ECIS technology reveals that monocytes isolated by CD14+ve selection mediate greater loss of BBB integrity than untouched monocytes, which occurs to a greater extent with IL-1β activated endothelium in comparison to TNFα

Abstract

BackgroundWe 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.MethodsMonocyte interaction was measured using the real time, label-free impedance based ECIS technology, to evaluate endothelial barrier integrity during monocyte attachment and transendothelial migration.ResultsECIS 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.ConclusionsWe 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 Importance of Multifrequency Impedance Sensing of Endothelial Barrier Formation Using ECIS Technology for the Generation of a Strong and Durable Paracellular Barrier

“…Contributing significantly to the differences in barrier resistance between Enriched Media and Minimal Media are the disparities in paracellular resistance R b ( Figure 4 C). Endothelial cells cultured in Enriched Media show R b values consistent with what we have observed previously, as per Kho et al [ 12 ]. However, removal of the growth-inducing compounds significantly impairs these interactions, leading to a much weaker paracellular barrier (R b ).…”

“…In order for these properties to be realised, brain endothelial cells must be cultured under suitable culture conditions to produce a strong and durable barrier [ 10 , 11 ]. Here, we demonstrate the power of ECIS biosensor technology to reveal that subtle differences in culture conditions have substantial effects on barrier formation of an immortalised brain endothelial cell line, which we have extensively characterised previously [ 6 , 7 , 8 , 12 ]. Importantly, the R b component of the barrier was the parameter affected greatest by the media modifications, and here, we show how rapidly these changes can occur.…”

Hypertonic saline mediates the NLRP3/IL‐1β signaling axis in microglia to alleviate ischemic blood‐brain barrier permeability by downregulating astrocyte‐derived VEGF in rats

“…51 It is well documented that activated microglia secrete an array of pro-inflammatory cytokines including TNF-α and IL-1β, and both are involved in disruption of BBB. [52][53][54][55] However, the lack of other cytokines (ie, TNF-a) being investigated in the present study is a limitation and an area for future research. Of course, our study did not prove the upstream mechanism by which HS inhibits NLRP3 inflammasome activation in ischemic stroke.…”

The functional and inflammatory response of brain endothelial cells to Toll-Like Receptor agonists

“…Inflammatory cytokine analysis revealed that Poly(I:C) and LPS were highly pro-inflammatory, whereas Imiquimod did not induce the secretion of any pro-inflammatory cytokines measured. Endothelial barrier function was measured using Electric-cell Substrate Impedance Sensing (ECIS) technology, considered the most sophisticated technology for in vitro barrier measurements 19 – 21 . ECIS demonstrated that Poly(I:C) and LPS both caused an acute reduction in barrier strength similar to our previous observations with TNFα and IL-1β 22 , whereas Imiquimod caused an immediate and sustained strengthening of the barrier.…”