Shulman Ziv, Alon Ronen
Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel.
Methods Enzymol. 2009;461:311-32. doi: 10.1016/S0076-6879(09)05414-7.
The mechanisms underlying leukocyte migration across endothelial barriers are still largely elusive. Integrin activation by chemokine signals is a key checkpoint in this process. Most of the current knowledge on transendothelial migration (TEM) of leukocytes has been derived from in vitro modified Boyden-chamber transfilter migration assays. In these assays, leukocyte migration toward chemokine gradients established across an endothelial barrier is measured under shear-free conditions. Consequently, these assays do not address the critical contribution of shear forces to dynamic integrin activation and redistribution at focal lymphocyte-endothelial contacts. Endothelial chemokines are displayed at high levels on blood vessel walls in vivo and play critical roles in both integrin activation and polarization of leukocytes on blood vessels, yet transwell assays do not assess the role of these chemokines in leukocyte TEM. To overcome these two drawbacks, several laboratories, including our group, developed assays based on in vitro live imaging microscopy to follow leukocyte migration across endothelial barriers that display defined compositions of integrin-stimulatory chemokines. These assays not only successfully simulate physiologic TEM processes but also enable the tracking and dissection of leukocyte adhesion, motility, and crossing of endothelial barriers in real time and under physiologic flow conditions. In addition, fluorescent tagging of membranes, adhesion molecules, and cytoskeletal regulatory elements on the endothelial barrier or the leukocyte can provide key spatial and temporal information on the mode of activity of these elements during distinct stages of leukocyte TEM. After fixation, subcellular changes in the redistribution of these key molecules can be further dissected by immunofluorescence tools and by ultrastructural analysis based on scanning and transmission electron microscopy.
白细胞穿越内皮屏障的潜在机制在很大程度上仍不清楚。趋化因子信号激活整合素是这一过程中的关键检查点。目前关于白细胞跨内皮迁移（TEM）的大多数知识来自体外改良的Boyden小室跨滤膜迁移试验。在这些试验中，在无剪切力的条件下测量白细胞向在内皮屏障上建立的趋化因子梯度的迁移。因此，这些试验没有考虑剪切力对局部淋巴细胞-内皮接触处动态整合素激活和重新分布的关键作用。内皮趋化因子在体内血管壁上高水平表达，在整合素激活和白细胞在血管上的极化中都发挥关键作用，但Transwell试验没有评估这些趋化因子在白细胞TEM中的作用。为了克服这两个缺点，包括我们小组在内的几个实验室开发了基于体外实时成像显微镜的试验，以跟踪白细胞穿越显示整合素刺激趋化因子特定组成的内皮屏障的迁移。这些试验不仅成功模拟了生理性TEM过程，还能够在生理流动条件下实时跟踪和剖析白细胞的黏附、运动以及穿越内皮屏障的过程。此外，对内皮屏障或白细胞上的膜、黏附分子和细胞骨架调节元件进行荧光标记，可以提供这些元件在白细胞TEM不同阶段活动模式的关键时空信息。固定后，这些关键分子重新分布的亚细胞变化可以通过免疫荧光工具以及基于扫描和透射电子显微镜的超微结构分析进一步剖析。
