Control of FcgammaR Triggered Macrophage Function

说明;我们的目的是了解如何介导的生物功能的巨噬细胞Fc受体的IgG（Fc γ R）的稳态控制。Fc γ R簇集触发多种巨噬细胞功能，如调理颗粒、炎性细胞因子的内吞作用和氧自由基的产生。信号级联始于聚集受体的胞质尾中基于酪氨酸的基序（ITAM）的磷酸化。这些磷酸化的ITAM反过来组装各种正作用激酶，如Syk和PI 13 K。必须严格调节级联反应以抑制炎症反应。至少有三个明显的控制水平。首先，ITIM-Fc γ RIIb，当与ITAM-受体共聚簇时，募集调节正向反应的肌醇磷酸酶SHIP。第二，Fc γ RIIb表达以及因此SHIP募集受到细胞因子的严格调节。第三，不仅ITIM-而且ITAM-承载受体如Fc γ RI和Fc γ RIIa（它们是已知的激动剂）也激活SHIP，整合来自单个ITAM的正信号和负信号。在多年的工作基础上，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Fc γ RIIb的新抗体，这将首次使我们能够研究这种受体在人细胞中的功能。第二，我们已经发现Fc γ RIIb表达被IL-4和培养密度大大增强。第三，我们发现不仅ITIM-而且ITAM-FcgammaR以及激活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-Fc γ R调节Fc γ R功能，而Fc γ RIIb的调节表达反过来调节SHIP对Fc γ R介导的应答的抑制。测试这些假设的几个预测，我们提出了三个目标。首先，我们探讨ITAM激活SHIP的概念的含义。其次，我们定义了IL-4增强Fc γ RIIb表达的分子细节，重点是其对SHIP活性的最终调节和两种Fc γ RIIb同种型的差异调节。第三，我们剖析了SHIP下游的分子事件，询问ITAM和ITIM介导的激活之间是否存在差异，下游SHP效应是否是酶促的或由于接头功能，以及多个Fc γ R介导的功能是否受到SHIP的差异调节。我们的工作重点是人类单核吞噬细胞谱系的细胞，虽然我们利用小鼠模型，在相关的。我们的技术方法的各种目标是广泛的基础和多学科，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，有望为调节抗体触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.