Role of caspases in cytokine-induced barrier breakdown in human brain endothelial cells.
During neuroinflammation, cytokines such as TNF-α and IFN-γ secreted by activated leukocytes and/or CNS resident cells have been shown to alter the phenotype and function of brain endothelial cells (BECs) leading to blood-brain barrier breakdown. In this study, we show that the human BEC line hCMEC/D3 expresses the receptors for TNF-α, TNF receptor 1 and TNF receptor 2, and for IFN-γ. BEC activation with TNF-α alone or in combination with IFN-γ induced endothelial leakage of paracellular tracers. At high cytokine concentrations (10 and 100 ng/ml), this effect was associated with caspase-3/7 activation and apoptotic cell death as evidenced by annexin V staining and DNA fragmentation (TUNEL) assays. In addition, inhibition of JNK and protein kinase C activation at these doses partially prevented activation of caspase-3/7, although only JNK inhibition was partially able to prevent the increase in BEC paracellular permeability induced by cytokines. By contrast, lower cytokine concentrations (1 ng/ml) also led to effector caspase activation, increased paracellular flux, and redistribution of zonula occludens-1 and VE-cadherin but failed to induce apoptosis. Under these conditions, specific caspase-3 and caspase-9, but not caspase-8, inhibitors partially blocked cytokine-induced disruption of tight and adherens junctions and BEC paracellular permeability. Our results suggest that the concentration of cytokines in the CNS endothelial microenvironment determines the extent of caspase-mediated barrier permeability changes, which may be generalized as a result of apoptosis or more subtle as a result of alterations in the organization of junctional complex molecules.
Department of Life, Health and Chemical Sciences, The Open University, Milton Keynes MK7 6AA, UK.
M Alejandro
MA
Lopez-Ramirez
Roman
R
Fischer
Claudia C
CC
Torres-Badillo
Heather A
HA
Davies
Karen
K
Logan
Klaus
K
Pfizenmaier
David K
DK
Male
Basil
B
Sharrack
Ignacio A
IA
Romero
D001812:Blood-Brain Barrier / Q000201:enzymology* / Q000276:immunology* / Q000473:pathology; D001921:Brain / Q000201:enzymology* / Q000276:immunology* / Q000473:pathology; D002460:Cell Line; D016207:Cytokines / Q000502:physiology*; D004730:Endothelium, Vascular / Q000201:enzymology* / Q000276:immunology* / Q000473:pathology; D006801:Humans; D018836:Inflammation Mediators / Q000378:metabolism / Q000502:physiology; D007371:Interferon-gamma / Q000378:metabolism; D008833:Microcirculation / Q000276:immunology; D017471:Receptors, Interferon / Q000096:biosynthesis; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000096:biosynthesis / Q000378:metabolism; D047889:Receptors, Tumor Necrosis Factor, Type II / Q000096:biosynthesis / Q000378:metabolism; D013347:Subcellular Fractions / Q000201:enzymology / Q000276:immunology / Q000473:pathology; D000096743:Interferon gamma Receptor
D016207:Cytokines; D018836:Inflammation Mediators; D007371:Interferon-gamma; D017471:Receptors, Interferon; D047888:Receptors, Tumor Necrosis Factor, Type I; D047889:Receptors, Tumor Necrosis Factor, Type II; D000096743:Interferon gamma Receptor