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Endogenous Galectn‐1 inhibits lymphocyte‐endothelial cell interactions as determined by small interference RNA
Author(s) -
Norling Lucy Victoria,
Cooper Dianne,
Perretti Mauro
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a647-a
Subject(s) - gene knockdown , transfection , small interfering rna , umbilical vein , endogeny , endothelial stem cell , endothelium , cell , microbiology and biotechnology , lymphocyte , rna interference , inflammation , cell culture , flow cytometry , biology , rna , immunology , biochemistry , in vitro , endocrinology , genetics , gene
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 (5x10 5 cells/ml; perfused at 1 dyne/cm 2 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)