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

Wedel-Parlow, Magdalena von; Schrot, Sebastian; Lemmen, Julia; Treeratanapiboon, Lertyot; Wegener, Joachim; Galla, Hans‐Joachim

doi:10.1016/j.brainres.2010.09.076

Cited by 50 publications

(34 citation statements)

References 50 publications

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“…TEER measurements using cells grown on porous filters, by either DC or AC techniques, are successfully applied for endothelia and epithelia that form very tight barriers, especially endothelia of the BBB [76,106,78,22], and renal and colon epithelial cells [82,45]. TEER values are typically normalized to a defined area of 1 cm 2 , to allow for comparisons between barrier function measurements performed with different filter sizes.…”

Section: Overview Of Endothelial Barrier Function Assaysmentioning

confidence: 99%

Impedance analysis of GPCR-mediated changes in endothelial barrier function: overview and fundamental considerations for stable and reproducible measurements

Stolwijk

Matrougui

Renken

et al. 2014

Pflugers Arch - Eur J Physiol

104

116

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The past 20 years have seen significant growth in using impedance-based assays to understand the molecular underpinning of endothelial and epithelial barrier function in response to physiological agonists, pharmacological and toxicological compounds. Most studies on barrier function use G protein coupled receptor (GPCR) agonists which couple to fast and transient changes in barrier properties. The power of impedance based techniques such as Electric Cell-Substrate Impedance Sensing (ECIS) reside in its ability to detect minute changes in cell layer integrity label-free and in real-time ranging from seconds to days. We provide a comprehensive overview of the biophysical principles, applications and recent developments in impedance-based methodologies. Despite extensive application of impedance analysis in endothelial barrier research little attention has been paid to data analysis and critical experimental variables, which are both essential for signal stability and reproducibility. We describe the rationale behind common ECIS data presentation and interpretation and illustrate practical guidelines to improve signal intensity by adapting technical parameters such as electrode layout, monitoring frequency or parameter (resistance versus impedance magnitude). Moreover, we discuss the impact of experimental parameters, including cell source, liquid handling and agonist preparation on signal intensity and kinetics. Our discussions are supported by experimental data obtained from human microvascular endothelial cells challenged with three GPCR agonists, thrombin, histamine and Sphingosine-1-Phosphate.

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Section: Overview Of Endothelial Barrier Function Assaysmentioning

confidence: 99%

Impedance analysis of GPCR-mediated changes in endothelial barrier function: overview and fundamental considerations for stable and reproducible measurements

Stolwijk

Matrougui

Renken

et al. 2014

Pflugers Arch - Eur J Physiol

104

116

View full text Add to dashboard Cite

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“…The mechanisms underlying the leukocyte’s decision to migrate through the endothelial cell body rather than the cell borders are unknown. Indirect evidence has suggested that at the BBB, leukocytes migrate transcellularly through the endothelial cell body (23, 24). We hypothesized that the tightness of endothelial junctions and the ability of leukocytes to breach them might dictate the pathway of TEM.…”

Section: Introductionmentioning

confidence: 99%

Rapid Remodeling of Tight Junctions during Paracellular Diapedesis in a Human Model of the Blood–Brain Barrier

Winger

Koblinski

Kanda

et al. 2014

The Journal of Immunology

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Leukocyte transendothelial migration (TEM; diapedesis) is a critical event in immune surveillance and inflammation. Most TEM occurs at endothelial cell borders (paracellular). However, there is indirect evidence to suggest that at the tight junctions of the blood-brain barrier (BBB), leukocytes migrate directly through the endothelial cell body (transcellular). Why leukocytes migrate through the endothelial cell body rather than the cell borders is unknown. To test the hypothesis that the tightness of endothelial cell junctions influences the pathway of diapedesis, we developed an in vitro model of the BBB that possessed ten-fold higher electrical resistance than standard culture conditions and strongly expressed the BBB tight junction proteins claudin-5 and claudin-3. We found that paracellular TEM was still the predominant pathway (≥98%) and TEM was dependent on PECAM-1 and CD99. We show that endothelial tight junctions expressing claudin-5 are dynamic and undergo rapid remodeling during TEM. Membrane from the endothelial lateral border recycling compartment (LBRC) is mobilized to the exact site of tight junction remodeling. This preserves the endothelial barrier by sealing the intercellular gaps with membrane and engaging the migrating leukocyte with unligated adhesion molecules (PECAM-1 and CD99) as it crosses the cell border. These findings provide new insights into leukocyte-endothelial interactions at the BBB and suggest that tight junctions are more dynamic than previously appreciated.

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“…In contrast, little is known about the signals that regulate neutrophil migration across the BBB in acute neuroinflammation. Studies performed to specifically address this issue are sparse (25), and although unique mechanisms of neutrophil extravasation across of the BBB have already been suggested (26), consideration of flow has been omitted in these previous studies.…”

mentioning

confidence: 99%

β2 Integrin–Mediated Crawling on Endothelial ICAM-1 and ICAM-2 Is a Prerequisite for Transcellular Neutrophil Diapedesis across the Inflamed Blood–Brain Barrier

Gorina

Lyck

Vestweber

et al. 2014

The Journal of Immunology

158

128

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In acute neuroinflammatory states such as meningitis, neutrophils cross the blood–brain barrier (BBB) and contribute to pathological alterations of cerebral function. The mechanisms that govern neutrophil migration across the BBB are ill defined. Using live-cell imaging, we show that LPS-stimulated BBB endothelium supports neutrophil arrest, crawling, and diapedesis under physiological flow in vitro. Investigating the interactions of neutrophils from wild-type, CD11a−/−, CD11b−/−, and CD18null mice with wild-type, junctional adhesion molecule-A−/−, ICAM-1null, ICAM-2−/− , or ICAM-1null/ICAM-2−/− primary mouse brain microvascular endothelial cells, we demonstrate that neutrophil arrest, polarization, and crawling required G-protein–coupled receptor–dependent activation of β2 integrins and binding to endothelial ICAM-1. LFA-1 was the prevailing ligand for endothelial ICAM-1 in mediating neutrophil shear resistant arrest, whereas Mac-1 was dominant over LFA-1 in mediating neutrophil polarization on the BBB in vitro. Neutrophil crawling was mediated by endothelial ICAM-1 and ICAM-2 and neutrophil LFA-1 and Mac-1. In the absence of crawling, few neutrophils maintained adhesive interactions with the BBB endothelium by remaining either stationary on endothelial junctions or displaying transient adhesive interactions characterized by a fast displacement on the endothelium along the direction of flow. Diapedesis of stationary neutrophils was unchanged by the lack of endothelial ICAM-1 and ICAM-2 and occurred exclusively via the paracellular pathway. Crawling neutrophils, although preferentially crossing the BBB through the endothelial junctions, could additionally breach the BBB via the transcellular route. Thus, β2 integrin–mediated neutrophil crawling on endothelial ICAM-1 and ICAM-2 is a prerequisite for transcellular neutrophil diapedesis across the inflamed BBB.

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

Cited by 50 publications

References 50 publications

Impedance analysis of GPCR-mediated changes in endothelial barrier function: overview and fundamental considerations for stable and reproducible measurements

Impedance analysis of GPCR-mediated changes in endothelial barrier function: overview and fundamental considerations for stable and reproducible measurements

Rapid Remodeling of Tight Junctions during Paracellular Diapedesis in a Human Model of the Blood–Brain Barrier

β2 Integrin–Mediated Crawling on Endothelial ICAM-1 and ICAM-2 Is a Prerequisite for Transcellular Neutrophil Diapedesis across the Inflamed Blood–Brain Barrier

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