Figure 2.

Slit2 inhibits neutrophil adhesion to activated endothelial cells. Freshly isolated human neutrophils were labeled with calcein and incubated with PBS, full-length hSlit2, or the bioactive N-hSlit2. Neutrophils (10⁵ cells/well) were incubated with confluent HUVEC monolayers and allowed to adhere for 30 minutes. Nonadherent cells were removed, and neutrophil adhesion was quantified using a fluorescent plate reader at excitation and emission wavelengths of 494 and 517 nm, respectively. (A) The effects of hSlit2 on neutrophil adhesion to HUVEC prestimulated with TNF-α. (B) To determine whether the observed effects of Slit2 result from its actions on neutrophils and/or endothelial cells, neutrophils were incubated with N-hSlit2 for 10 minutes, and unbound N-hSlit2 was washed away before performing the adhesion assays. In parallel experiments, endothelial cells were incubated with N-hSlit2, and unbound N-hSlit2 was washed away before performing the adhesion assays. (C) Hypoxic conditions were induced by exposing HUVEC to 1% oxygen at 37°C. Cells were exposed to 2 hours of hypoxia followed by the indicated periods of reoxygenation, and neutrophil adhesion assays were performed as in A. *P<0.05 versus basal. ^†P<0.05 versus TNF-α stimulation. ^‡P<0.05 versus HUVEC exposed to 2 hours of hypoxia followed by 0.5 hours of reoxygenation. ^§P<0.05 versus HUVECs exposed to 2 hours of hypoxia followed by 3 hours of reoxygenation. Mean values ± SEM from three to five independent experiments.