Control of FcgammaR Triggered Macrophage Function

描述;我们的目的是了解巨噬细胞Fc受体介导的IgG （FcgammaR）的生物学功能是如何被稳态控制的。fgammar聚类可触发巨噬细胞的多种功能，如活化颗粒的内吞作用、炎性细胞因子的内吞作用、氧自由基的产生等。信号级联始于簇状受体细胞质尾部酪氨酸基基基序（ITAM）的磷酸化。这些磷酸化的ITAM依次组装各种正作用激酶，如Syk和PI13K。必须严格调节级联反应以抑制炎症反应。至少三个层次的控制是明显的。首先，当itam - FcgammaRIIb与itam -受体共聚集时，招募肌醇磷酸酶SHIP，从而缓和正向反应。其次，FcgammaRIIb的表达以及SHIP的募集受到细胞因子的严格调控。第三，不仅是ITAM，还有携带ITAM的受体，如FcgammaRI和FcgammaRIIa，它们是已知的激动剂，也可以激活SHIP，整合来自单个ITAM的阳性和阴性信号。在几年的工作基础上，我们为这个项目带来了三项创新。首先，我们应用了一种识别人类FcgammaRIIb的新抗体，这将首次使我们能够研究该受体在人类细胞中的功能。其次，我们发现IL-4和培养密度对FcgammaRIIb的表达有很大的促进作用。第三，我们发现不仅ITAM-FcgammaR，而且ITAM-FcgammaR也激活SHIP，为fcgammar介导的下游功能调节提供了新的范例。因此，我们的两个主要假设是，SHIP通过ITAM-和ITIM-FcgammaR调节FcgammaR功能，而FcgammaRIIb的表达调节反过来调节SHIP对FcgammaR介导的反应的抑制。为了检验这些假设的几种预测，我们提出了三个目标。首先，我们探讨ITAM激活SHIP这一概念的含义。其次，我们定义了围绕IL-4增强FcgammaRIIb表达的分子细节，重点关注其对SHIP活性的最终调控以及两种FcgammaRIIb亚型的差异调控。第三，我们剖析了SHIP下游的分子事件，询问ITAM-和itim -介导的激活是否存在差异，下游的SHP效应是酶促的还是由于适配器功能，以及多种fcgammar介导的功能是否受到SHIP的差异调节。我们的工作重点是单核吞噬细胞谱系的人类细胞，尽管我们在相关的地方利用小鼠模型。我们的技术方法是广泛的和多学科的，包括分子和细胞生物学，生物化学和免疫学的工具。这项工作的意义在于，在分子水平上理解这些调节机制，有望为调节抗体引发的炎症和杀伤提供新的治疗方法。

Description; Our aim is to understand how the biologic functions mediated by macrophage Fc receptors for IgG (FcgammaR) are homeostatically controlled. FcgammaR clustering triggers a variety of macrophage functions such as endocytosis of opsonized particles, of inflammatory cytokines, and the generation of oxygen radicals. The signal cascade begins with phosphorylation of tyrosine based motifs (ITAM) in the cytoplasmic tails of the clustered receptors. These phosphorylated ITAM in turn assemble a variety of positive-acting kinases such as Syk and PI13K. The cascade must be tightly regulated to contain the inflammatory response. At least three levels of control of apparent. First, ITIM-being FcgammaRIIb, when co-clustered with ITAM-receptors, recruit the inositol phosphatase SHIP which tempers the forward response. Second, FcgammaRIIb expression, and thus SHIP recruitment, is tightly regulated by cytokines. Third, not only ITIM-but ITAM-bearing receptors such as FcgammaRI and FcgammaRIIa, which are known agonists, activate SHIP as well, integrating positive and negative signals from a single ITAM. Building on several years of work we bring to this project three innovations. First, we apply a new antibody recognizing human FcgammaRIIb that for the first time will allow us to study the function of this receptor in human cells. Second, we have found that FcgammaRIIb expression is greatly enhanced by IL-4 and culture density. Third, we show that not only ITIM-but ITAM-FcgammaR as well as activate SHIP, providing a new paradigm for FcgammaR-mediated regulation of downstream functions. Our two major hypotheses, then, are that SHIP wo4rks through both ITAM- and ITIM-FcgammaR to regulate FcgammaR function and that the regulated expression of FcgammaRIIb in turn modulates SHIP inhibition of FcgammaR-mediated responses. Testing several predictions of these hypotheses we propose three aims. First, we explore implications of the notion that ITAM activates SHIP. Second, we define the molecular details surrounding IL-4 enhancement of FcgammaRIIb expression focusing on its ultimate regulation of SHIP activity and differential regulation of the two FcgammaRIIb isoforms. Third, we dissect the molecular events downstream of SHIP asking whether there are differences between ITAM- and ITIM-mediated activation, whether the downstream SHP effects are enzymatic or due to adaptor functions, and whether the multiple FcgammaR-mediated functions are differentially regulated by SHIP. Our work focuses on human cells of the mononuclear phagocyte lineage although we take advantage of murine models where relevant. Our technical approaches to the various aims are broad based and multi- disciplinary and include the tools of molecular and cellular biology, biochemistry, and immunology. The significance of this work is that understanding these regulatory mechanisms at the molecular level holds the promise of novel therapeutic approaches for modulating antibody- triggered inflammation and killing.