Endogenous Galectin-1 inhibits lymphocyte-endothelial cell interactions as determined by small interference RNA

LV Norling, D Cooper, M Perretti. William Harvey Research Institute, Bart’s and the London, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ.

Galectin-1 (Gal-1), a β-galactoside-binding protein, is highly expressed throughout the cytosol, nucleus and membrane compartments of endothelial cells. Previous research has shown Gal-1 to play a pivotal role in controlling experimental inflammation, modulating leukocyte-endothelial cell interactions (La et al, 2003). These studies have however focused on the role of exogenous human recombinant Gal-1. The primary objective of this study was to investigate the role of endogenous Gal-1 on lymphocyte-endothelial cell interactions under flow conditions.

To achieve this, a genetic approach using 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 and protein expression monitored by western blotting. 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 superfusion with freshly isolated lymphocytes (5x105 cells/ml; perfused at 1 dyne/cm2 for 8 min then 10 random fields of view were recorded for off-line analysis). Finally, potential changes in HUVEC phenotype were monitored by quantifying adhesion molecule expression on resting and TNF -α -stimulated HUVEC. To do so, cells were labelled with anti-ICAM-1, VCAM-1, PECAM and E-selectin monoclonal antibodies and analysed by flow cytometry. Data were analysed by ANOVA followed by Dunnett’s test.

Application of siRNA to HUVEC suppressed Gal-1 levels by 75% at 48h post-transfection. Knockdown of Gal-1 expression led to a significant increase (86%; 50.6 ± 3.7 compared with 27.2 ± 2.1, p < 0.01, n=5) in the initial capture of lymphocytes to the endothelium and a subsequent increase in cell rolling and adhesion. Basal levels of ICAM-1, VCAM and E-Selectin were higher in transfected cells compared with non-transfected HUVEC, with no significant change in PECAM expression. Levels of ICAM-1 and E-Selectin were increased in all cells after stimulation with TNF-α (4h, 10 ng/ml), while PECAM expression remained unaltered. VCAM-1 expression was increased in both the non-transfected and control transfected cells following TNF-α stimulation whereas levels remained unchanged in Gal-1 siRNA transfected cells.

These data are strongly indicative that the absence of endothelial Gal-1 augments the initial steps of the inflammatory response. It is likely that the differences observed in adhesion molecule levels post-transfection do not account for these effects of Gal-1 siRNA. In conclusion, knockdown of endothelial Gal-1 increases lymphocyte recruitment under flow, indicating that endogenous Gal-1 may act to limit such recruitment during inflammation. These findings further elucidate some of the anti-inflammatory properties of Gal-1.

La et al. Am J Pathol 163:1505-1515, 2003.

This work was funded by the Research Advisory Board of Bart’s and the London Special Trustees (RAB03/Mres).