The co-involvement of tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) during blood-brain barrier (BBB) injury has been reported in various models of neuroinflammation, although the precise functional interplay between these archetypal proinflammatory cytokines remains largely undefined within this context. In the current paper, we tested the hypothesis that TNF-α-mediated BBB disruption is measurably attributable in-part to induction of microvascular endothelial IL-6 production. In initial experiments, we observed that treatment of human brain microvascular endothelial cells (HBMvECs) with TNF-α (0-100 ng/ml, 0-24 hrs) robustly elicited both time- and dose-dependent induction of IL-6 expression and release, as well as expression of the IL-6 family receptor, GP130. Further experiments demonstrated that the TNF-α-dependent generation of reactive oxygen species (ROS), downregulation of adherens/tight junction proteins, and concomitant elevation of HBMvEC permeability, were all significantly attenuated by blockade of IL-6 signaling using either an anti-IL-6 neutralizing antibody or an IL-6 siRNA. Based on these observations, we conclude that TNF-α treatment of HBMvECs in vitro activates IL-6 production and signaling, events that were shown to synergize with TNF-α actions to elicit HBMvEC permeabilization. These novel findings offer a constructive insight into the specific contribution of downstream cytokine induction to the injurious actions of TNF-α at the BBB microvascular endothelium interface. This article is protected by copyright. All rights reserved.