Introduction:
The blood-brain barrier (BBB) represents a restrictive interface between the circulation and CNS. Within the multicellular neurovascular unit (NVU), the microvascular endothelium resides at the forefront of this dynamic barrier in direct contact with blood. Following cerebrovascular injury (e.g. ischemic stroke, trauma, infection), inflammatory cytokines are released by NVU cells (neurons, astrocytes, pericytes, endothelial cells) and circulating immune cells, often leading to elevated microvessel permeability and pathology. Much has yet to be uncovered however about how cytokines specifically impact endothelial BBB phenotype at the molecular level. In the present study, we investigated how TNF Alpha and IL-6 affect adherens/tight junction assembly using a human brain microvascular endothelial cell (HBMVEC) model
in vitro
.
Methods:
Cultured HBMVECs were exposed to either TNF Alpha or IL-6 over a range of doses (1-100 ng/ml) and times (6 and 18 hours). Cytokine levels were monitored by both standard and multiplex ELISA. Endothelial barrier phenotype was routinely monitored with respect to paracellular permeability (transwell monolayer assay) and junctional protein expression (qRT-PCR & Western blotting).
Results:
(i) Both TNF Alpha and IL6 treatments increased HBMVEC permeability in a dose- and time-dependent manner. Treatments also increased cell proliferation whilst having minimal impact on cell viability (as monitored by FACS, MTS, LDH and xCelligence assays); (ii) In parallel with permeability increases, cytokine treatments dose-/time-dependently decreased mRNA and protein levels for tight junction-associated occludin, zonnula occludens-1 and claudin-5, as well as for adherens junction-associated VE-cadherin; (iii) TNF Alpha treatment was also seen to dose-/time-dependently induce IL-6 expression and release from HBMVECs.
Conclusions:
Inflammatory cytokines (TNF Alpha and IL-6) reduce microvascular endothelial BBB integrity in-part through a mechanism involving reduced synthesis of junctional proteins. Cytokine-mediated post-translational modification of junctional proteins (e.g. occludin phosphorylation and ubiquitination) is also suspected in these events. Moreover, the TNF Alpha-stimulated production/release of IL-6 may reflect a putative mechanism through which the former cytokine elicits BBB compromise.
A passive diffusion model of fluorescein derivatives in an in vitro human brain microvascular endothelial cell (HBMEC) monolayer
