Abstract
BackgroundThe blood–cerebrospinal fluid barrier (BCSFB) established by the choroid plexus (CP) epithelium has been recognized as a potential entry site of immune cells into the central nervous system during immunosurveillance and neuroinflammation. The location of the choroid plexus impedes in vivo analysis of immune cell trafficking across the BCSFB. Thus, research on cellular and molecular mechanisms of immune cell migration across the BCSFB is largely limited to in vitro models. In addition to forming contact-inhibited epithelial monolayers that express adhesion molecules, the optimal in vitro model must establish a tight permeability barrier as this influences immune cell diapedesis.MethodsWe compared cell line models of the mouse BCSFB derived from the Immortomouse® and the ECPC4 line to primary mouse choroid plexus epithelial cell (pmCPEC) cultures for their ability to establish differentiated and tight in vitro models of the BCSFB.ResultsWe found that inducible cell line models established from the Immortomouse® or the ECPC4 tumor cell line did not express characteristic epithelial proteins such as cytokeratin and E-cadherin and failed to reproducibly establish contact-inhibited epithelial monolayers that formed a tight permeability barrier. In contrast, cultures of highly-purified pmCPECs expressed cytokeratin and displayed mature BCSFB characteristic junctional complexes as visualized by the junctional localization of E-cadherin, β-catenin and claudins-1, -2, -3 and -11. pmCPECs formed a tight barrier with low permeability and high electrical resistance. When grown in inverted filter cultures, pmCPECs were suitable to study T cell migration from the basolateral to the apical side of the BCSFB, thus correctly modelling in vivo migration of immune cells from the blood to the CSF.ConclusionsOur study excludes inducible and tumor cell line mouse models as suitable to study immune functions of the BCSFB in vitro. Rather, we introduce here an in vitro inverted filter model of the primary mouse BCSFB suited to study the cellular and molecular mechanisms mediating immune cell migration across the BCSFB during immunosurveillance and neuroinflammation.
Highlights
The blood–cerebrospinal fluid barrier (BCSFB) established by the choroid plexus (CP) epithelium has been recognized as a potential entry site of immune cells into the central nervous system during immunosurveillance and neuroinflammation
Isolation and culture of highly purified primary mouse choroid plexus epithelial cells In order to provide a suitable in vitro model of the mouse BCSFB to investigate the cellular and molecular mechanisms mediating immune cell migration across the BCSFB, we established a procedure for the isolation and culture of highly purified primary mouse choroid plexus epithelial cells by adapting a previously-published protocol for the isolation and culture of rat choroid plexus epithelial cells [20]
A suitable tool for reproducible high throughput investigations of the molecular and cellular mechanisms that mediate the migration of specific immune cells into the central nervous system (CNS) via the mouse BCSFB is missing to date
Summary
The blood–cerebrospinal fluid barrier (BCSFB) established by the choroid plexus (CP) epithelium has been recognized as a potential entry site of immune cells into the central nervous system during immunosurveillance and neuroinflammation. In addition to forming contact-inhibited epithelial monolayers that express adhesion molecules, the optimal in vitro model must establish a tight permeability barrier as this influences immune cell diapedesis. Methods: We compared cell line models of the mouse BCSFB derived from the Immortomouse® and the ECPC4 line to primary mouse choroid plexus epithelial cell (pmCPEC) cultures for their ability to establish differentiated and tight in vitro models of the BCSFB. Results: We found that inducible cell line models established from the Immortomouse® or the ECPC4 tumor cell line did not express characteristic epithelial proteins such as cytokeratin and E-cadherin and failed to reproducibly establish contact-inhibited epithelial monolayers that formed a tight permeability barrier. Paracellular diffusion of molecules is prevented by unique tight junctions (TJs) constituted by the oligodendrocyte specific protein (OSP/claudin-11) that runs in parallel strands around the entire circumference of the CP epithelial cells [2, 3]. The adherens junctions (AJ) of the BCSFB are formed by homophilic interactions of the transmembrane epithelial cadherin (E-cadherin) that is linked to the actin cytoskeleton via α- and β-catenin [5, 6]
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