Oligomerized, filamentous surface presentation of RANTES/CCL5 on vascular endothelial cells.

Inger Øynebråten,Guttorm Haraldsen,Trygve Bergeland,Nicolas Barois,Axel M Küchler,Oddmund Bakke

doi:10.1038/srep09261

Abstract

Vascular endothelial cells present luminal chemokines that arrest rolling leukocytes by activating integrins. It appears that several chemokines must form higher-order oligomers to elicit proper in vivo effects, as mutants restricted to forming dimers have lost the ability to recruit leukocytes to sites of inflammation. Here, we show for the first time that the chemokine RANTES/CCL5 binds to the surface of human endothelial cells in a regular filamentous pattern. Furthermore, the filaments bound to the surface in a heparan sulfate-dependent manner. By electron microscopy we observed labeling for RANTES on membrane projections as well as on the remaining plasma membrane. Mutant constructs of RANTES restricted either in binding to heparin, or in forming dimers or tetramers, appeared either in a granular, non-filamentous pattern or were not detectable on the cell surface. The RANTES filaments were also present after exposure to flow, suggesting that they can be present in vivo. Taken together with the lacking in vivo or in vitro effects of RANTES mutants, we suggest that the filamentous structures of RANTES may be of physiological importance in leukocyte recruitment.

Highlights

Vascular endothelial cells present luminal chemokines that arrest rolling leukocytes by activating integrins
RANTES is organized in filament-like structures on the endothelial cell surface
Analysis at different time points after exposing Human umbilical vein endothelial cells (HUVECs) to TNFa and IFNc revealed that RANTES was distributed in puncta and short elongated structures after 12 h

Summary

Introduction

Vascular endothelial cells present luminal chemokines that arrest rolling leukocytes by activating integrins. Mutagenesis studies on RANTES have shown that the interaction of RANTES and heparin is mainly mediated through the highly basic BBXB motif 44RKNR47, located on the surface exposed 40s loop of the protein[11,12] Replacement of these three basic residues with alanines (44AANA47) results in a variant with a substantially reduced heparin affinity[11,12] and a complete inability to recruit inflammatory cells to the peritoneal cavity of mice[13,14]. The filamentous expression of RANTES was not found when expressing mutants that are restricted to form dimers or tetramers, or when expressing a mutant with reduced GAG affinity These data add further support to the merging view that formation of higher order oligomers of RANTES is crucial to elicit its fully biological function in activation of leukocytes

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