The role of PP2A in an inflammatory induced increase in Brain Microvascular Permeability

抄録

Introduction

Inflammation modulates permeability of the endothelial layer of the blood¯brain barrier (BBB) through alteration of junctional proteins. Although the latter are regulated through their phosphorylation status [1, 2], the role of phosphatases remains to be determined. Therefore, the aims of this study were to investigate the effect of macrophage phenotype on: 1) brain microvascular permeability, 2) expression of key junctional proteins, 3) PP2A activity and 4) ascertain if their effect is mimicked by inhibition of PP2A.

Methods

Human peripheral blood mononuclear cells were polarised to an M1 phenotype using LPS (100 ng/mL) + IFN¯γ; (20 ng/mL). Human brain microvascular endothelial cells (HBMECs) were cultured in the absence and presence of macrophages in transwell plates. HBMEC were also exposed to okadaic acid (OA; 10 nM). Transendothelial permeability was determined using FITC-dextran, while expression of junctional proteins was determined using real-time RT-PCR and immunoblotting. PP2Ac activity was measure using an immunoprecipitation assay (Millipore). Data (normalised where appropriate) are presented as mean ± S.E.M., analysed by one-way ANOVA with post hoc (Bonferroni). *P<0.05.

Results

M1 macrophages had higher (TNF¯α: 175±25.87 pg/mL, IL¯1β: 139.8±9.14 pg/mL) (P<0.05) expression levels of proinflammatory cytokines compared to Mø (TNF¯α: 14±0.96 pg/mL, IL¯1β: 7.89±2.81 pg/mL) macrophages. Co-culture of HBMEC with M1 macrophages increased HBMEC monolayer permeability by ～3 fold (P<0.05) compared to the Mø and untreated groups. M1 and Mø macrophages differentially altered mRNA and protein expression of Ve-cadherin and Pecam-1 mRNA (Table1) compared to control. Co¯culture of HBMECs with M1 macrophages decreased PP2A activity by 68±8.6% (P<0.05), while Mø macrophages had no effect. OA, inhibitor of PP2A, reduced abundance of Ve¯Cadherin and Pecam¯1 (Table1) while eliciting a fold increase in permeability of the HBMEC monolayer.

Conclusion

Pro¯inflammatory macrophages (M1) increase permeability of the BBB while decreasing expression of Ve¯cadherin and Pecam¯1, and PP2Ac activity in an in vitro model. The effects on protein abundance, permeability and PP2A activity are mirrored by pharmacological inhibition of PP2A. These data indicate that targeting PP2A may be used to counteract the detrimental effects of inflammation on BBB function.

References

1. Bertocchi C et al. (2012).

2. Rao R (2009).