The regulation of transendothelial migration: new knowledge and new questions

Abstract

Leucocyte transendothelial migration (TEM) involves a co-operative series of interactions between surface molecules on the leucocyte and cognate counter-ligands on the endothelial cell. These interactions set up a cascade of signalling events inside the endothelial cell that both allow for the junctions to loosen and for membrane to be recruited from the lateral border recycling compartment (LBRC). The LBRC is thought to provide an increased surface area and unligated receptors to the leucocyte to continue the process. The relative importance of the individual adhesion/signalling molecules that promote transmigration may vary depending on the type of leucocyte, the vascular bed, the inflammatory stimulus, and the stage of the inflammatory response. However, the molecular interactions between leucocyte and endothelial cell activate signalling pathways that disengage the adherens and tight junctions and recruit the LBRC to the site of transmigration. With the exception of disengaging the junctions, similar molecules and mechanisms promote transcellular migration as paracellular migration of leucocytes. This review will discuss the molecular interactions and signalling pathways that regulate transmigration, and the common themes that emerge from studying TEM of different leucocyte subsets under different inflammatory conditions. We will also raise some unanswered questions in need of future research.

Keywords: Adhesion molecules; Cell junctions; Diapedesis; Lateral border recycling compartment; Transendothelial migration.

Figure 1

PECAM, CD99, and JAM-A, but not VE-cadherin, are in the LBRC. Endothelial cells were incubated with HRP-conjugated mAb specific for PECAM, CD99, JAM-A, or VE-cadherin for 1 h at 37°C, then fixed, and reacted with diaminoabenzidine-H₂O₂ as described.^– As a control for non-specific labelling, free HRP was added at the concentration present on the antibodies. En face sections were cut for EM analysis. In addition to being present along the cell border (dark electron-dense staining), PECAM, CD99, and JAM-A are all present in interconnected vesicular structures of the LBRC. VE-cadherin is present at the cell border (arrowhead), but not in the interconnected vesicular structures adjacent to it (arrows). Arrowheads indicate cell borders; arrows indicate the LBRC. Scale bar: 200 nm. Reproduced from Mamdouh et al. with permission.

Figure 2

Targeted trafficking of membrane from the LBRC to surround leucocytes undergoing transcellular migration. PECAM in the LBRC was labelled with Fab fragments of a non-blocking mAb as a surrogate marker for LBRC trafficking to the endothelial surface, as described.^– Staining of recycled PECAM as a surrogate for recycling LBRC (left panel, red in overlay) surrounding monocytes (upper panel) and a neutrophil (PMN) (lower panel) demonstrates movement of membrane from the LBRC to the endothelial surface. Recycled PECAM is seen around the leucocytes migrating transcellularly (thin arrows) far from the endothelial border (arrowheads). Recycling LBRC enrichment is as great around the monocyte migrating transcellularly as it is around the monocyte migrating paracellularly (thick arrow). Actin staining (middle panel, green in merge) shows relative positions of the cells. Orthogonal (X–Z) projection demonstrates leucocytes in the act of transcellular migration. Arrows indicate recycled PECAM around leucocytes. Bar = 10 μm. Reproduced from Mamdouh et al. with permission.

Figure 3

Schematic model of some molecular events that regulate TEM. A highly oversimplified diagram of a leucocyte (top) engaging endothelial cells at the start of TEM. Events relevant to weakening of adherens junctions are shown in the left endothelial cell; those relevant to the LBRC are shown in the right endothelial cell. In reality, these events are taking place simultaneously in both cells. Similarly, the clustering of ICAM-1 and VCAM-1 is separated, but these molecules intermix. Clustering of ICAM-1 by leucocyte integrins LFA-1 and Mac-1 and of VCAM-1 by leucocyte integrin VLA-4 leads to the signalling cascades described in the text. These pathways intersect and both lead to increase in actin–myosin tension and phosphorylation of VE-cadherin, which facilitates its clearance for the adherens junction. Homophilic interactions between PECAM (hook-shaped surface molecules) on the leucocyte and PECAM on the endothelial cell lead to activation of kinesin and recruitment of the LBRC membrane along microtubules to the site of TEM. Clustering of ICAM-1 activates src, which is required for TEM and brings SHP2 to the site of TEM, perhaps to dephosphorylate PECAM and PVR and reset the system. Other abbreviations: ↑[Ca⁺²]_i , increase in cytosolic-free calcium ion; CaM, calmodulin; MLCK, myosin light chain kinase; MPase, myosin phosphatase; ROCK, Rho kinase; ROS, reactive oxygen species; circled P, phosphorylated state.