MOLECULAR AND FORCE DYNAMICS IN NEUTROPHIL RECRUITMENT

描述（提案中的逐字表述）：在急性炎症部位与内皮细胞相互作用的白细胞通过由白细胞向血管壁边集引发的多步级联事件，并继续迁移到组织中。虽然选择素家族的成员介导中性粒细胞在内皮上的束缚和滚动，但细胞停滞依赖于β 2-整联蛋白家族的成员。这一过程对于启动宿主对细菌和真菌病原体的免疫应答至关重要，并且是炎症治疗干预的常见靶标。从细胞束缚到稳定粘附和迁移的转变的中心是激活白细胞整联蛋白以结合发炎内皮上上调的细胞间粘附分子-1（ICAM-1）。足够数量的整联蛋白键的形成提供了平衡流动血液的阻力的细胞粘附强度。这种多步骤的迁移过程也适用于嗜中性粒细胞-中性粒细胞聚集。我们已经确定，同型聚集也需要L-选择素拴系和β 2-整联蛋白结合。目前发表的数据表明，选择素受体也可以作为信号转导分子，促进白细胞活化。在这个应用程序中，我们将测试的假设，通过选择素的束缚和信号传递，以增加中性粒细胞的边缘化，牢固的粘附和迁移的效率。提出了两个具体目标。1)确定在确定的剪切力下从选择素拴系到整合素依赖性牢固粘附转变的生物物理机制。2)探讨L-选择素的束缚作用如何影响中性粒细胞活化和整合素依赖性粘附。我们的实验策略是研究从人类血液中分离并保持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术技术来测量快速的分子识别和信号事件下定义的流体动力学剪切场毫秒分辨率。将在天然细胞和转染以表达特异性白细胞和血管粘附分子的稳定细胞系中测量中性粒细胞-中性粒细胞和嗜中性粒细胞-内皮细胞粘附。这些研究的总体目标是揭示中性粒细胞中选择素在束缚和信号粘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.