FGD5 is necessary to prevent neutrophil-induced leakiness in vitro and in vivo. (A) Paracellular permeability for 40 kDa FITC-dextran was determined for TNF-α–stimulated HUVEC that were pretreated with control (Ctrl) or FGD5 siRNA in the presence or absence of human neutrophils freshly isolated from whole blood (as indicated). Values were normalized to Ctrl siRNA monolayers without PMNs. (B) Relative transmigration of human PMNs across HUVEC monolayers treated with Ctrl or FGD5 siRNA. (C) HUVEC transfected with Ctrl or FGD5-targeting siRNA were immunoblotted for FGD5 and α-tubulin. (D) Mice were injected intrascrotally with FGD5 or Ctrl siRNA and received anti–Gr-1 antibodies or control IgG intraperitoneally the following day to deplete neutrophils. Twenty-four hours later, mice were stimulated with IL-1β for 3 hours before fluorescent microspheres were injected IV, and mice were euthanized 5 minutes later. Whole mounts of the cremaster muscle were stained with antibodies against PECAM-1 and MRP14. Arrowheads indicate microsphere leakage. Scale bars, 40 µm (left panels), 15 µm (right panels). Quantification of microspheres (E) and extravasated neutrophils (F) per vessel area. (G) Total lung lysates of mice treated with Ctrl siRNA or FGD5 siRNA were immunoblotted for the indicated antigens. Data are mean ± SEM. Results are representative of (C,D,G) or pooled from 4 independent experiments (A-B) or pooled from 4 independent experiments, with a total of 35 to 40 vessels analyzed per condition (E-F). *P < .05, **P < .01, ***P < .001, Student t test (B), 2-way ANOVA (A,E-F). n.s., not significant.