Transendothelial migration: unifying principles from the endothelial perspective

Abstract

Transendothelial migration (TEM) of polymorphonuclear leukocytes (PMN) involves a carefully orchestrated dialog of adhesion and signaling events between leukocyte and endothelial cell. This article focuses on the contribution of endothelial cells to transmigration. The initiation of TEM itself generally requires interaction of PECAM on the leukocyte with PECAM at the endothelial cell border. This is responsible for the transient elevation of cytosolic-free calcium ions in endothelium that is required for TEM and for recruitment of membrane from the lateral border recycling compartment (LBRC). TEM requires LBRC to move to the site at which TEM will take place and for VE-cadherin to move away. Targeting of the LBRC to this site likely precedes movement of VE-cadherin and may play a role in clearing VE-cadherin from the site of TEM. The process of TEM can be dissected into steps mediated by distinct pairs of PMN/endothelial interacting molecules. CD99 regulates a step at or close to the end of TEM. CD99 signals through soluble adenylyl cyclase to activate PKA to trigger ongoing targeted recycling of the LBRC. Paracellular transmigration predominates (≥90% of events) in the cremaster muscle circulation, but transcellular migration may be more important at sites such as the blood-brain barrier. Both processes involve many of the same molecules and recruitment of the LBRC.

Keywords: CD99; endothelial cell junctions; lateral border recycling compartment (LBRC); platelet/endothelial cell adhesion molecule 1 (PECAM); transmigration.

Figure 1. Enrichment of endothelial cell adhesion molecules around leukocytes migrating transcellularly

Representative confocal or deconvolved images of monocytes or PMN caught in the act of transcellular migration are shown stained with mAb for the indicated cell adhesion molecule (CAM, left panel, green in merge), phalloidin to mark actin (middle panel, red in merge), and DAPI to mark nuclei (blue in merge). Rings of enrichment of ICAM-1, PECAM, CD99, and JAM-A were consistently seen surrounding the site at which leukocytes migrated through the endothelial cell (arrows in left panel). Each event was verified by scanning through the monolayer. Orthogonal (X-Z) projections along the plane indicated by the white line (XZ) shown below the merged images demonstrate that the leukocyte was indeed crossing the endothelial cell (arrows in right panel). Thin white line underlies the basal surface of the endothlelial cell. The JAM-A panels show a monocyte (upper panel) and a PMN (lower panel). Note in the PECAM panel and upper JAM-A panel that several leukocytes are present on the endothelial cell, but only the site of transcellular migration stains for PECAM or JAM-A, respectively, demonstrating that the PECAM and JAM-A stained around the leukocyte belongs to the endothelial cell. (*) indicates leukocytes not in the act of transmigration and not stained for PECAM or JAM-A. Long arrow in PECAM and VE-cadherin panels point to the site of transcellular migration where endothelial cell actin is pushed aside. Arrowheads mark cell borders. Scale bar = 10 μm. Reproduced from (14) with permission.

Figure 2. Schematic diagram of the sequential block assay

Molecules A and B on the EC regulate TEM in that order. (Top) If TEM is blocked by antibody against molecule A, and that block is released, antibody against B can still arrest TEM. (Bottom) If TEM is blocked by antibody against molecule B, and that block is released, TEM can no longer be blocked by antibody against A.

Figure 3. Targeted trafficking of membrane from the LBRC to surround leukocytes undergoing transcellular migration

Staining of recycled PECAM as a surrogate for recycling LBRC (left panel, red in overlay) surrounding monocytes (upper panel) and a PMN (lower panel) demonstrates movement of membrane from the LBRC to the endothelial surface. Recycled LBRC is seen around the leukocytes 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 leukocytes in the act of transcellular migration. Arrows indicate recycled PECAM around leukocytes. Bar = 10 μm. Reproduced from (14) with permission.