Menadione inhibits attenuation of the paracellular permeability of a brain endothelioma cell monolayer by interferon-beta in vitro

Background: Breakdown of the blood-brain barrier (BBB) is an essential part of the pathogenesis of multiple sclerosis (MS). BBB function can be impaired by free radicals in vitro, and free radicals are highly elevated in patients with acute exacerbations of MS. Menadione can cross the lipid bilayer and is therefore considered as an intracellular oxyradical producing agent. We have shown previously that low-dose interferon-β (IFN-b) attenuates the permeability of a monolayer of brain endothelioma cells in vitro.

Objective: We examined, whether exposure of a monolayer of brain endothelioma cells to menadione inhibits the attenuation of paracellular permeability after incubation of these cells with IFN-b.

Methods: The immortalized endothelioma cell line bEnd5 from mouse brain capillaries was grown to confluence for ten days on semipermeable filters precoated with rat tail collagen. From the tenth day on, endothelioma cells were treated with 100 and 1000 IU/ml IFN-b for another ten days. After a total of twenty days, culture media was replaced with Ringer-HEPES solution, and we determined the paracellular permeability of radioactive [14C]-sucrose and [3H]-inulin across the endothelioma monolayers after exposure to 300µM menadione during the assay period. Permeability was compared to controls that had not been treated with IFN-b.

Results: After exposure to 300µM menadione of the bEnd5 cells that had been treated with 100 IU/ml IFN-b for ten days, the paracellular permeability of the treated monolayers was not attenuated as compared to untreated controls. Treatment with 1000 IU/ml IFN-b even increased paracellular permeability as compared to controls.

Conclusion: The results show that exposure of a monolayer of brain endothelioma cells to menadione, which produces intracellular free radicals, inhibits the attenuation of the permeability of these cells achieved at a low IFN-b concentration in vitro. It remains to be established, whether the production of intracellular free radicals during acute exacerbations of MS reduces the beneficial effects of IFN-b therapy in vivo.