Involvement of endothelial ephrin-B2 in adhesion and transmigration of EphB-receptor-expressing monocytes

Abstract

The vascular endothelium is a crucial interface that controls the recruitment of circulating leukocytes. Based on the luminal expression of the ephrin-B2 ligand by endothelial cells (ECs) and the expression of EphB receptors (EphBRs) by circulating monocytes, we hypothesized that EphBR-ephrinB interactions are involved in monocyte adhesion. Adhesion experiments with monocytic cells were performed on ECs that overexpressed either full-length ephrin-B2 or cytoplasmically truncated ephrin-B2 (ΔC-ephrin-B2). Atomic force microscopy confirmed similar adhesive strengths of EphBR-expressing J774 cells to ECs that either overexpressed full-length ephrin-B2 or truncated ΔC-ephrin-B2 (1-minute interaction). Yet, adhesion experiments under static or flow conditions for 30 minutes demonstrated the preferential adhesion of monocytic cells to ECs that overexpressed full-length ephrin-B2 but not to ΔC-ephrin-B2 or to ECs that had been mock transduced. Adhesion was blocked by ephrin-B2-specific and EphBR-specific antibodies. Correspondingly, adhesion of EphB4-receptoroverexpressing monocytes to ephrin-B2-positive ECs was further augmented. Trafficking experiments of cell-surface molecules revealed that, prior to internalization, the resulting EphB4-receptor-ephrin-B2 complex translocated from the luminal surface to inter-endothelial junctions. Lastly, fulllength ephrin-B2 in ECs was also involved in monocyte transmigration. Collectively, our study identifies a role of EphBR-ephrinB interactions as a new step in the cascade of events leading to monocyte adhesion and transmigration through the vascular endothelium. Supplementary material available online at

EphB4 Promotes Site-Specific Metastatic Tumor Cell Dissemination by Interacting with Endothelial Cell–Expressed EphrinB2

“…We have also shown that selected resting endothelial cell populations express ephrinB2 on their luminal surface and that circulating leukocytes express EphB receptors (28). These findings have shown that bidirectional signaling-dependent EphB4/ephrinB2 interactions control monocyte adhesion to and transmigration through endothelial cells (29). Correspondingly, EphB4 plays a pivotal role in the recruitment of monocytes to ephrinB2-positive endothelial cells to sites of ischemic arteriogenic vascular remodeling (30).…”

The Role of EphB4 and Ephrin-B2 Interactions in Prostate Cancer Models of Intravasation and Extravasation

“…Two rare, events were observed where HUVEC cells formed a circular enclosure around a 22Rv1 EphB4 over-expressing cell, and a similar event was also found for 22 Rv1 (2013) shows is that knockdown of EphB4 inhibits the invasiveness and migratory capability of tumour cells, which has been shown in multiple studies to date [16][17][118][119]135]. However, we show that CIL is restored not because the invasiveness of EphB4 overexpressing tumour cells has been suppressed, but that the interaction of with EphB4-Fc [242]. They found that between 30 min and 2.5 h the ligand is internalised and accumulated in the perinuclear region, and 6.5 hours after treatment ephrin-B2 is almost entirely degraded [242].…”

“…[242]. This study also showed that EphB expressing monocytes preferentially adhered to and transmigrated through ephrin-B2 expressing endothelium [242]. These studies corroborate the pro-adhesive effect of forward EphB4 signalling through ephrin-B2 expressing and LNCaP display an adhesive attraction to an ephrin-B2 over-expressing HUVEC endothelium, through cell-cluster formation.…”

“…However, we show that CIL is restored not because the invasiveness of EphB4 overexpressing tumour cells has been suppressed, but that the interaction of with EphB4-Fc [242]. They found that between 30 min and 2.5 h the ligand is internalised and accumulated in the perinuclear region, and 6.5 hours after treatment ephrin-B2 is almost entirely degraded [242]. Therefore, stimulation of vSMCs with ephrin-B2-Fc or EphB4-Fc leads to temporary period of signalling followed by a longer phase of protein depletion and degradation [242].…”

“…[242]. This study also showed that EphB expressing monocytes preferentially adhered to and transmigrated through ephrin-B2 expressing endothelium [242].…”