Enhanced ICAM-1 expression in HUVECs directs transcellular TEM. (A) PMN accumulation and TEM on 4-hour TNF-α-activated iHUVECs, GFP iHUVECs, or ICAM-1GFP iHUVECs were assessed as described in “Materials and methods” and Figure 1D legend. (B) Increasing numbers of PMNs (as indicated) were perfused across HUVEC monolayers under lower shear stress (1 dyne/cm²). Accumulation of PMNs (left) and the route of TEM (right) were determined. (C) PMNs (10⁶/mL) were perfused across HUVEC monolayers at the estimated shear stress indicated. The route of TEM (right) was determined as in “Materials and methods.” n = 3 experiments. (D) Electron microscopy of PMNs undergoing transcellular TEM of 4-hour TNF-α-activated ICAM-1GFP iHUVECs under static conditions. In the top panel, 2 PMNs have extended granule-containing and, in one case, nuclear-lobe-containing cell processes, completely through the endothelial cell thickness from their apical to basilar surfaces. These endothelial cell holes are bound by endothelial cell plasma membranes and are located fewer then 5 μm (the cutoff used to group migrating cells in the “junctional route” category) from adjacent closed junctions (J1 and J2) and apposed lateral borders (arrows) of individual endothelial cells in the HUVEC monolayer. In lower panel, another PMN is nearly completely beneath the HUVEC layer after passing through an endothelial cell hole fewer than 5 μm from the adjacent closed junction (arrow) between 2 endothelial cells (EC1 and EC2). An organelle-free, actin-rich, cellular process (arrowhead) still spans the endothelial cell hole. Magnification top, × 18 000; lower, × 20 500. Bar, 1 μm. (E) Live-cell imaging of ICAM-1 GFP iHUVEC monolayers was performed as detailed³⁵  to monitor redistribution of ICAM-1 during PMN transcellular TEM under shear flow (1 dyne/cm²). Arrows identify 2 PMNs that undergo transcellular TEM (see Video S2). Bars, 20 μm. ^*P < .05.