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

Dennis Pfaff,Maria Riedel,Thomas Korff,Thomas Ludwig,MéLanie HéRoult,Hellmut G Augustin,Robert Kirmse,Yvonne Reiss,Markus Hecker

doi:10.1242/jcs.030627

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 (DeltaC-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 DeltaC-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 DeltaC-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-receptor-overexpressing 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, full-length 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.

Highlights

The vascular endothelium forms an interface between the circulation and the different body compartments
Expression of EphB receptors (EphBRs) in hematopoietic cells and monocytic cell lines Guided by our previous finding that ephrin-B2 is expressed on the luminal side of endothelial cells (ECs) and is upregulated during angiogenesis (Korff et al, 2006), as well as the arteriogenesis-associated upregulation of ephrin-B2 with enhanced monocyte transmigration (Korff et al, 2008), we set out to systematically analyze the cellular mechanisms of ephrin-B2- and EphBR-mediated adhesive interactions between ECs and leukocytes
Ephrin-B2 expression in ECs mediates the adhesion of monocytes Previous research has established attractive functions of ECexpressed ephrin-B2 (Füller et al, 2003; Hamada et al, 2003), supporting the hypothesis that ephrin-B2-positive ECs exert proadhesive effects on EphBR-positive circulating mononuclear cells

Summary

Introduction

The vascular endothelium forms an interface between the circulation and the different body compartments. As such, it controls the trafficking of leukocytes. The sequential activities of selectins, integrins and IgG-superfamily molecules in concert with chemokines control leukocyte tethering, rolling, firm adhesion and subsequent transmigration through the vascular endothelium (Vestweber, 2007). The specific repertoire of adhesion molecules and chemotactic signals establishes the site and cell-type-selective vascular ‘area code’ necessary for spatiotemporal control of leukocyte trafficking (Vestweber, 2007). Whereas the major players of leukocyte trafficking have been well established, additional adhesive signals may be involved in the fine tuning of the vascular address code. The steps leading from firm leukocyte adhesion to transmigration are still poorly understood

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