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

Background We have previously shown that TNF alpha and IL-1 beta differentially regulate the inflammatory phenotype of human brain endothelial cells (hCMVECs). In this regard, IL-1 beta treatment was considerably more potent than TNFa 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-1 beta or TNF alpha differed in their interaction with monocytes. Methods 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. Results ECIS technology revealed that there was a greater loss of barrier integrity with IL-1 beta 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 similar to 70% enrichment on average. These two enrichment methods were compared as it was thought that the presence of non-classical CD16(hi) monocytes in the untouched enrichment may mediate greater compromise than the classical CD14(hi) 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. Conclusions We conclude that IL-1 beta and TNF alpha differentially influence monocyte interaction with brain endothelial cells and moreover, the enrichment method also influences the monocyte response as revealed using ECIS technology.