Galectins are structurally related proteins characterised by an affinity for β-galactosides. Galectin-1 (Gal-1), the prototype member of this family, decreases neutrophil recruitment to sites of inflammation in vivo (1) and inhibits neutrophil chemotaxis in vitro (2). Endothelial cells express Gal-1 in both cytosolic and membrane compartments, as well as releasing it into culture medium. This expression is increased upon exposure to pro-inflammatory cytokines (3). Thus endothelial-derived Gal-1 may function to limit leukocyte recruitment during inflammation. The objectives of this study were to investigate the effects of exogenous and endogenous Gal-1 on neutrophil recruitment under flow in vitro and leukocyte recruitment in the inflamed cremaster in vivo. To investigate the effects of exogenous Gal-1, human umbilical vein endothelial cells (HUVEC) were stimulated with TNF-α (10ng/ml) for 4h. Freshly isolated neutrophils were incubated with human recombinant Gal-1 (0.04-4μg/ml) and perfused over the endothelial monolayers at a rate of 1 dyn/cm². To investigate the role of endothelial Gal-1, small interference RNA (siRNA) was used to deplete Gal-1 in HUVEC. Cells were transfected with non-targeting or Gal-1 specific siRNAs. Gal-1 expression, monitored by Western blotting was maximally suppressed by 75% at 48h post-transfection. HUVEC were then stimulated with TNF-α as described above and neutrophils were perfused over the monolayers at a rate of 1 dyn/cm². Six random fields were examined and the numbers of captured, rolling and adherent neutrophils were quantified off-line. To determine the effect of Gal-1 on leukocyte trafficking in vivo, Gal-1 null mice and their wild-type counterparts were treated with 30ng IL-1β (intra-scrotal) for 2-6 h followed by intra-vital microscopy of the cremaster (mice were anaesthetised with xylazine (7.5 mg/kg) and ketamine (150 mg/kg) i.p.). The numbers of rolling, adherent and emigrated leukocytes were quantified in a minimum of three vessels/mouse. Exogenous Gal-1 produced concentration-dependent effects with the lowest concentration (0.04μg/ml) significantly decreasing all three parameters measured (p<0.05, n=4). At 0.4μg/ml, neutrophil capture was significantly decreased by 49% (p<0.05, n=4), while at 4μg/ml, rolling was decreased by 94% (p<0.001, n=4). Conversely knock-down of endothelial Gal-1 resulted in significant increases in neutrophil capture and rolling by 89% and 66%, respectively (p<0.05, n=3). In vivo, a lack of Gal-1 resulted in significant increases in leukocyte emigration at 2 and 6h post IL-1 injection (p<0.05, n=4-7/group). These data indicate a role for both exogenous and endogenous Gal-1 in limiting leukocyte recruitment during inflammation.