Neutrophils both reduce and increase permeability in a cell culture model of the blood–brain barrier

Abstract

This study was carried out to determine the effects that human neutrophils have on permeability across a model of the blood–brain barrier (BBB) formed by primary cultures of bovine brain microvessel endothelial cells (BBMEC). Transendothelial electrical resistance (TEER) was used to measure changes in permeability across BBMEC monolayers in a dual compartment system, during neutrophil interactions. When neutrophils (5×10 6/ml) were applied to monolayers, TEER increased (permeability decreased). Adenosine was implicated, since the TEER increase was blocked by adenosine deaminase (1 U/ml) and the adenosine A 2 receptor antagonist ZM 241385 (at 10 −6 M but not 10 −8 M, implicating A 2B receptors). Oxygen free radicals were implicated as the TEER increase was blocked by combined catalase (100 U/ml) and superoxide dismutase (60 U/ml). When a gradient of the bacterial chemoattractant peptide formyl methionyl leucine phenylalanine (fMLP, 10 −7 M) was applied to neutrophils, the TEER decreased (permeability increased), concurrent with migration. When fMLP (10 −7 M) was added to the neutrophils, without migration, no change occurred. The TEER decrease was blocked by loading endothelium with the calcium buffer BAPTA (10 μM) and partially blocked by the serine protease inhibitor aprotinin (20 μg/ml). Measures to block the potential extracellular triggers heparin binding protein, glutamate, oxygen free radicals and binding to intercellular cell adhesion molecule-1 (ICAM-1) were ineffective. These data indicate that neutrophils both reduce and increase permeability in a cell culture model of the BBB, correlated to their proximity and migration through the endothelium. They explore the role of neutrophils in BBB breakdown, and the formation or amelioration of vasogenic cerebral edema.