Shulman Ziv, Alon Ronen
Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel.
Methods Mol Biol. 2012;757:31-45. doi: 10.1007/978-1-61779-166-6_3.
The role of integrins in leukocyte migration across endothelial barriers is widely accepted. In contrast, the contribution of integrins to interstitial motility of leukocytes is still elusive. Chemokine binding to G-protein-coupled receptors expressed on the surface of leukocytes plays key roles in both of these processes by directly activating integrin conformations favorable for ligand binding and integrin microclustering. Chemokines can also serve as weak adhesive ligands and potent inducers of actin cytoskeleton remodeling. Real-time assays utilizing live imaging microscopy have been implemented to dissect these versatile roles of chemokines in different leukocyte migration processes. Here, we review several in vitro assays useful for exploring the contribution of chemokine signals and shear forces to integrin activation and function during various stages of leukocyte transendothelial migration. In addition, we describe a new assay that assesses the contribution of chemokines to integrin-independent interstitial leukocyte motility. These assays can also follow the outcome of specific genetic or biochemical manipulations of either the leukocyte or the endothelial barrier on distinct migratory steps. Following fixation, subcellular changes in the distribution of integrin subsets and of specific integrin-associated adaptors can be further dissected by immunofluorescence tools and by ultrastructural electron microscopic analysis.
整合素在白细胞穿越内皮屏障迁移过程中的作用已被广泛认可。相比之下，整合素对白细胞间质运动的贡献仍不明确。趋化因子与白细胞表面表达的G蛋白偶联受体结合，通过直接激活有利于配体结合和整合素微簇形成的整合素构象，在这两个过程中都发挥着关键作用。趋化因子还可作为弱黏附配体和肌动蛋白细胞骨架重塑的强效诱导剂。利用实时成像显微镜的实时检测方法已被用于剖析趋化因子在不同白细胞迁移过程中的这些多样作用。在此，我们综述了几种体外检测方法，这些方法有助于探究趋化因子信号和剪切力在白细胞跨内皮迁移不同阶段对整合素激活和功能的作用。此外，我们描述了一种新的检测方法，该方法可评估趋化因子对不依赖整合素的白细胞间质运动的作用。这些检测方法还可追踪白细胞或内皮屏障的特定基因或生化操作对不同迁移步骤的影响。固定后，可通过免疫荧光工具和超微结构电子显微镜分析进一步剖析整合素亚群及特定整合素相关衔接蛋白分布的亚细胞变化。
