Figure 4.

Polymorphonuclear (PMN) cell transmigration and vascular endothelial (VE)-cadherin gap formation require lateral border recycling compartment (LBRC) trafficking. Targeted recycling assay was performed on cytokine-activated human umbilical endothelial cell (HUVEC) monolayers using PMN cells. A: Under control (CTL) conditions, LBRCs are enriched around a PMN cell (red arrows). There is a corresponding VE-cadherin gap at this site (green arrows). The merged image indicates a transmigrating PMN cell is present at this site (blue color; XZ view orthogonal). In contrast, in endothelial cells that were pretreated with 1 μmol/L of demecolcine (DCN) (right panels), PMN cells do not transmigrate (orthogonal), there is no enriched LBRC immunofluorescence at this site (red arrows), and there is no VE-cadherin gap (green arrows). The horizontal dotted lines in the merged view represent the plane of the orthogonal image, and the dotted lines in the XZ view indicate the plane of the endothelial monolayer. B: Quantification of the targeted recycling assay illustrates that under control conditions, targeted recycling occurs and is associated with VE-cadherin gaps, whereas in HUVECs treated with DCN, targeted recycling is markedly inhibited and is associated with absence of VE-cadherin gaps. C: HUVECs were treated with pharmacologic agents as above. PMN cells were allowed to transmigrate for 20 minutes at 37°C. Data in B and C are expressed as means ± SEM and represent data from three independent experiments from >100 PMN/endothelial interactions; each condition repeated in triplicates. ^∗∗∗P < 0.001, ^∗∗∗∗P < 0.0001, t-test. Scale bars = 10 μm. XZ, orthogonal view displays the XZ plane of a three-dimensional image at a given point.