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.
Studies will be performed to identify and understand the physiological expression pattern of the ligand for the transmembrane receptor TLT2, which is expressed on neutrophils, monocytes/macrophages and B cells, and plays a critical role in regulation of the innate immune response against microbial pathogens. Studies will initially determine how expression of the ligand for TLT2 is regulated in response to acute inflammation using whole animal imaging and this information will be used to clone the gene encoding the ligand for TLT2. Identification of the ligand for TLT2 will provide important information about the innate immune response and how it responds to microbial infection or acute inflammation.