Culture models to study leukocyte trafficking across the choroid plexus

Tobias Tenenbaum,Jürgen Berger,Christian Schwerk,Corinna Friedrich,Horst Schroten,Ulrike Steinmann

doi:10.1186/2045-8118-10-1

Abstract

BackgroundA critical point during the course of central nervous system infection is the influx of leukocytes from the blood into the brain across the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB). However, experimental in vitro models to investigate leukocyte transmigration across cultured choroid plexus epithelial cells have been lacking so far.MethodsWe have developed a porcine and human “inverted” culture insert system that enables leukocyte transmigration specifically from the physiologically relevant basolateral side. The models use primary porcine choroid plexus epithelial cells (PCPEC) and human choroid plexus papilloma cells (HIBCPP). As a prerequisite for a functional barrier, we optimized culture conditions in which cells are maintained in serum-containing medium until high barrier function is reached. Leukocyte transmigration through the plexus epithelial cells is analysed by three-dimensional Apotome®-imaging and electron microscopy, and the route of transmigration through the plexus epithelial cells, i.e. transcellular as well as paracellular, can be determined.DiscussionAs a functionally relevant porcine and human BCSFB model, PCPEC and HIBCPP respectively, offer a wide range of options for analysis of disease-related mechanisms at the choroid plexus epithelium, especially involving human pathogens. Moreover, our in vitro models facilitate the investigation of leukocyte entry into the CNS via the blood-CSF barrier.

Highlights

A critical point during the course of central nervous system infection is the influx of leukocytes from the blood into the brain across the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB)
One crucial step in the pathogenesis of inflammatory diseases of the central nervous system (CNS) is the excessive infiltration of leukocytes into the CSF bearing severe consequences
Leukocytes isolated from peripheral blood can be added to the upper chamber of the culture insert and stimulated to migrate toward a chemoattractant [e.g., n-formyl-methionyl-leucyl-pheny- lalanine, interleukin (IL)-8, C5a, leukotriene B4, IL1β, Monocyte chemotactic protein-1 (MCP-1), CXCL-12] applied to the lower reservoir

Summary

Methods

Cells are concentrated in 100 μl medium, using a Figure 1 Experimental setup of the analysis of leukocyte transmigration in porcine and human inverted culture system with PCPEC and HIBCPP, respectively. To the lower cell culture insert compartment (CSF-side) 30 min before starting the transmigration experiments. After 4 h of transmigration the cell culture inserts are removed and the 24-well plates are centrifuged (5 min, 300 x g) to ensure that all PMN are attached to the bottom of the wells. On the following day the cells are washed again, incubated for 60 min with the secondary antibody 1:1000 (Alexa fluorW 594 goat anti-chicken; Molecular probes, Karlsruhe, Germany), with Phalloidin Alexa fluorW 660 (Invitrogen, Paisley, UK) for staining the actin cytoskeleton and with 4‘-6-diamidino-2-phenylindole dihydrochloride (DAPI) (Calbiochem, Merck KGaA, Darmstadt, Germany) (1:25.000) for staining nuclei. Before a bacterial infection experiment is initiated, the filters have to be thoroughly washed with antibiotic-free medium

Discussion

Background