Endogenous Galectn‐1 inhibits lymphocyte‐endothelial cell interactions as determined by small interference RNA

Abstract

It has recently been suggested that, besides its role on T cell life span, the β-galactoside binding protein Galectin-1 (Gal-1) may play a pivotal role in controlling experimental inflammation by modulating leukocyte-endothelial cell interactions. These studies have so far focused on the effects of exogenously administered Gal-1. The primary objective of this study was to investigate the function of endothelial Gal-1 on lymphocyte-endothelial cell interactions under flow conditions. To achieve this, small interference RNA (siRNA) synthesised by Santa Cruz Biotechnology was used to deplete Gal-1 in human umbilical vein endothelial cells (HUVEC). Cells were transfected with or without non-targeting or Gal-1 specific siRNAs. In all cases, HUVECs were stimulated with TNF-α (10 ng/ml) for 4h, and for 15 min with SDF-1α (20ng/ml) prior to perfusion with freshly isolated lymphocytes (5x105 cells/ml; perfused at 1 dyne/cm2 for 8 min then 10 random frames recorded for off-line analysis). Application of siRNA suppressed Gal-1 levels by 75% at 48h post-transfection as monitored by western blotting. Knockdown of Gal-1 led to a significant increase (86%) in the initial capture of lymphocytes to the endothelium and a subsequent increase in cell rolling and adhesion. These data are strongly indicative that the absence of endothelial Gal-1 favours the initial steps of the inflammatory response. To conclude, knockdown of Gal-1 increases lymphocyte recruitment under flow, indicating that endogenous Gal-1 may act to limit such recruitment during inflammation. This work was funded by the Bart’s and the London Special Trustees (RAB03/Mres)