Abstract
The pleiotropic cytokine interleukin 2 (IL2) disrupts the blood-brain barrier and alters brain microcirculation, underlying vascular leak syndrome that complicates cancer immunotherapy with IL2. The microvascular effects of IL2 also play a role in the development of multiple sclerosis and other chronic neurological disorders. The mechanism of IL2-induced disruption of brain microcirculation has not been determined previously. We found that both human and murine brain microvascular endothelial cells express constituents of the IL2 receptor complex. Then we established that signaling through this receptor complex leads to activation of the transcription factor, nuclear factor κB, resulting in expression of proinflammatory interleukin 6 and monocyte chemoattractant protein 1. We also discovered that IL2 induces disruption of adherens junctions, concomitant with cytoskeletal reorganization, ultimately leading to increased endothelial cell permeability. IL2-induced phosphorylation of vascular endothelial cadherin (VE-cadherin), a constituent of adherens junctions, leads to dissociation of its stabilizing adaptor partners, p120-catenin and β-catenin. Increased phosphorylation of VE-cadherin was also accompanied by a reduction of Src homology 2 domain-containing protein-tyrosine phosphatase 2, known to maintain vascular barrier function. These results unravel the mechanism of deleterious effects induced by IL2 on brain microvascular endothelial cells and may inform the development of new measures to improve IL2 cancer immunotherapy, as well as treatments for autoimmune diseases affecting the central nervous system.
Highlights
The pleiotropic cytokine interleukin 2 (IL2) disrupts the blood-brain barrier and alters brain microcirculation, underlying vascular leak syndrome that complicates cancer immunotherapy with IL2
The stability of AJs depends on complex formation between vascular endothelial cadherin
Characterization of brain microvascular endothelial cells (BMECs) and Their Expression of the IL2 Receptor Subunits—In an effort to ensure that the endothelial cell phenotype was maintained in this study, we analyzed two well known markers specific for endothelial cells, von Willebrand factor and platelet endothelial cell adhesion molecule 1 (PECAM1) [26]
Summary
Cell Culture—The human BMEC line hCMEC/D3 was provided through the courtesy of Professor Babette Weksler (Weill Cornell Medical College, New York, NY) [22]. hCMEC/D3 cells were cultured at 37 °C in 5% CO2 in EBM-2 (Lonza) supplemented with 5% heat-inactivated fetal bovine serum (HI-FBS), 1% penicillin/streptomycin (Mediatech), 1.4 M hydrocortisone (Sigma), 5 g/ml ascorbic acid (Fisher Scientific), chemically defined lipid concentrate (Invitrogen) diluted 1:100, 10 mM HEPES (Mediatech), and 1 ng/ml human basic FGF (Sigma). During time- and concentration-dependent titration experiments, supernatant samples were taken following stimulation as indicated. Cytokine and chemokine expression was analyzed in medium from BMEC monolayers cultured in Transwell chambers for permeability experiments. Immunofluorescence Staining and Fluorescence Microscopy—Human hCMEC/D3 or murine bEnd. cells grown to confluence on Transwell permeability supports were stimulated with species-specific IL2 (BD Biosciences, Prometheus) or TNF␣ (Invitrogen) as indicated. Following 24-h culture in low serum medium, cells were left unstimulated or stimulated for 24 h with species-specific 300 kilounits/ml IL2 or 30 ng/ml TNF␣. Cytokine and chemokine levels in cultured cell supernatants and Western blotting quantification of comparative controls were analyzed using an unpaired t test with Welch’s correction or Student’s t test as indicated. A p value of Ͻ0.05 was considered significant
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