HYDRODYNAMIC INTERACTIONS/CELL DEFORMATION IN NEUTROPHIL

许多物理和生化因素结合在一起控制微循环中炎性白细胞的募集过程。虽然分子介质和血流动力学在白细胞黏附和渗出中的作用或多或少是单独理解的，但这些因素之间复杂的、非线性的相互作用却不那么清楚。本项目致力于将影响白细胞募集率的多种因素，如微尺度血流动力学、白细胞变形和黏附受体的空间分布，整合到仿生实验和最先进的计算机模拟流动下的细胞黏附中。在目标1中，我们将进行人中性粒细胞流经具有圆形截面和选择素涂层表面的微细制造的分支管道的流动黏附实验，以了解 白细胞在微血管网络中形成边缘并滚动。这些结果将与多粒子黏附动力学改进版本的理论预测进行比较，并最终与小鼠炎症模型的活体实验进行比较。在目标2中，我们将进一步扩展计算机模拟，以考虑粘弹性变形的中性粒细胞以及细胞扁平化在稳定选择素和整合素介导的内皮黏附中的作用。理论模型将通过不同细胞骨架修饰剂下的中性粒细胞压缩的微吸管实验进行验证，然后用于解释流室对选择素和整合素呈递表面的粘附性实验，其中接触面积在 滚动的中性粒细胞是通过从侧面观察相互作用来测量的。这些研究将解决我们的假设，即细胞扁平化作用于稳定滚动黏附，而下调中性粒细胞或底物黏附受体可以破坏这种稳定。最后，在目标3中，我们将使用微接触打印方法系统地研究模型内皮上选择素和整合素配体分子的相对空间分布如何作用于控制白细胞黏附的动力学和白细胞牢固停滞的最终位置。该项目的拟议工作将使用工程方法将目前关于白细胞-内皮细胞相互作用的知识整合到更完整的体内炎症图景中。

Many physical and biochemical factors combine to control the process of inflammatory leukocyte recruitment in the microcirculation. While the roles of molecular mediators and hemodynamics on leukocyte adhesion and extravasation are more or less understood individually, the complex, nonlinear interaction between these factors is less so. This project focuses on integrating multiple factors affecting rates of leukocyte recruitment such as microscale hemodynamics, leukocyte deformation, and spatial distributions of adhesion receptors, into biomimetic experiments and state-of-the-art computer simulations of cell adhesion under flow. In Aim 1 we will conduct flow adhesion experiments with human neutrophils flowing through microfabricated branching conduits with circular cross-section and selectin-coated surfaces, to understand the physics of leukocyte margination and rolling throughout the microvascular network. These results will be compared to theoretical predictions of an improved version of multiparticle adhesive dynamics and ultimately to in vivo experiments in mouse models of inflammation. In Aim 2 we will further extend the computer simulation to consider viscoelastically deforming neutrophils and the role of cell flattening in stabilizing selectin and integrin-mediated adhesion to the endothelium. The theoretical model will be validated with micropipette experiments of neutrophil compression under various cytoskeletal modifiers, and then used to help interpret flow chamber adhesion experiments to selectin and integrin-presenting surfaces where the contact area under rolling neutrophils is measured by viewing interactions from the side. These studies will address our hypothesis that cell flattening acts to stabilize rolling adhesion and that downregulation of either neutrophil or substrate adhesion receptors can disrupt this stabilization. Finally, in Aim 3 we will use microcontact printing methods to systematically study how relative spatial distributions of selectin and integrin ligand molecules on model endothelium act to control the dynamics of leukocyte adhesion and the ultimate location of leukocyte firm arrest. The proposed work of this project will use engineering methods to integrate current knowledge of leukocyte-endothelial interactions into a more complete picture of inflammation in vivo.