Effects of Interleukin-6 on the Expression of Tight Junction Proteins in Isolated Cerebral Microvessels from Yearling and Adult Sheep

Abstract

Objectives: The blood-brain barrier is a selective diffusion barrier between brain parenchyma and the intravascular compartment. Tight junctions are integral components of the blood-brain barrier. Pro-inflammatory cytokines are important in the pathogenesis of brain injury and could modify the protein constituents of tight junctions. We hypothesized that interleukin-6 (IL-6) downregulates key protein constituents of endothelial tight junctions (e.g. occludin and claudin-5). Methods: We examined the effects of IL-6 on tight junction protein expression using an in vitro blood-brain barrier model. We isolated microvessels from yearling and adult ovine cerebral cortex and placed them into culture with IL-6 concentrations of 0 (control, phosphate-buffered saline), 1, 10, and 100 ng/ml for 24 h. Cerebral microvessels were harvested, Western immunoblot performed for occludin and claudin-5, densitometry performed, and results expressed as a ratio to control values. Results: Western immunoblot analysis showed that treatment with 100 ng/ml of IL-6, but not the lower concentrations, reduced (p < 0.05) occludin expression in microvessels from yearling and adult sheep and claudin-5 in microvessels from adult sheep. However, treatment with 10 ng/ml of IL-6 increased claudin-5 in microvessels from yearling sheep. The percent of lactate dehydrogenase released from the microvessels into the surrounding media was not increased by IL-6 treatment, suggesting that the reductions in tight junction proteins did not result from cell death. Treatment of adult cerebral cortical microvessels with IL-6 preincubated with anti-IL-6 monoclonal antibodies partially attenuated the reduction in claudin-5. Conclusion: We conclude that IL-6 modulates tight junction protein expression in cerebral cortical microvessels from yearling and adult sheep.

Effects of neuromyelitis optica–IgG at the blood–brain barrier in vitro

“…Blocking IL-6 signaling removed the stimulatory effect of A4 cell exposure to NMO-IgG, whereas inducing IL-6 signaling in ECs was sufficient to mediate downstream consequences of abluminal NMO-IgG for astrocyte/EC co-cultures. These observations are consistent with reports that astrocytes express IL-6 in neuroinflammation 26 and that IL-6 downregulates EC tight junction molecules, 27 decreases barrier function, 28,29 and induces CCL2 and CXCL8. 30,31 In vitro, increased barrier permeability and enhanced leukocyte migration can also be induced via complement-dependent astrocytopathy, a mechanism associated with destructive lytic lesions in patients.…”

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

“…Similarly, Cohen et al have demonstrated the ability of IL-6 to decrease occludin and claudin-5 expression in ovine cerebral microvessels ex vivo [31], whilst a role for TNF-α in BBB permeabilization in an in vivo mouse model has recently been reported by Wilson et al [32]. In contrast to our findings however, the aforementioned study by Cohen et al demonstrated that IL-6 concentrations below 100 ng/ml did not reduce protein expression, whilst 10 ng/ml of IL-6 was actually seen to increase claudin-5 expression in cerebral microvessels from yearling sheep [31]. In other contrasting studies, a lack of effect on VE-cadherin expression has been reported for hCMEC/D3 cells treated with similar concentrations of TNF-α [30], whilst a recent study by Aveleira et al demonstrates significant upregulation of occludin protein expression in bovine retinal microvascular endothelial cells following TNF-α treatment [11].…”

Transitory loss of glia and the subsequent modulation in inflammatory cytokines/chemokines regulate paracellular claudin-5 expression in endothelial cells

“…3&4). Other in vitro studies have also shown pro-inflammatory cytokines to increase endothelial and epithelial cell monolayer permeability (Desai et al, 2002; Capaldo and Nusrat, 2009; Cohen et al, 2013). TNF-α impairs the role of tight junction proteins in a number of cell types including human umbilical vascular endothelial cells (HUVEC) and human colonic adenocarcinoma (Caco-2) cells.…”