Examination of MCP-1 (CCL2) partitioning and presentation during transendothelial leukocyte migration.

Abstract

It is proposed that a chemokine concentration gradient promotes vectorial leukocyte migration across the vascular endothelium during inflammation. In this study, monocyte migration across a model endothelial monolayer was assessed at defined time-points after the addition of MCP-1 (CCL2). At each time-point transendothelial migration was quantified, medium from the apical and basal surface was collected for ELISA and monolayers were stained to detect both heparan sulfate and MCP-1. Statistically significant monocyte migration was observed within 60 min of chemokine addition to the basal surface of the endothelium and an asymmetric distribution of MCP-1 across the monolayer was observed at all time-points. Dual color immunofluorescence analysis demonstrated that MCP-1 was focused into heparan sulfate-containing domains on the apical surface of some of the endothelial cells. Furthermore, no uniform concentration gradient of chemokine was observed within the space between adjacent endothelial cells with apical MCP-1 application resulting in a staining pattern identical to that observed after basal application. The addition of a functional, monomeric form of MCP-1 produced a staining pattern identical to that observed using the wild-type protein, suggesting that localized chemokine oligomerization is not responsible for generating the focal chemokine distribution. Together, these data suggest that apical presentation of concentrated, chemokine-containing domains provides sufficient stimulus to promote transendothelial leukocyte migration in the absence of the formation of a formal haptotactic concentration gradient between endothelial cells.