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Updated: Jul 17, 2026
Isolation of Primary Murine Brain Microvascular Endothelial Cells
Published on: November 14, 2014
Adhesion and migration of polymorphonuclear leukocytes across human brain microvessel endothelial cells are
Donald Wong1, Rukmini Prameya, Katerina Dorovini-Zis
1Department of Pathology and Laboratory Medicine, Division of Neuropathology, Vancouver, General Hospital and the University of British Columbia, Vancouver, BC, Canada.
Abstract:
The mechanisms by which polymorphonuclear leukocytes (PMN) cross the human blood-brain barrier have not been fully elucidated. Using a well characterized in vitro model of the human BBB, we examined the role of endothelial cell adhesion molecules on the adhesion and transendothelial migration of PMN across primary cultures of human brain microvessel endothelial cells (HBMEC). A small number of PMN (0.06%) adhered to unstimulated HBMEC, and the basal adhesion was not affected by anti-adhesion molecule antibodies. Treatment of HBMEC with tumor necrosis factor (TNF)-alpha resulted in increased PMN adhesion that was significantly inhibited by blocking antibodies to E-selectin and ICAM-1, but not VCAM-1 or PECAM-1. A very small number of adherent PMN migrated across unstimulated HBMEC monolayers. Migration increased 2 to 20 fold following stimulation of HBMEC with TNF-alpha. Monoclonal antibody blocking studies showed that PMN used ICAM-1, but not VCAM-1, E-selectin or PECAM-1 to move across activated monolayers. Anti-adhesion molecule antibodies did not diminish the basal PMN migration. Ultrastructurally, PMN often aggregated on top and between adjacent endothelial cells and adhered by first extending pseudopodia along the apical endothelial surface. They then flattened and inserted themselves between endothelial cells in order to migrate across the monolayers. At the end of the migration period, the cultures resumed their continuity with no evidence of disruption. Transendothelial migration of PMN decreased the transendothelial electrical resistance and increased the permeability to horseradish peroxidase, which penetrated alongside the migrating leukocytes. A blocking antibody to ICAM-1 that greatly decreased migration, had no effect on the permeability changes. These studies provide insights into the mechanisms that regulate the entry of PMN into the brain and the increased permeability of the BBB in CNS inflammation.
Insights
Polymorphonuclear leukocytes (PMN) use ICAM-1 to cross the human blood-brain barrier (BBB) when inflamed. This migration increases BBB permeability, offering insights into central nervous system inflammation.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- The precise mechanisms of polymorphonuclear leukocyte (PMN) transmigration across the human blood-brain barrier (BBB) remain incompletely understood.
- Understanding these pathways is crucial for addressing central nervous system (CNS) inflammation.
Purpose of the Study:
- To investigate the role of endothelial cell adhesion molecules in PMN adhesion and transendothelial migration across primary human brain microvessel endothelial cells (HBMEC).
- To elucidate the molecular mechanisms governing PMN entry into the brain and subsequent BBB permeability changes.
Main Methods:
- Utilized an in vitro model of the human BBB using primary HBMEC cultures.
- Employed tumor necrosis factor-alpha (TNF-α) to stimulate HBMEC and assessed PMN adhesion and migration.
- Utilized blocking antibodies against specific adhesion molecules (E-selectin, ICAM-1, VCAM-1, PECAM-1) to determine their roles.
- Performed ultrastructural analysis of PMN migration and measured changes in transendothelial electrical resistance and permeability.
Main Results:
- TNF-α stimulation significantly increased PMN adhesion and migration, primarily mediated by E-selectin and ICAM-1 for adhesion, and ICAM-1 for migration.
- PMN transmigration occurred without disrupting endothelial monolayer integrity but increased permeability to horseradish peroxidase.
- Blocking ICAM-1 reduced PMN migration but did not affect the associated permeability increases.
Conclusions:
- ICAM-1 is a key mediator for PMN migration across the activated human BBB.
- PMN transmigration contributes to increased BBB permeability during CNS inflammation, independent of ICAM-1's role in migration itself.
- These findings provide critical insights into the cellular and molecular basis of neuroinflammation.
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