Activation of HUVEC monolayers by IL-1 beta or TNF-alpha induces migration of eosinophils across the endothelial monolayer (i.e., transendothelial migration) in vitro. In the present study, we demonstrate that culture of freshly isolated eosinophils in the presence of IL-5 for 24 to 48 h before use in the assay dramatically potentiated CD18-dependent eosinophil transendothelial migration through unstimulated endothelial monolayers. Granulocyte macrophage (GM)-CSF induced eosinophil transendothelial migration but did not induce neutrophil transendothelial migration. When IL-1 beta-activated endothelial cells were used, GM-CSF, IL-3, or IL-5 caused only modest potentiation of eosinophil transendothelial migration. Since activated endothelial cells are known to produce GM-CSF, we hypothesized that endothelial-derived GM-CSF might play a role in the process of IL-1 beta-induced eosinophil transendothelial migration. IL-1 beta-activated endothelial monolayers grown on the permeable supports produced 0.3 +/- 0.1 ng/ml of GM-CSF during a 4-h incubation and neutralizing Ab against GM-CSF inhibited eosinophil transendothelial migration by 48%, suggesting that endothelial-derived GM-CSF may participate in the response. Eosinophils purified from bronchoalveolar lavage 18 to 20 h after experimental Ag challenge in the lungs of allergic volunteers showed enhanced transendothelial migration, indicating that the cells may have undergone cytokine activation in vivo. Eosinophil-active cytokines may contribute to the preferential accumulation of eosinophils in vivo in part via potentiation of eosinophil transendothelial migration.