HUMAN BLOOD MONOCYTE RECEPTOR EXPRESSION AND MODULATION

单核细胞/巨噬细胞通过其Fc-γ受体的刺激诱导导致吞噬作用的信号转导。在Fc-γ受体中，Fc-γ-RI是独特的，因为它具有以高亲和力结合配体的胞外结构域（EC）和缺乏酪氨酸的独特的61个氨基酸胞质结构域（CY）。我们已经确定，在上皮细胞和鼠巨噬细胞转染子中，所有3种人Fc-γ受体类别的成员都可以在不存在任何其他人Fc-γ受体的情况下诱导吞噬作用，但只有Fc-γ- RI通过其自身的α链CY和相关γ亚基的CY传递信号。我们还确定了它们链CY对于Fc-γ-RI的吞噬作用是必需的，并且与单核细胞/巨噬细胞中的γ链相关的Syk激酶将Fc-γ-RI的吞噬作用增强数倍。除了确定将特异性Fc-γ受体和嵌合Fc-γ受体转染到诸如CHO细胞和肝细胞的上皮细胞中使这些细胞能够吞噬之外，我们还观察到体内用Fc-γ受体转染的肝细胞具有吞噬IgG包被的细胞的潜力。然而，尽管吞噬作用在生理和病理生理过程中的重要性，但在吞噬作用期间Fc-γ受体及其相关信号传导分子的细胞内命运并不明确。具体而言，我们将探索以下内容：1）在吞噬过程中Fc-γ-RI α、γ链和Syk激酶之间相互作用的动力学及其在细胞内运输中的作用。将使用荧光蛋白标签、荧光成像和能量转移技术探测Fc-γ-RI α、γ和Syk的关联。我们的假设是γ链在确定Fc-γ-RI α的细胞内路线中起作用，并且γ而不是Syk激酶是吞噬体成熟所需的。我们还将检查Fc-γ-RIIA（一种单链Fc-γ受体，其也与Syk激酶相互作用，但在不存在γ链的情况下诱导吞噬作用）的运输; 2）网格蛋白、网格蛋白包被的凹坑的其他组分和泛素化在Fc-γ受体的路由中的作用;和3）用于产生在IgG包被的细胞的清除中高效的肝细胞的方法。我们提案的一个主要主题是进一步研究体外（CHO和肝细胞）和体内（肝细胞）上皮细胞中Fc-γ受体的吞噬作用，以赋予肝细胞清除IgG包被细胞的能力。我们的长期目标是将Fc-γ受体系统地插入肝细胞，从而建立增强宿主防御和增强免疫复合物清除的潜力。

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.