Downregulation of Blood-Brain Barrier Phenotype by Proinflammatory Cytokines Involves NADPH Oxidase-Dependent ROS Generation: Consequences for Interendothelial Adherens and Tight Junctions

Abstract

Background and ObjectivesBlood-brain barrier (BBB) dysfunction is an integral feature of neurological disorders and involves the action of multiple proinflammatory cytokines on the microvascular endothelial cells lining cerebral capillaries. There is still however, considerable ambiguity throughout the scientific literature regarding the mechanistic role(s) of cytokines in this context, thereby warranting a comprehensive in vitro investigation into how different cytokines may cause dysregulation of adherens and tight junctions leading to BBB permeabilization.MethodsThe present study employs human brain microvascular endothelial cells (HBMvECs) to compare/contrast the effects of TNF-α and IL-6 on BBB characteristics ranging from the expression of interendothelial junction proteins (VE-cadherin, occludin and claudin-5) to endothelial monolayer permeability. The contribution of cytokine-induced NADPH oxidase activation to altered barrier phenotype was also investigated.ResultsIn response to treatment with either TNF-α or IL-6 (0–100 ng/ml, 0–24 hrs), our studies consistently demonstrated significant dose- and time-dependent decreases in the expression of all interendothelial junction proteins examined, in parallel with dose- and time-dependent increases in ROS generation and HBMvEC permeability. Increased expression and co-association of gp91 and p47, pivotal NADPH oxidase subunits, was also observed in response to either cytokine. Finally, cytokine-dependent effects on junctional protein expression, ROS generation and endothelial permeability could all be attenuated to a comparable extent using a range of antioxidant strategies, which included ROS depleting agents (superoxide dismutase, catalase, N-acetylcysteine, apocynin) and targeted NADPH oxidase blockade (gp91 and p47 siRNA, NSC23766).ConclusionA timely and wide-ranging investigation comparing the permeabilizing actions of TNF-α and IL-6 in HBMvECs is presented, in which we demonstrate how either cytokine can similarly downregulate the expression of interendothelial adherens and tight junction proteins leading to elevation of paracellular permeability. The cytokine-dependent activation of NADPH oxidase leading to ROS generation was also confirmed to be responsible in-part for these events.

[Ca2+] Oscillations and IL-6 Release Induced by α-Hemolysin from Escherichia coli Require P2 Receptor Activation in Renal Epithelia

“…Here interaction with the epithelial barrier is likely to take precedence. In this context, it is interesting that both E. coli (46) and aerolysin from Aeromonas hydrophila have been shown to modify tight junctions (47) and several cytokines including IL-6 have indeed been shown to modulate the expression pattern of occludins and claudins as well as modulate tight junctional function in certain epithelia/ endothelia (48,49). Thus, it is likely that HlyA and the following ATP-dependent cytokine release may interfere with the barrier function of the epithelia and thus, its ability to keep the bacteria from invasion.…”

Drugs of abuse and blood-brain barrier endothelial dysfunction: A focus on the role of oxidative stress

“…77,78 In this inflammatory landscape where cytokines activate NADPH oxidase-dependent oxidative stress, 79 TNFa release mediates both paracellular and transcellular (vesicular) permeability of BBB following acute METH exposure at relatively low doses (1 mM), which was blocked by NF-kB inhibitor. 68 METH administration was also shown to differentially modulate the expression of BBB gatekeepers belonging to ABC superfamily of efflux transporters through stimulation of apolipoprotein E signaling at BBB endothelium.…”

Glial regulation of the blood-brain barrier in health and disease

“…The activation of microglia in MS lesions leads to the secretion of ROS, proinflammatory cytokines, and chemokines, all of which have the capacity to contribute to BBB disruption and immune cell infiltration [160]. For example, reactive microglia were shown to express NADPH oxidase in active MS lesions [168] and blocking of NADPH oxidase could protect the BBB-ECs from tight junction disruption in vitro [169].…”