Endothelial cell E- and P-selectin and vascular cell adhesion molecule-1 function as signaling receptors.

P Lorenzon,E Vecile,F Tedesco,E Nardon,A Dobrina,J.M Harlan,E Ferrero

doi:10.1083/jcb.142.5.1381

Abstract

Previous studies have shown that polymorphonuclear leukocyte (PMN) adherence to endothelial cells (EC) induces transient increases in EC cytosolic free calcium concentration ([Ca2+]i) that are required for PMN transit across the EC barrier (Huang, A.J., J.E. Manning, T. M. Bandak, M.C. Ratau, K.R. Hanser, and S.C. Silverstein. 1993. J. Cell Biol. 120:1371-1380). To determine whether stimulation of [Ca2+]i changes in EC by leukocytes was induced by the same molecules that mediate leukocyte adherence to EC, [Ca2+]i was measured in Fura2-loaded human EC monolayers. Expression of adhesion molecules by EC was induced by a pretreatment of the cells with histamine or with Escherichia coli lipopolysaccharide (LPS), and [Ca2+]i was measured in single EC after the addition of mAbs directed against the EC adhesion proteins P-selectin, E-selectin, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), or platelet/endothelial cell adhesion molecule-1 (PECAM-1). Both anti-P- and anti-E-selectin mAb, as well as anti-VCAM-1 mAb, induced transient increases in EC [Ca2+]i that were comparable to those induced by 200 microM histamine. In contrast, no effect was obtained by mAbs directed against the endothelial ICAM-1 or PECAM-1. PMN adherence directly stimulated increases in [Ca2+]i in histamine- or LPS-treated EC. mAbs directed against leukocyte CD18 or PECAM-1, the leukocyte counter-receptors for endothelial ICAM-1 and PECAM-1, respectively, did not inhibit PMN-induced EC activation. In contrast, mAb directed against sialyl Lewis x (sLex), a PMN ligand for endothelial P- and E-selectin, completely inhibited EC stimulation by adherent PMN. Changes in EC [Ca2+]i were also observed after adherence of peripheral blood monocytes to EC treated with LPS for 5 or 24 h. In these experiments, the combined addition of mAbs to sLex and VLA-4, the leukocyte counter-receptor for endothelial VCAM-1, inhibited [Ca2+]i changes in the 5 h-treated EC, whereas the anti-VLA-4 mAb alone was sufficient to inhibit [Ca2+]i changes in the 24 h-treated EC. Again, no inhibitory effect was observed with an anti-CD18 or anti-PECAM-1 mAb. Of note, the conditions that induced changes in EC [Ca2+]i, i.e. , mAbs directed against endothelial selectins or VCAM-1, and PMN or monocyte adhesion to EC via selectins or VCAM-1, but not via ICAM-1 or PECAM-1, also induced a rearrangement of EC cytoskeletal microfilaments from a circumferential ring to stress fibers. We conclude that, in addition to their role as adhesion receptors, endothelial selectins and VCAM-1 mediate endothelial stimulation by adhering leukocytes.

Highlights

The process of leukocyte extravasation at sites of inflammation involves two main events: (1) adherence of circulating leukocytes to the endothelial wall of post-capillary venules, and (2) passage of leukocytes across the endothelium
In an effort to analyze comprehensively the involvement of endothelial adhesion proteins in transmembrane signaling, we examined the role of E-selectin, P-selectin, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and platelet/endothelial cell adhesion molecule-1 (PECAM-1) in mediating intracellular changes resulting from leukocyte adherence
After incubation of Endothelial cells (ECs) in the presence or absence of histamine or LPS, two agents known to induce expression of adhesion molecules on Human umbilical vein endothelial cells (HUVEC) (Schleimer and Rutledge, 1986; Lorant et al, 1991), EC were exposed to leukocyte adherence-blocking mAbs directed against endothelial P- and E-selectins, ICAM-1, VCAM-1, and PECAM-1

Summary

Introduction

The process of leukocyte extravasation at sites of inflammation involves two main events: (1) adherence of circulating leukocytes to the endothelial wall of post-capillary venules, and (2) passage (diapedesis) of leukocytes across the endothelium. Endothelial stimulation by adhering leukocytes was investigated by Huang et al (1993) They found that there was a transient increase in endothelial [Ca2ϩ]i during transendothelial migration of polymorphonuclear leukocytes (PMNs). Yoshida et al (1996) suggested that E-selectin, an inducible endothelial adhesion molecule that binds to complex carbohydrate ligands on leukocytes, may have a role in outside-in signaling. Durieur-Trautmann et al (1994) demonstrated that clustering of intercellular adhesion molecule-1 (ICAM-1), the endothelial ligand for the leukocyte CD11/ CD18, induced tyrosine phosphorylation of cytoskeletal proteins potentially involved in the assembly of adherence junctions. In an effort to analyze comprehensively the involvement of endothelial adhesion proteins in transmembrane signaling, we examined the role of E-selectin, P-selectin, ICAM-1, vascular cell adhesion molecule-1 (VCAM-1), and platelet/endothelial cell adhesion molecule-1 (PECAM-1) in mediating intracellular changes resulting from leukocyte adherence. We questioned whether engagement of endothelial adhesion receptors by leukocytes would provoke cytoskeletal changes similar to those induced when thrombin binds to its receptor, and whether these would account for changes in EC shape during leukocyte diapedesis

Methods

Results

Conclusion