Date Published: July 21, 2017
Publisher: Public Library of Science
Author(s): Dan Ting Kho, Rebecca Johnson, Laverne Robilliard, Elyce du Mez, Julie McIntosh, Simon J. O’Carroll, Catherine E. Angel, E. Scott Graham, Stefan Liebner.
We have previously shown that TNFα and IL-1β differentially regulate the inflammatory phenotype of human brain endothelial cells (hCMVECs). In this regard, IL-1β treatment was considerably more potent than TNFα at increasing expression of inflammatory chemokines and leukocyte adhesion molecules. We therefore hypothesised that interaction of the hCMVECs with human monocytes would also be dependent on the activation status of the endothelium. Therefore, the primary aim of this study was to assess whether brain endothelial cells activated by IL-1β or TNFα differed in their interaction with monocytes.
Monocyte interaction was measured using the real time, label-free impedance based ECIS technology, to evaluate endothelial barrier integrity during monocyte attachment and transendothelial migration.
ECIS technology revealed that there was a greater loss of barrier integrity with IL-1β activation and this loss lasted for longer. This was expected and consistent with our hypothesis. However, more striking and concerning was the observation that the method of monocyte enrichment greatly influenced the extent of endothelial barrier compromise. Importantly, we observed that positively isolated monocytes (CD14+ve) caused greater reduction in barrier resistance, than the negatively selected monocytes (untouched). Analysis of the isolated monocyte populations revealed that the CD14+ve isolation consistently yields highly pure monocytes (>92%), whereas the untouched isolation was much more variable, yielding ~70% enrichment on average. These two enrichment methods were compared as it was thought that the presence of non-classical CD16hi monocytes in the untouched enrichment may mediate greater compromise than the classical CD14hi monocytes. This however, was not the case and these observations raise a number of important considerations pertaining to the enrichment strategy, which are essential for generating reliable and consistent data.
We conclude that IL-1β and TNFα differentially influence monocyte interaction with brain endothelial cells and moreover, the enrichment method also influences the monocyte response as revealed using ECIS technology.
The blood brain barrier (BBB) is a selectively permeable physical barrier composed of vascular endothelial cells, pericytes and astrocytes. The brain is highly vascularised and the BBB endothelial cells tightly regulate transport of nutrients, secretion of waste/toxins, and exclude entry to bacterial and viral particles (reviewed by ). In addition, the BBB endothelium is critical in controlling the migration of immune cells between the blood and the brain . Brain vascular endothelial cells are characterized by the high density of tight junction and adherens junction complexes, thus providing brain endothelial cells with a low paracellular permeability to cells and solutes (reviewed by ). The tightness of the BBB is vital in maintaining the brain’s immune specialised status.
The breakdown of the BBB is a feature of neuroinflammation occurring in a range of neurological conditions (reviewed by ). The key cellular component of the BBB is the endothelium, which expresses high levels of tight junction and adherens junction. These molecules provide a paracellular barrier to the movement of immune cells from the blood into the brain. Immune cell trafficking into the central nervous system is very low under normal physiological conditions and this confers the brain’s immune specialised status. However, this dynamic changes greatly during neuro-inflammation. The BBB endothelium can be activated resulting in the innate ability to secrete a variety of inflammatory mediators and to up-regulate expression of adhesion molecules capable of recruiting a range of blood borne leukocytes . We have previously shown that human brain endothelial cells (hCMVECs) respond differentially to the pro-inflammatory cytokines IL-1β and TNFα . In this regard, IL-1β induced higher expression of key leukocyte adhesion molecules CD54 and CD106. IL-1β was more potent than TNFα at inducing secretion of IL6, IP10, MCP-1, GCSF and GMCSF. Whereas TNFα induced a greater level of IL-8 and RANTES secretion. Taken together this suggests differential regulation of the brain vascular endothelial cells under these inflammatory conditions. These observations prompted us to investigate whether endothelial monocyte migration and barrier integrity is also differentially regulated by these two cytokines. ECIS technology currently represents the gold standard for monitoring of endothelial barrier integrity due to its autonomous real time capacity, which therefore can measure both acute and chronic changes in barrier functions . This study would simply not be possible without the real-time nature of the ECIS recordings and capacity to measure 96 wells simultaneously.