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TLT 2配体在急性炎症反应中的生理表达模式。该信息将用于产生消减cDNA文库以克隆编码TLT 2配体的基因。TLT 2是在中性粒细胞、单核细胞/巨噬细胞和B细胞上表达的跨膜受体。研究表明，TLT 2连接增强了嗜中性粒细胞对结合G蛋白偶联受体的激动剂的反应，导致ROS产生、脱粒和趋化性增强。TLT 2表达在中性粒细胞和巨噬细胞上响应于炎症而上调，并增强中性粒细胞向体内炎症部位的募集。此外，体内TLT 2连接导致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，以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，TLT 2驱动正前馈回路，其涉及调节中性粒细胞活化和趋化性的因子的产生，并且同时增强中性粒细胞对这些因子（即KC和MIP-2）的应答。这些发现支持了TLT 2在调节对微生物挑战的先天免疫应答中起重要作用的假设。与此同时，TLT 2可能驱动导致急性炎症反应的过程。TLT 2依赖性过程在体内的操纵为这两种假设提供了直接支持。静脉内给予激动性抗TLT 2 mAb可保护小鼠免受S.肺炎相反，静脉内施用阻断性TLT 2：Fc重组融合蛋白减弱了响应于LPS的气管内施用的嗜中性粒细胞向肺的募集。因此，TLT 2在调节先天免疫应答中起重要的、非冗余的作用。为了更充分地了解TLT 2在先天免疫应答期间的生理作用，鉴定TLT 2的配体是至关重要的。为了实现这一目标，提出了实验来分析响应于急性炎症的TLT 2配体表达（具体目标1）。这些研究旨在证明TLT 2配体表达在炎症反应中以空间和时间方式上调。后续研究将利用该信息生成用于表达克隆的消减cDNA文库，以鉴定编码TLT 2配体的基因（特异性目的2）。所获得的信息将显著扩展对控制先天免疫应答的分子过程的理解，并将鉴定用于开发调节急性炎症应答的治疗干预的新靶点（即TLT 2配体）。

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.