Neutrophils cross the BBB primarily on transcellular pathways: An in vitro study

Abstract

The cerebral microcapillary endothelium forms a highly important barrier between the blood and the interstitial fluid of the brain (blood–brain barrier) that controls the passage of molecules and cells in and out of the CNS. Several CNS diseases include leukocyte extravasation through the endothelium via two mechanistically distinct routes, the paracellular and the transcellular pathway. We established a new in vitro model of the inflamed blood–brain barrier consisting of primary cultured porcine brain capillary endothelial cells which express a tight endothelial barrier even under inflammatory conditions. By means of this specialized blood–brain barrier model we extensively studied the transmigration of neutrophils. Electron and scanning force microscopy as well as immunofluorescence imaging captured the penetrating neutrophil on the endothelial cellular body in between the junctions clearly suggesting a transcellular migration pathway. Electric cell−substrate impedance sensing and transendothelial electrical resistance measurements in combination with expression analysis of tight junction proteins demonstrate that the neutrophil–endothelial interaction does not disrupt the barrier. In conclusion, this study, based on an in vitro model of the blood-brain barrier under inflammatory conditions, evidently implicates that neutrophils preferentially migrate across the BBB via the transcellular route without impairing endothelial barrier function whereas paracellular transmigration plays only a minor role if the barrier is strongly expressed.