巨噬细胞中没有任何其他人Fc-γ受体的情况下，所有3个人Fc-Gamma受体类的成员都可以诱导吞噬作用，但只有Fc-Gamma-RI通过其自身的阿尔法链CY和相关伽马亚单位的CY传递信号。我们还确定了它们链CY是Fc-Gamma-RI吞噬细胞所必需的，而Syk激酶与Fc-Gamma-RI的伽马链相关，使Fc-Gamma-RI的吞噬能力提高了几倍。除了证实将特异性的Fc-γ受体和嵌合的Fc-γ受体导入CHO细胞和肝细胞使其具有吞噬功能外，我们还观察到体内转Fc-γ受体的肝细胞具有吞噬包被免疫球蛋白的细胞的能力。然而，尽管吞噬作用在生理和病理生理过程中很重要，但吞噬过程中Fc-γ受体及其相关信号分子在细胞内的命运还没有很好的确定。具体地说，我们将探索以下内容：1)吞噬过程中Fc-Gamma-RIpha、Gamma链和Syk激酶之间相互作用的动力学及其在细胞内转运中的作用。将使用荧光蛋白质标签、荧光成像和能量转移技术来探索Fc-Gamma-RIpha、Gamma和Syk之间的关联。我们的假设是，Gamma链在决定Fc-Gamma-RIpha的细胞内路线中发挥作用，吞噬小体成熟需要Gamma，而不是Syk Kinase。我们还将研究Fc-Gamma-RIIA的运输，Fc-Gamma-RIIA是一种单链Fc-Gamma受体，它也与Syk激酶相互作用，但在没有Gamma链的情况下诱导吞噬；2)笼蛋白、笼蛋白包裹的凹坑的其他成分以及泛素化在Fc-Gamma受体的路线中的作用；以及3)生产肝细胞的方法，以高效清除被IgG覆盖的细胞。我们建议的一个主要主题是进一步研究Fc-γ受体在体外(CHO和肝细胞)和体内(肝细胞)上皮细胞的吞噬作用，以赋予肝细胞清除包被免疫球蛋白的细胞的能力。我们的长期目标是系统地将Fc-Gamma受体插入肝细胞，从而建立增强宿主防御和增强免疫复合体清除的可能性。

Monocyte/macrophage stimulation by their Fc-gamma receptors induces signal transduction leading to phagocytosis. Among the Fc-gamma receptors, Fc-gamma-RI is unique in that it has an extracellular domain (EC) which binds ligand with high affinity and a distinct 61 amino acid cytoplasmic domain (CY) which lacks tyrosines. We have established that members of all 3 human Fc-gamma receptor classes can induce phagocytosis in the absence of any other human Fc-gamma receptor in epithelial cell and murine macrophage transfectants, but only Fc-gamma- RI transmits a signal through both its own alpha chain CY and the CY of an associated gamma subunit. We have also determined that they chain CY is necessary for phagocytosis by Fc-gamma-RI and that Syk kinase, associated with the gamma chain in monocytes/macrophages, enhances phagocytosis by Fc-gamma-RI several fold. In addition to establishing that transfection of specific Fc-gamma receptors and chimeric Fc-gamma receptors into such epithelial cells as CHO cells and hepatocytes renders these cells capable of phagocytosis, we have observed that hepatocytes transfected with Fc-gamma receptors in vivo have the potential to phagocytose IgG coated cells. However, despite the importance of phagocytosis in physiologic and pathophysiologic processes, the intracellular fate of Fc-gamma receptors and their associated signaling molecules during phagocytosis is not well defined. Specifically, we will explore the following: 1) The kinetics of the interactions between Fc-gamma-RIalpha, the gamma chain and Syk kinase during phagocytosis and their role in intracellular trafficking. The association of Fc-gamma-RIalpha, gamma and Syk will be probed using fluorescent protein tags, fluorescence imaging and energy transfer techniques. Our hypothesis is that the gamma chain plays a role in determining the intracellular routing of Fc-gamma-RIalpha, and that gamma, but not Syk kinase, is required for phagosomal maturation. We will also examine the trafficking of Fc-gamma-RIIA, a single chain Fc- gamma receptor which also interacts with Syk kinase, but induces phagocytosis in the absence of the gamma chain; 2) The role of clathrin, other components of the clathrin coated pit and ubiquitination in the routing of Fc-gamma receptors; and 3) Approaches for producing hepatocytes highly efficient in the clearance of IgG coated cells. A major theme of our proposal is to further study Fc-gamma receptor phagocytosis in epithelial cells in vitro (CHO and hepatocytes) and in vivo (hepatocytes) in order to impart to hepatocytes the ability to clear IgG coated cells. Our long term goal is to systematically insert Fc-gamma receptors into hepatocytes thus establishing the potential to enhance host defense and to enhance the clearance of immune complexes.