NEUTROPHIL TRANSENDOTHELIAL MIGRATION IS INDEPENDENT OF TIGHT JUNCTIONS AND OCCURS PREFERENTIALLY AT TRICELLULAR CORNERS

Since macromolecular permeability between endothelial cells is regulat ed by tight junctions (zonula occludens), we wished to determine wheth er they also regulate neutrophil transendothelial migration. HUVEC mon olayers, a commonly used model for studying leukocyte transmigration, were characterized using electric cell substrate impedance sensing and transmission electron microscopy. We show that culture medium contain ing endothelial cell growth supplement (50 mu g/ml) was sufficient and necessary for the development of endothelial tight junctions. The fre quency with which tight junctions were observed by transmission electr on microscopy was further increased (twofold) by culturing HUVEC monol ayers in a 1:1 mixture of endothelial medium and astrocyte-conditioned medium. These astrocyte-conditioned HUVEC monolayers showed a >1.5-fo ld increase in transcellular electrical resistance. The extent of neut rophil migration across IL-1-treated (10 U/ml for 4 h) HUVEC monolayer s was the same whether tight junctions were present or absent, and the molecular requirements for neutrophil transmigration (CD18 and interc ellular adhesion molecule-1) were unaffected by culturing in astrocyte -conditioned medium. Immunostaining for proteins associated with the i ntercellular junctional domain (occludin, ZO-1, cadherin, beta-catenin , gamma-catenin, and platelet-endothelial cell adhesion molecule-1) wa s localized to the endothelial borders, regardless of the culture cond itions. Discontinuities were observed in the border staining for occlu din, ZO-1, cadherin, and beta-catenin at the tricellular corner where the borders of three endothelial cells intersected. Significantly, 75% of neutrophil migration across IL-1-treated HUVEC monolayers occurred at tricellular corners. It appears that neutrophils preferentially mi grate around endothelial tight junctions by crossing at tricellular co rners rather than passing through the tight junctions that tie between two endothelial cells.