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Trafficking of Endogenous Immunoglobulins by Endothelial Cells at the Blood-Brain Barrier.

ABSTRACT: The Blood-Brain Barrier (BBB) restricts access of large molecules to the brain. The low endocytic activity of brain endothelial cells (BECs) is believed to limit delivery of immunoglobulins (IgG) to the brain parenchyma. Here, we report that endogenous mouse IgG are localized within intracellular vesicles at steady state in BECs in vivo. Using high-resolution quantitative microscopy, we found a fraction of endocytosed IgG in lysosomes. We observed that loss of pericytes (key components of the BBB) in pdgf-b(ret/ret) mice affects the intracellular distribution of endogenous mouse IgG in BECs. In these mice, endogenous IgG was not detected within lysosomes but instead accumulate at the basement membrane and brain parenchyma. Such IgG accumulation could be due to reduced lysosomal clearance and increased sorting to the abluminal membrane of BECs. Our results suggest that, in addition to low uptake from circulation, IgG lysosomal degradation may be a downstream mechanism by which BECs further restrict IgG access to the brain.

SUBMITTER: Villasenor R

PROVIDER: S-EPMC4858719 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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Trafficking of Endogenous Immunoglobulins by Endothelial Cells at the Blood-Brain Barrier.

Villaseñor Roberto R Ozmen Laurence L Messaddeq Nadia N Grüninger Fiona F Loetscher Hansruedi H Keller Annika A Betsholtz Christer C Freskgård Per-Ola PO Collin Ludovic L

Scientific reports 20160506

The Blood-Brain Barrier (BBB) restricts access of large molecules to the brain. The low endocytic activity of brain endothelial cells (BECs) is believed to limit delivery of immunoglobulins (IgG) to the brain parenchyma. Here, we report that endogenous mouse IgG are localized within intracellular vesicles at steady state in BECs in vivo. Using high-resolution quantitative microscopy, we found a fraction of endocytosed IgG in lysosomes. We observed that loss of pericytes (key components of the BB ...[more]

PMID: 27149947

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Project description:BackgroundPrimary brain capillary endothelial cells (BCECs) are a promising tool to study the blood-brain barrier (BBB) in vitro, as they maintain many important characteristics of the BBB in vivo, especially when co-cultured with pericytes and/or astrocytes. A novel strategy for drug delivery to the brain is to transform BCECs into protein factories by genetic modifications leading to secretion of otherwise BBB impermeable proteins into the central nervous system. However, a huge challenge underlying this strategy is to enable transfection of non-mitotic BCECs, taking a non-viral approach. We therefore aimed to study transfection in primary, non-mitotic BCECs cultured with defined BBB properties without disrupting the cells' integrity.MethodsPrimary cultures of BCECs, pericytes and astrocytes were generated from rat brains and used in three different in vitro BBB experimental arrangements, which were characterised based on a their expression of tight junction proteins and other BBB specific proteins, high trans-endothelial electrical resistance (TEER), and low passive permeability to radiolabeled mannitol. Recombinant gene expression and protein synthesis were examined in primary BCECs. The BCECs were transfected using a commercially available transfection agent Turbofect™ to express the red fluorescent protein HcRed1-C1. The BCECs were transfected at different time points to monitor transfection in relation to mitotic or non-mitotic cells, as indicated by fluorescence-activated cell sorting analysis after 5-and 6-carboxylfluorescein diacetate succinidyl ester incorporation.ResultsThe cell cultures exhibited important BBB characteristics judged from their expression of BBB specific proteins, high TEER values, and low passive permeability. Among the three in vitro BBB models, co-culturing with BCECs and astrocytes was well suited for the transfection studies. Transfection was independent of cell division and with equal efficacy between the mitotic and non-mitotic BCECs. Importantly, transfection of BCECs exhibiting BBB characteristics did not alter the integrity of the BCECs cell layer.ConclusionsThe data clearly indicate that non-viral gene therapy of BCECs is possible in primary culture conditions with an intact BBB.

Dataset Information

Trafficking of Endogenous Immunoglobulins by Endothelial Cells at the Blood-Brain Barrier.

Publications

Trafficking of Endogenous Immunoglobulins by Endothelial Cells at the Blood-Brain Barrier.

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