MOLECULAR AND FORCE DYNAMICS IN NEUTROPHIL RECRUITMENT

描述(原文逐字)：在急性炎症部位，白细胞与内皮细胞相互作用，通过一系列由白细胞边集到血管壁并进入组织的多步骤级联反应。虽然选择素家族的成员介导了中性粒细胞在内皮上的捆绑和滚动，但细胞停滞依赖于β2整合素家族的成员。这一过程对启动宿主对细菌和真菌病原体的免疫反应至关重要，也是炎症治疗干预的常见靶点。从细胞系留到稳定的黏附和迁移的中心是激活白细胞整合素与炎症内皮上上调的细胞间黏附分子-1(ICAM-1)结合。足够数量的整合素键的形成提供了细胞黏附强度，以平衡流动的血液的阻力。这种多步骤的迁移过程也适用于中性粒细胞-中性粒细胞聚集。我们已经确定同型聚集还需要L-选择素链系和β2-整合素结合。最近发表的数据表明，选择素受体也可以作为信号转导分子促进白细胞的激活。在这一应用中，我们将检验这样的假设，即通过选择素的拴系和信号传递有助于提高中性粒细胞的边集、牢固黏附和迁移的效率。提出了两个具体目标。1)确定在特定剪切力下从选择素链系向整合素依赖的牢固黏附转变的生物物理机制。2)研究L-选择素对中性粒细胞活化和整合素依赖性黏附的影响。我们的实验策略是研究从人类血液中分离并保持在原始状态的中性粒细胞。只有这样，我们才能尝试应用粘度计和流式细胞仪技术来测量定义的流体力学剪切场下毫秒分辨率的快速分子识别和信号事件。在天然细胞和稳定的细胞系中检测中性粒细胞-中性粒细胞和中性粒细胞-内皮细胞的黏附，以表达特定的白细胞和血管黏附分子。这些研究的总体目标是揭示选择素在中性粒细胞系留和信号黏附功能中的双重功能。

DESCRIPTION (verbatim from the proposal): Leukocytes interacting with the endothelium at sites of acute inflammation pass through a multistep cascade of events initiated by leukocyte margination to the vessel wall and proceeding to transmigration into the tissue. While members of the selectin family mediate the tethering and rolling of PMNs on the endothelium, cell arrest is dependent on members of the beta2-integrin family. This process is critical for initiation of the host immune response to bacterial and fungal pathogens, and is a common target for therapeutic intervention in inflammation. Central to the transition from cell tethering to stable adhesion and transmigration is the activation of leukocyte integrins to bind to intercellular adhesion molecule-1 (ICAM-1) upregulated on inflamed endothelium. The formation of integrin bonds in sufficient number provides the cell adhesion strength that balances the drag forces of flowing blood. This multistep process of emigration also applies to neutrophil-neutrophil aggregation. We have established that homotypic aggregation also requires L-selectin tethering and beta2-integrin binding. Current published data suggest that selectin receptors can also function as signal transduction molecules in facilitating leukocyte activation. In this application we will test the hypothesis that tethering and signaling through selectins serves to increase the efficiency of margination, firm adhesion, and transmigration of neutrophils. Two specific aims are proposed. 1) To determine the biophysical mechanisms underlying the transition from selectin tethering to integrin-dependent firm adhesion under defined shear. 2) To examine how tethering through L-selectin signals PMN activation and integrin-dependent adhesion. Our experimental strategy is to study neutrophils isolated from human blood and maintained in a pristine state. Only in this manner can we attempt to apply viscometry and flow cytometric techniques to measure the rapid molecular recognition and signaling events under defined hydrodynamic shear fields with millisecond resolution. Neutrophil-neutrophil, and neutrophil-endothelial adhesion will be measured in native cells and stable cell lines transfected to express specific leukocyte and vascular adhesion molecules. The overall objective of these studies is to reveal the dual functions of the selectins in tethering and signaling adhesion functions in neutrophils.