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J. Cell Biol.,
Volume 142, Number 5, September 7, 1998 1381-1391
* Department of Physiology and Pathology, University of Trieste, Trieste, Italy 34127; 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 µM 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.
Laboratory of Tumor Immunology, San
Raffaele Scientific Institute, Milan, Italy 20132; and § Department of Medicine, University of Washington, Seattle, Washington 98195
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