ICAM-1 stimulated NO production and PMN transmigration are dependent on Src, Akt, and eNOS. (A) ICAM-1 crosslinking (XL) induced NO production in HUVECs. NO production was measured in the medium during secondary antibody incubation (30 minutes) using a porphyrinic NO electrode. (B) ICAM-1 XL-induced NO production was sensitive to both PP2 and wortmannin (Wort). Data are mean ± SD of triplicates from a representative experiment that was repeated 3 times with equivalent results. (C) ICAM-1–mediated PMN transendothelial cell migration was assessed in HUVECs expressing empty vector, WT–ICAM-1, or Y518F–ICAM-1 (top panels) or after treatment with pharmacologic inhibitors (bottom panels). Mouse PMNs labeled with fluorescent dye were added to transfected HUVEC monolayers grown to confluence in tissue culture wells for adhesion assays or Transwell filter inserts to assess transmigration. Migration was triggered by the addition of 10nM fMLP to the bottom chamber, and then the PMN fluorescence that accumulated was measured after 3 hours. (D) HUVEC monolayers were pretreated with L-NAME (1mM), PP2 (10μM), or wortmannin (100nM) for 30 minutes. Later, 10nM fMLP was added to the bottom chamber of Transwell plates to initiate migration. ICAM-1–mediated PMN adhesion was increased by L-NAME and wortmannin, whereas transmigration was blocked by PP2, wortmannin, and L-NAME. Data are the mean ± SD of 4 or 5 experiments. *P < .05 versus Ctrl (A-D); P < .05 versus WT (C-D).