Figure 3.

Slit2 inhibits neutrophil capture by, adhesion to, and transendothelial migration across activated endothelial cells under shear flow conditions. HUVEC grown to confluence in channels of the Bioflux microfluidic system were incubated with TNF-α for 4 hours. Calcein-labeled human neutrophils were preincubated with PBS or hSlit2 and then perfused through the channels at a shear rate of 0.5 dynes/cm². A Nikon TE2000 inverted microscope and Hamamatsu video camera were used to video record neutrophil–HUVEC interactions. Neutrophil adhesion was quantified using Bioflux Montage software. (A) Neutrophil–endothelial interactions were recorded after 1 minute of flow at 0.5 dynes/cm², and the number of neutrophils that were rolling and firmly arrested was determined. (B) Neutrophil adhesion to the endothelial monolayer was determined after 12 minutes of shear flow at 0.5 dynes/cm², and representative images were taken from three to five independent experiments using a 20× objective. (C) Experiments were performed as in B, and the number of neutrophils was quantified. (D) To assess the effects of Slit2 on neutrophil transendothelial migration, HUVEC were grown to confluence on fibronectin-coated polyester transwell inserts placed in a 24-well plate and exposed to hypoxia (2 hours) followed by reoxygenation (0.5 hours). Calcein-labeled human neutrophils were incubated with PBS or Slit2 (4.5 µg/ml) for 10 minutes and placed in the upper well of the transwell chamber, and the chemokine IL-8 (50 ng/ml) was added to the lower well. Neutrophils were allowed to migrate for 3 hours at 37°C. Neutrophils that had migrated from the upper to the lower well were permeabilized with 1% Triton, and the fluorescence emitted was read using a fluorescent plate reader. Mean values ± SEM from three independent experiments. *P<0.05 versus TNF-α–stimulated HUVECs. ^†P<0.05 versus basal HUVEC. ^‡P<0.05 versus IL-8 treatment and HUVEC exposure to 2 hours of hypoxia followed by 0.5 hours of reoxygenation.