Identification and Analysis of the Physiological Ligand for TLT2

描述(申请人提供)：本提案的目的是阐明TLT2的配体在急性炎症反应中的生理表达模式。这些信息将被用来产生一个消减的cDNA文库，以克隆编码TLT2配体的基因。TLT2是一种跨膜受体，表达于中性粒细胞、单核/巨噬细胞和B细胞上。研究表明，TLT2结扎增强了中性粒细胞对激动剂的反应，激动剂与G蛋白偶联受体结合，导致ROS产生、脱颗粒和趋化作用增强。炎症反应中性粒细胞和巨噬细胞上的TLT2表达上调，并增强中性粒细胞在体内炎症部位的募集。此外，体内TLT2结扎导致G-CSF和趋化因子KC和MIP-2的产生。这些因子作用于中性粒细胞，增加它们从骨髓中的动员，并引导它们向感染或炎症部位迁移。因此，研究表明，TLT2驱动一个正向前馈循环，涉及调节中性粒细胞激活和趋化的因子的产生，同时增强中性粒细胞对这些因子(即KC和MIP-2)的反应。这些发现支持TLT2在调节对微生物攻击的先天免疫反应中发挥重要作用的假说。同时，TLT2可能推动了导致急性炎症反应的过程。体内TLT2依赖过程的操纵为这两种假说提供了直接支持。静脉注射激动型抗TLT2单抗可保护小鼠免受肺炎链球菌致死性气管内攻击。相反，静脉注射阻断TLT2：Fc重组融合蛋白可减少中性粒细胞因气管内注射脂多糖而重新聚集到肺内。因此，TLT2在调节先天免疫反应中起着重要的、非冗余的作用。为了更全面地了解TLT2在先天免疫反应中的生理作用，确定TLT2的配体是至关重要的。为此，提出了分析TLT2配体在急性炎症反应中的表达的实验(特定目标1)。这些研究旨在证明TLT2配体的表达在炎症反应中以空间和时间的方式上调。后续的研究将利用这些信息来生成消减的cDNA文库，用于表达克隆，以确定编码TLT2配体的基因(特定目标2)。所获得的信息将大大扩展对控制先天性免疫反应的分子过程的理解，并将确定一个新的靶点(即TLT2配体)，用于开发调节急性炎症反应的治疗干预措施。

DESCRIPTION (provided by applicant): The goal of this proposal is to elucidate the physiological expression pattern of the ligand for TLT2 in response to acute inflammation. This information will be used to generate a subtracted cDNA library to clone the gene encoding the ligand for TLT2. TLT2 is a transmembrane receptor that is expressed on neutrophils, monocytes/macrophages and B cells. Studies have demonstrated that TLT2 ligation potentiates the response of neutrophils to agonists that bind to G protein coupled receptors leading to enhanced ROS production, degranulation and chemotaxis. TLT2 expression is upregulated on neutrophils and macrophages in response to inflammation and potentiates neutrophil recruitment to sites of inflammation in vivo. Additionally, TLT2 ligation in vivo leads to the production of G-CSF and the chemokines KC and MIP-2. These factors act on neutrophils to increase their mobilization from the bone marrow and to direct their migration to sites of infection or inflammation. Thus, studies demonstrate that TLT2 drives a positive feed-forward loop that involves the production of factors that modulate neutrophil activation and chemotaxis, and at the same time potentiates the neutrophil response to those factors (i.e. KC and MIP-2). These findings support the hypothesis that TLT2 plays an important role in regulating the innate immune response to microbial challenge. At the same time, TLT2 may drive processes that contribute to the acute inflammatory response. Manipulation of TLT2- dependent processes in vivo provides direct support for both hypotheses. Intravenous administration of agonistic anti-TLT2 mAb protects mice from a lethal intratracheal challenge with S. pneumonia. Conversely, intravenous administration of blocking TLT2:Fc recombinant fusion protein attenuates recruitment of neutrophils to the lung in response to intratracheal administration of LPS. Thus, TLT2 serves an important, non-redundant role in regulating the innate immune response. To more fully appreciate the physiological role of TLT2 during the innate immune response, it is critical to identify the ligand for TLT2. Towards this goal, experiments are proposed to analyze TLT2 ligand expression in response to acute inflammation (Specific Aim 1). These studies are designed to demonstrate that TLT2 ligand expression is upregulated in a spatial and temporal manner in response to inflammation. Subsequent studies will utilize this information to generate subtracted cDNA libraries to be used in expression cloning to identify the gene that encodes the TLT2 ligand (Specific Aim 2). The information obtained will significantly extend understanding of molecular processes that control the innate immune response and will identify a novel target (i.e. the TLT2 ligand) for the development of therapeutic interventions to modulate the acute inflammatory response.