Endothelial Regulation of Leukocyte Transmigration

白细胞迁移的内皮调节

• 批准号: 7249469
• 负责人: FRANCIS W. LUSCINSKAS
• 金额: $ 40.67万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-08-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7249469
• 关键词: Actins; Acute; Address; Adherent Culture; Adhesions; Adhesives; Amino Acids; Arteries; Atherosclerosis; Binding; Biochemical; Biological Assay; Biomechanics; Blood Vessels; Blood flow; Buffers; CXCR4 gene; Cell model; Cells; Characteristics; Chemotactic Factors; Chronic; Condition; Cytoplasmic Protein; Cytoplasmic Tail; Cytoskeleton; Data; Defect; Disease; E-Selectin; Endothelial Cells; Endothelium; Environment; Exhibits; Failure; Fluorescence; Fluorescence Recovery After Photobleaching; Funding; Goals; Green Fluorescent Proteins; Human; Image; Immune; In Vitro; Inflammation; Inflammatory; Injury; Intercellular Junctions; Intercellular adhesion molecule 1; Leukocytes; Life; Link; Location; Measurement; Mediating; Memory; Mesentery; Modeling; Mononuclear Leukocytes; Mus; Mutagenesis; Mutation; Neutrophil Infiltration; Organ; P-selectin ligand protein; Pathway interactions; Permeability; Phenotype; Physiological; Play; Protein Binding; Protein Overexpression; Proteins; Range; Reaction; Regulation; Reporting; Research Personnel; Retinal Cone; Role; Route; Signal Pathway; Signal Transduction; Site; Spatial Behavior; System; T memory cell; T-Lymphocyte; T-Lymphocyte Subsets; Tail; Techniques; Testing; Therapeutic Intervention; Tissues; Transgenic Mice; Vascular Endothelium; Vascular Permeabilities; Vascular System; Venous; Wild Type Mouse; Work; cellular imaging; cremaster muscle; cytokine; design; fluorescence imaging; in vitro Assay; in vitro Model; in vivo; in vivo Model; intravital microscopy; monocyte; monolayer; mutant; neutrophil; novel; programs; shear stress; trafficking

DESCRIPTION (provided by applicant): The interaction of neutrophils and mononuclear leukocytes with endothelium plays a pivotal role in leukocyte recruitment in acute and chronic inflammation, immune reactions, vascular injury and atherosclerosis. In the previous funding period, four major accomplishments occurred. (1) We identified a novel E-selectin dependent adhesion pathway in T cells subsets. (2) We determined that t-bet T cells have a defect in trafficking in vivo and in vitro related to impaired PSGL-1 function and failure to express CXCR4. (3) We developed a live cell fluorescence assay to resolve the temporal and spatial behavior of LFA-1 and ICAM-1 during leukocyte transmigration. (4) We developed an in vitro assay for non-junctional neutrophil transmigration. This proposal builds on this scientific progress and will consist of three interrelated specific aims that will focus on the role of endothelial cell ICAM-1 tail in leukocyte transmigration. Our working hypothesis is that during leukocyte adhesion, LFA-1 binds ICAM-1, triggering its association with the cytoskeleton, and this step functions in "outside in" signaling to promote ICAM-1 clustering around transmigrating leukocytes. Specific Aim 1 will elucidate the role of ICAM-1 cytoplasmic tail in leukocyte transmigration using biochemical and biophysical techniques as well as a newly developed live cell fluorescence imaging system and vascular endothelium that support both junctional and non-junctional transmigration. Specific Aim 2 will test the role of the ICAM-1 tail in chemoattractant driven leukocyte transmigration in an in vivo model of inflammation. Confocal intravital fluorescence imaging will visualize the sites of leukocyte transmigration (junctional or non-junctional route) using fluorescent markers of cell-cell junctions, and in parallel, determine whether the route of transmigration adversely effects vascular permeability. Specific Aim 3 will study the role of the ICAM-1 tail in an arterial endothelial cell model of inflammation in vitro. Endothelial monolayers of an arterial or venous phenotype will be created in vitro by applying a pulsatile shear stress in an arterial or venous waveform using a recently described cone-plate apparatus. The adhesive interactions and sites of transmigration of blood monocytes and memory T cells with the above conditioned endothelium will be examined. The information gained from these studies will help delineate the ICAM-1 dependent mechanisms underlying leukocyte transmigration and may thus define new targets for therapeutic intervention in immune and inflammatory diseases.

描述（由申请人提供）：中性粒细胞和单核白细胞与内皮细胞的相互作用在急性和慢性炎症、免疫反应、血管损伤和动脉粥样硬化中的白细胞募集中起着关键作用。在上一个资助期间，取得了四项重大成就。 (1) 我们在 T 细胞亚群中发现了一种新的 E-选择素依赖性粘附途径。 (2) 我们确定 t-bet T 细胞在体内和体外运输方面存在缺陷，这与 PSGL-1 功能受损和不能表达 CXCR4 有关。 (3)我们开发了一种活细胞荧光测定法来解析LFA-1和ICAM-1在白细胞迁移过程中的时间和空间行为。 (4) 我们开发了一种非连接性中性粒细胞迁移的体外测定方法。该提案建立在这一科学进展的基础上，由三个相互关联的具体目标组成，重点关注内皮细胞 ICAM-1 尾部在白细胞迁移中的作用。我们的工作假设是，在白细胞粘附过程中，LFA-1 结合 ICAM-1，触发其与细胞骨架的关联，并且此步骤在“由外向内”信号传导中发挥作用，促进 ICAM-1 聚集在迁移的白细胞周围。具体目标 1 将利用生物化学和生物物理技术以及新开发的活细胞荧光成像系统和支持连接和非连接轮回的血管内皮来阐明 ICAM-1 细胞质尾部在白细胞轮回中的作用。具体目标 2 将测试 ICAM-1 尾部在体内炎症模型中趋化剂驱动的白细胞迁移中的作用。共聚焦活体荧光成像将使用细胞-细胞连接的荧光标记来可视化白细胞迁移的部位（连接或非连接途径），并同时确定迁移途径是否对血管通透性产生不利影响。具体目标 3 将研究 ICAM-1 尾部在体外炎症动脉内皮细胞模型中的作用。通过使用最近描述的锥板装置在动脉或静脉波形中施加脉动剪切应力，可以在体外产生动脉或静脉表型的内皮单层。将检查血液单核细胞和记忆T细胞与上述条件内皮的粘附相互作用和迁移位点。从这些研究中获得的信息将有助于描述白细胞迁移背后的 ICAM-1 依赖性机制，从而可能确定免疫和炎症疾病治疗干预的新目标。