Diapedesis of monocytes is associated with MMP-mediated occludin disappearance in brain endothelial cells.
The blood-brain barrier (BBB), a selective barrier formed by endothelial cells and dependent on the presence of tight junctions, is compromised during neuroinflammation. A detailed study of tight junction dynamics during transendothelial migration of leukocytes has been lacking. Therefore, we retrovirally expressed green fluorescent protein (GFP) fused to the N-terminus of the tight junction protein occludin in the rat brain endothelial cell line GP8/3.9. Confocal microscopy analyses revealed that GFP-occludin colocalized with the intracellular tight junction protein, ZO-1, localized at intercellular connections, and was absent at cell borders lacking apposing cells. Using live cell imaging we found that monocytes scroll over the brain endothelial cell surface toward cell-cell contacts, induce gap formation, which is associated with local disappearance of GFP-occludin, and subsequently traverse the endothelium paracellularly. Immunoblot analyses indicated that loss of occludin was due to protein degradation. The broad spectrum matrix metalloproteinase (MMP) inhibitor BB-3103 significantly inhibited endothelial gap formation, occludin loss, and the ability of monocytes to pass the endothelium. Our results provide a novel insight into the mechanism by which leukocytes traverse the BBB and illustrate that therapeutics aimed at the stabilization of the tight junction may be beneficial to resist a neuroinflammatory attack.
Reijerkerk
Arie
A
Neuroimmunology Research Group, Molecular Cell Biology and Immunology, VU University Medical Center, P.O. Box 7057, 1007 MB Amsterdam, The Netherlands. a.reijerkerk@vumc.nl
Kooij
Gijs
G
van der Pol
Susanne M A
SM
Khazen
Shadi
S
Dijkstra
Christine D
CD
de Vries
Helga E
HE
D000818:Animals; D001812:Blood-Brain Barrier; D001921:Brain; D002460:Cell Line; D042783:Endothelial Cells; D005786:Gene Expression Regulation; D006877:Hydroxamic Acids / Q000494:pharmacology; D008264:Macrophages; D061965:Matrix Metalloproteinase Inhibitors; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009000:Monocytes / Q000378:metabolism*; D062793:Occludin; D010750:Phosphoproteins / Q000378:metabolism; D021381:Protein Transport; D051381:Rats; D011917:Rats, Inbred Lew; D062826:Zonula Occludens-1 Protein
C108282:BB 3103; D006877:Hydroxamic Acids; D061965:Matrix Metalloproteinase Inhibitors; D008565:Membrane Proteins; D062793:Occludin; C568987:Ocln protein, rat; D010750:Phosphoproteins; C569044:Tjp1 protein, rat; D062826:Zonula Occludens-1 Protein