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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 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 mo...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5521748/ https://www.ncbi.nlm.nih.gov/pubmed/28732059 http://dx.doi.org/10.1371/journal.pone.0180267 |
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author | Kho, Dan Ting Johnson, Rebecca Robilliard, Laverne du Mez, Elyce McIntosh, Julie O’Carroll, Simon J. Angel, Catherine E. Graham, E. Scott |
author_facet | Kho, Dan Ting Johnson, Rebecca Robilliard, Laverne du Mez, Elyce McIntosh, Julie O’Carroll, Simon J. Angel, Catherine E. Graham, E. Scott |
author_sort | Kho, Dan Ting |
collection | PubMed |
description | BACKGROUND: 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. 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-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 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-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. |
format | Online Article Text |
id | pubmed-5521748 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-55217482017-08-07 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 Kho, Dan Ting Johnson, Rebecca Robilliard, Laverne du Mez, Elyce McIntosh, Julie O’Carroll, Simon J. Angel, Catherine E. Graham, E. Scott PLoS One Research Article BACKGROUND: 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. 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-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 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-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. Public Library of Science 2017-07-21 /pmc/articles/PMC5521748/ /pubmed/28732059 http://dx.doi.org/10.1371/journal.pone.0180267 Text en © 2017 Kho et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Kho, Dan Ting Johnson, Rebecca Robilliard, Laverne du Mez, Elyce McIntosh, Julie O’Carroll, Simon J. Angel, Catherine E. Graham, E. Scott 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 |
title | 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 |
title_full | 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 |
title_fullStr | 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 |
title_full_unstemmed | 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 |
title_short | 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 |
title_sort | 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 |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5521748/ https://www.ncbi.nlm.nih.gov/pubmed/28732059 http://dx.doi.org/10.1371/journal.pone.0180267 |
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