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

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.

Regulation of major efflux transporters under inflammatory conditions at the blood-brain barrier in vitro.

ATP-driven efflux transport proteins at the blood-brain barrier protect the healthy brain but impede pharmacotherapy of the disordered CNS. To investigate the question how ATP-binding cassette (ABC)-transporters are regulated during inflammation or infection we analysed the effects of the cytokines tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) on the expression of brain multidrug resistance proteins in primary cultures of porcine brain capillary endothelial cells. We found that TNF-alpha and IL-1beta rapidly decrease Abcg2 (BMDP/BCRP) mRNA expression within 6 h. After 24 and 48 h the mRNA level came back to control values. The mRNA reduction at 6 h was counter-regulated by the anti-inflammatory glucocorticoid hydrocortisone. Abcg2 protein levels were suppressed at prolonged stimulations but not after 6 h of stimulation which correlates with Abcg2 specific substrate uptake measurements. Abcb1 (p-glycoprotein) protein expression was transiently increased after TNF-alpha addition within 6 h of incubation followed by a reduction after 24 and 48 h whereas the Abcb1 mRNA levels were not changed. IL-1beta caused a continuous decrease in protein expression of both ABC-transporters. Long-term treatment with an assumed TNF-alpha-downstream agent, the vasoconstrictor endothelin-1, induced Abcg2 protein expression but suppressed Abcb1. Other efflux pumps like multidrug resistance-associated proteins/Abcc were rarely affected. The present results imply a complex regulation of the two most abundant ABC-transporters at the blood-brain barrier during early inflammation stages suggesting that Abcb1 (p-glycoprotein) is an early target of TNF-alpha-signalling counterbalanced by Abcg2.