Migration Of Primed Human Eosinophils Across Cytokine-Activated Endothelial-Cell Monolayers

Eosinophils are known to adhere to cytokine-activated endothelium. Whereas transendothelial migration for neutrophils is an inevitable consequence of this endothelial-dependent adherence, this has not yet been shown for eosinophils. By means of human umbilical vein endothelial cells (HUVE) grown to confluence on microporous filters as an in vitro model of leukocytic migration across postcapillary venules, we have characterized the conditions leading to endothelium-driven transmigration of blood eosinophils from normals and from patients with allergic asthma. Freshly isolated eosinophils from nonallergic donors adhered to interleukin-1 (IL-1) and tumor necrosis factor-activated HUVE, but did not penetrate these monolayers. In contrast, eosinophils from allergic asthma patients showed an increased adherence and transmigration capacity. This increased functional competence was not caused by a difference in density phenotype, because the eosinophils from both groups showed a comparable density distribution over discontinuous Percoll gradients. Moreover, no difference existed within one group among eosinophils harvested from the Percoll density bands 1.080, 1.085, and 1.090 g/mL in terms of transendothelial migration. In vitro cultivation of freshly isolated eosinophils from nonallergic individuals in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-3 induced a stepwise decrease of the density distribution over such gradients. In contrast, eosinophils from patients with allergic asthma directly shifted to a final density of 1.075 g/mL within 24 hours of culture. Notwithstanding the kinetics of density changes, eosinophils from nonallergic donors already expressed the capacity to transmigrate IL-1-activated HUVE monolayers 20 hours after cultivation with different combinations of GM-CSF, IL-3, and IL-5. Inhibition studies with monoclonal antibodies showed that endothelium-driven transmigration of eosinophils predominantly implicates CD11/CD18 structures on the eosinophil surface, whereas no significant inhibition was found with the anti-VLA-4 monoclonal antibody HP2/1. From cytofluorometric studies, we conclude that spontaneous transmigration of eosinophils from allergic asthma patients is not accompanied by quantitative upregulation of these antigens. Taken together, these results allow the conclusion that blood eosinophils from allergic asthma patients have undergone in vivo priming, mimicked in vitro by cytokines such as GM-CSF, IL-3, and IL-5, leading to induction of the capacity to migrate across cytokine-activated HUVE monolayers.