ABSTRACT
T cells are known for their contribution to the inflammatory element of atherosclerosis. Recently, it has been demonstrated that the Th17 derived cytokine IL-17 is involved in the pro-inflammatory response of vascular smooth muscle cells (VSMC). The aim of the present study was to examine whether reactive oxygen species (ROS) might be involved in this context. The effect of IL-17A on ROS generation was examined using the fluorescent dye 2'7'-dichlorodihydrofluorescein (H(2)DCF) in primary murine VSMC. IL-17A induced an increase in H(2)DCF fluorescence in VSMC, and this effect was blocked by the NAD(P)H-oxidase inhibitor apocynin and siRNA targeting Nox2. The p38-MAPK inhibitors SB203580 and SB202190 dose-dependently reduced the IL-17A induced ROS production. The IL-17A induced release of the pro-inflammatory cytokines IL-6, G-CSF, GM-CSF and MCP-1 from VSMC, as detected by the Luminex technology, was completely abolished by NAD(P)H-oxidase inhibition. Taken together, our data indicate that IL-17A causes the NAD(P)H-oxidase dependent generation of ROS leading to a pro-inflammatory activation of VSMC.
Subject(s)
Interleukin-17/metabolism , Membrane Glycoproteins/metabolism , Muscle, Smooth, Vascular/enzymology , Muscle, Smooth, Vascular/immunology , NADPH Oxidases/metabolism , Reactive Oxygen Species/metabolism , Acetophenones/pharmacology , Animals , Aorta, Thoracic/cytology , Cell Differentiation/drug effects , Cell Differentiation/physiology , Cell Movement/drug effects , Cell Movement/physiology , Cells, Cultured , Cytokines/metabolism , Enzyme Inhibitors/pharmacology , Interleukin-17/pharmacology , Membrane Glycoproteins/antagonists & inhibitors , Membrane Glycoproteins/genetics , Mice , Mice, Inbred C57BL , Muscle, Smooth, Vascular/cytology , NADPH Oxidase 2 , NADPH Oxidase 4 , NADPH Oxidases/antagonists & inhibitors , NADPH Oxidases/genetics , Oxidative Stress/drug effects , Oxidative Stress/physiology , RNA, Small Interfering , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Recently T-helper 17 (Th17) cells were demonstrated to disrupt the blood-brain barrier (BBB) by the action of IL-17A. The aim of the present study was to examine the mechanisms that underlie IL-17A-induced BBB breakdown. Barrier integrity was analyzed in the murine brain endothelial cell line bEnd.3 by measuring the electrical resistance values using electrical call impedance sensing technology. Furthermore, in-cell Western blots, fluorescence imaging, and monocyte adhesion and transendothelial migration assays were performed. Experimental autoimmune encephalomyelitis (EAE) was induced in C57BL/6 mice. IL-17A induced NADPH oxidase- or xanthine oxidase-dependent reactive oxygen species (ROS) production. The resulting oxidative stress activated the endothelial contractile machinery, which was accompanied by a down-regulation of the tight junction molecule occludin. Blocking either ROS formation or myosin light chain phosphorylation or applying IL-17A-neutralizing antibodies prevented IL-17A-induced BBB disruption. Treatment of mice with EAE using ML-7, an inhibitor of the myosin light chain kinase, resulted in less BBB disruption at the spinal cord and less infiltration of lymphocytes via the BBB and subsequently reduced the clinical characteristics of EAE. These observations indicate that IL-17A accounts for a crucial step in the development of EAE by impairing the integrity of the BBB, involving augmented production of ROS.-Huppert, J., Closhen, D., Croxford, A., White, R., Kulig, P., Pietrowski, E., Bechmann, I., Becher, B., Luhmann, H. J., Waisman, A., Kuhlmann, C. R. W. Cellular mechanisms of IL-17-induced blood-brain barrier disruption.
Subject(s)
Blood-Brain Barrier/metabolism , Encephalomyelitis, Autoimmune, Experimental/metabolism , Endothelial Cells/metabolism , Interleukin-17/metabolism , Reactive Oxygen Species/metabolism , T-Lymphocytes, Helper-Inducer/metabolism , Animals , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/pharmacology , Azepines/pharmacology , Blood-Brain Barrier/immunology , Blood-Brain Barrier/pathology , Cell Line, Transformed , Down-Regulation/drug effects , Down-Regulation/immunology , Encephalomyelitis, Autoimmune, Experimental/immunology , Encephalomyelitis, Autoimmune, Experimental/pathology , Endothelial Cells/immunology , Endothelial Cells/pathology , Enzyme Inhibitors/pharmacology , Interleukin-17/antagonists & inhibitors , Interleukin-17/immunology , Interleukin-17/pharmacology , Membrane Proteins/immunology , Membrane Proteins/metabolism , Mice , Mice, Inbred BALB C , Myosin-Light-Chain Kinase , NADPH Oxidases/immunology , NADPH Oxidases/metabolism , Naphthalenes/pharmacology , Occludin , Oxidative Stress/drug effects , Oxidative Stress/immunology , Reactive Oxygen Species/immunology , T-Lymphocytes, Helper-Inducer/immunology , T-Lymphocytes, Helper-Inducer/pathology , Xanthine Oxidase/immunology , Xanthine Oxidase/metabolismABSTRACT
N-methyl-d-aspartate receptor (NMDA-R)-mediated oxidative stress has been implicated in blood-brain barrier (BBB) disruption in a variety of neuropathological diseases. Although some interactions between both phenomena have been elucidated, possible influences of reactive oxygen species (ROS) on the NMDA-R itself have so far been neglected. The objective of this study was to examine how the cerebroendothelial NMDA-R is affected by exposure to oxidative stress and to assess possible influences on BBB integrity. RT-PCR confirmed several NMDA-R subunits (NR1, NR2B-D) expressed in the bEnd3 cell line (murine cerebrovascular endothelial cells). NR1 protein expression after exposure to ROS was observed via in-cell Western. The functionality of the expressed NMDA-R was determined by measuring DiBAC fluorescence in ROS-preexposed cells upon stimulation with the specific agonist NMDA. Finally, the effects on barrier integrity were evaluated using the ECIS system to detect changes in monolayer impedance upon NMDA-R stimulation after exposure to ROS. The expression of NR1 significantly (p<0.001) increased 72 h after 30 min exposure to superoxide (+33.8+/-7.5%), peroxynitrite (+84.9+/-10.7%), or hydrogen peroxide (+92.8+/-7.6%), resulting in increased cellular response to NMDA-R stimulation and diminished monolayer impedance. We conclude that oxidative stress upregulates NMDA-R on cerebrovascular endothelium and thus heightens susceptibility to glutamate-induced BBB disruption.
Subject(s)
Endothelium, Vascular/metabolism , Oxidative Stress , Receptors, N-Methyl-D-Aspartate/metabolism , Animals , Apoptosis , Blood-Brain Barrier/metabolism , Blotting, Western , Cells, Cultured , Cerebrovascular Circulation , Endothelium, Vascular/cytology , Glutamic Acid/pharmacology , Immunoenzyme Techniques , Mice , N-Methylaspartate/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reactive Oxygen Species/metabolism , Receptors, N-Methyl-D-Aspartate/genetics , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Functional and structural alterations of clustered postsynaptic ligand gated ion channels in neuronal cells are thought to contribute to synaptic plasticity and memory formation in the human brain. Here, we describe a novel molecular mechanism for structural alterations of NR1 subunits of the NMDA receptor. In cultured rat spinal cord neurons, chronic NMDA receptor stimulation induces disappearance of extracellular epitopes of NMDA receptor NR1 subunits, which was prevented by inhibiting matrix metalloproteinases (MMPs). Immunoblotting revealed the digestion of solubilized NR1 subunits by MMP-3 and identified a fragment of about 60 kDa as MMPs-activity-dependent cleavage product of the NR1 subunit in cultured neurons. The expression of MMP-3 in the spinal cord culture was shown by immunoblotting and immunofluorescence microscopy. Recombinant NR1 glycine binding protein was used to identify MMP-3 cleavage sites within the extracellular S1 and S2-domains. N-terminal sequencing and site-directed mutagenesis revealed S542 and L790 as two putative major MMP-3 cleavage sites of the NR1 subunit. In conclusion, our data indicate that MMPs, and in particular MMP-3, are involved in the activity dependent alteration of NMDA receptor structure at postsynaptic membrane specializations in the CNS.
Subject(s)
Glycine/chemistry , Matrix Metalloproteinase 3/chemistry , N-Methylaspartate/chemistry , Receptors, N-Methyl-D-Aspartate/metabolism , Synapses/metabolism , Animals , Central Nervous System/metabolism , Neuronal Plasticity , Neurons/metabolism , Protein Kinase C/metabolism , Rats , Rats, Sprague-Dawley , Rats, Wistar , Receptors, N-Methyl-D-Aspartate/chemistry , Recombinant Proteins/chemistry , Spinal Cord/metabolismABSTRACT
The synthesis and characterization of novel core-shell macromolecules consisting of a fluorescent perylene-3,4,9,10-tetracarboxdiimide chromophore in the center surrounded by a hydrophobic polyphenylene shell as a first and a flexible hydrophilic polymer shell as a second layer was presented. Following this strategy, several macromolecules bearing varying polymer chain lengths, different polymer shell densities, and increasing numbers of positive and negative charges were achieved. Because all of these macromolecules reveal a good water solubility, their ability to cross cellular membranes was investigated. In this way, a qualitative relationship between the molecular architecture of these macromolecules and the biological response was established.
