Identification and Analysis of the Physiological Ligand for TLT2

描述（由申请人提供）：本提案的目的是阐明TLT2配体在急性炎症反应中的生理表达模式。该信息将用于生成一个减cDNA文库，以克隆编码TLT2配体的基因。TLT2是一种跨膜受体，在中性粒细胞、单核/巨噬细胞和B细胞上表达。研究表明，TLT2连接增强了中性粒细胞对与G蛋白偶联受体结合的激动剂的反应，从而增强了ROS的产生、脱颗粒和趋化性。TLT2在中性粒细胞和巨噬细胞中表达上调，以响应炎症，并增强中性粒细胞向体内炎症部位的募集。此外，TLT2在体内的结扎导致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，增加其从骨髓的动员，并引导其迁移到感染或炎症部位。因此，研究表明，TLT2驱动一个正前馈循环，该循环涉及调节中性粒细胞活化和趋化性的因子的产生，同时增强中性粒细胞对这些因子（即KC和MIP-2）的反应。这些发现支持了TLT2在调节微生物攻击的先天免疫反应中起重要作用的假设。同时，TLT2可能会驱动急性炎症反应的过程。在体内对TLT2依赖过程的操纵为这两种假设提供了直接的支持。静脉注射激动性抗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，静脉给药阻断TLT2:Fc重组融合蛋白会减弱中性粒细胞在气管内给药LPS的作用下向肺的募集。因此，TLT2在调节先天免疫应答中起着重要的、非冗余的作用。为了更充分地了解TLT2在先天免疫应答中的生理作用，鉴定TLT2的配体是至关重要的。为此，我们提出了分析TLT2配体在急性炎症反应中的表达的实验（Specific Aim 1）。这些研究旨在证明TLT2配体表达在炎症反应中以空间和时间方式上调。后续的研究将利用这些信息生成缺失的cDNA文库，用于表达克隆，以鉴定编码TLT2配体的基因（Specific Aim 2）。所获得的信息将大大扩展对控制先天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.