A real time in vitro assay for studying leukocyte transendothelial migration under physiological flow conditions.

The mechanisms underlying leukocyte migration across endothelial barriers are largely elusive. 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 towards chemokine gradients constructed across the endothelial barrier is measured under shear-free conditions. These assays do not incorporate the contribution of shear flow to leukocyte adherence and migration across the endothelial barrier. Furthermore, transfilter assays do not reconstitute the physiological distribution of endothelial chemokines shown to be displayed in vivo at high levels on vessel walls. To overcome these two drawbacks, we have recently developed a novel in vitro assay to follow real time leukocyte migration across endothelial barriers under physiological flow conditions. Using this assay, we have found that apically displayed endothelial chemokines could trigger robust lymphocyte TEM through signaling to lymphocyte-expressed G-protein coupled receptors. This migration required continuous exposure of lymphocytes, adherent to the endothelial barrier, to fluid shear, but did not require a chemotactic gradient across the barrier. In the present review, we describe this new flow-based migration assay and discuss future applications for investigating TEM processes of different types of leukocytes across distinct endothelial barriers.