title: Modeling immune functions of the mouse blood-cerebrospinal fluid barrier in vitro: primary rather than immortalized mouse choroid plexus epithelial cells are suited to study immune cell migration across this brain barrier.
abstract: BACKGROUND
  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. 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.
  
  
  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. 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.
  
  
  CONCLUSIONS
  Our 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.
authors.affiliation: Theodor Kocher Institute, University of Bern, Freiestrasse 1, 3012, Bern, Switzerland. ivana.lazarevic@tki.unibe.ch.
authors.forename: Ivana
authors.initials: I
authors.lastname: Lazarevic
authors.affiliation: Theodor Kocher Institute, University of Bern, Freiestrasse 1, 3012, Bern, Switzerland. bengel@tki.unibe.ch.
authors.forename: Britta
authors.initials: B
authors.lastname: Engelhardt
mesh_terms: D000818:Animals; D001812:Blood-Brain Barrier / Q000276:immunology*; D002460:Cell Line; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000276:immunology*; D002555:Cerebrospinal Fluid / Q000276:immunology*; D002831:Choroid Plexus / Q000166:cytology* / Q000276:immunology*; D005260:Female; D008297:Male; D051379:Mice; D008954:Models, Biological
references.citation: J Neurosci. 1999 Aug 1;19(15):6275-89
references.pmid: 10414957
references.citation: J Cell Biol. 2015 May 25;209(4):493-506
references.pmid: 26008742
references.citation: Lab Invest. 2005 Jun;85(6):734-46
references.pmid: 15908914
references.citation: Microsc Res Tech. 1998 Apr 15;41(2):124-57
references.pmid: 9579599
references.citation: Brain Res. 2002 Dec 27;958(2):371-80
references.pmid: 12470873
references.citation: J Immunol. 2010 Oct 15;185(8):4846-55
references.pmid: 20861356
references.citation: Microsc Res Tech. 2001 Jan 1;52(1):112-29
references.pmid: 11135454
references.citation: Fluids Barriers CNS. 2012 Aug 07;9(1):15
references.pmid: 22870943
references.citation: Microsc Res Tech. 2001 Jan 1;52(1):137-52
references.pmid: 11135456
references.citation: Cerebrospinal Fluid Res. 2010 Aug 12;7:11
references.pmid: 20704740
references.citation: Am J Physiol Gastrointest Liver Physiol. 2009 Mar;296(3):G455-60
references.pmid: 19109407
references.citation: Neurosci Lett. 2008 Apr 4;434(3):310-4
references.pmid: 18329172
references.citation: Cerebrospinal Fluid Res. 2007 Dec 27;4:11
references.pmid: 18162136
references.citation: J Cereb Blood Flow Metab. 2009 Sep;29(9):1503-16
references.pmid: 19471279
references.citation: Front Cell Neurosci. 2015 Mar 12;9:80
references.pmid: 25814932
references.citation: Am J Pathol. 1996 Jun;148(6):1819-38
references.pmid: 8669469
references.citation: J Cereb Blood Flow Metab. 2011 Jan;31(1):315-27
references.pmid: 20606687
references.citation: Eur J Cell Biol. 1997 Sep;74(1):68-78
references.pmid: 9309392
references.citation: J Neuroinflammation. 2013;10:31
references.pmid: 23448224
references.citation: J Neurochem. 1981 Oct;37(4):845-54
references.pmid: 6119356
references.citation: Brain. 2013 Nov;136(Pt 11):3427-40
references.pmid: 24088808
references.citation: Immunity. 2013 Mar 21;38(3):555-69
references.pmid: 23477737
references.citation: Brain Res Brain Res Rev. 2003 Jun;42(3):221-42
references.pmid: 12791441
references.citation: Immunol Lett. 2015 Oct;167(2):57-62
references.pmid: 26235132
references.citation: Virchows Arch A Pathol Anat Histopathol. 1983;400(2):201-11
references.pmid: 6412433
references.citation: PLoS One. 2015;10(3):e0121738
references.pmid: 25815836
references.citation: Pharm Res. 1996 Jun;13(6):952-6
references.pmid: 8792439
references.citation: AIDS. 2003 Jul 4;17(10):1473-85
references.pmid: 12824785
references.citation: J Neurochem. 1997 Feb;68(2):726-31
references.pmid: 9003063
references.citation: J Pharmacol Exp Ther. 2002 Jun;301(3):820-9
references.pmid: 12023509
references.citation: PLoS One. 2012;7(1):e30069
references.pmid: 22253884
references.citation: Biomaterials. 2015 Jun;54:9-20
references.pmid: 25907035
references.citation: Eur J Immunol. 2015 Apr;45(4):1043-58
references.pmid: 25545837
references.citation: Biochem Biophys Res Commun. 2005 Feb 18;327(3):734-41
references.pmid: 15649408
references.citation: Brain Res. 2000 Jan 31;854(1-2):197-206
references.pmid: 10784122
references.citation: Exp Cell Res. 1999 Aug 1;250(2):576-80
references.pmid: 10413610
references.citation: Acta Neuropathol. 2010 Jan;119(1):75-88
references.pmid: 20033190
references.citation: Toxicol In Vitro. 2008 Feb;22(1):190-9
references.pmid: 17825520
references.citation: Jpn J Cancer Res. 1996 Sep;87(9):893-9
references.pmid: 8878450
references.citation: In Vitro Cell Dev Biol Anim. 1998 Jan;34(1):40-5
references.pmid: 9542634
references.citation: Int J Dev Neurosci. 1990;8(5):575-88
references.pmid: 2177951
references.citation: Brain Res Mol Brain Res. 1994 Jan;21(1-2):67-74
references.pmid: 7513042
references.citation: PLoS One. 2011;6(10):e27058
references.pmid: 22046444
references.citation: Pharm Res. 2001 Jan;18(1):16-22
references.pmid: 11336348
references.citation: J Neuropathol Exp Neurol. 2000 Jul;59(7):561-74
references.pmid: 10901227
references.citation: Cell Microbiol. 2009 Feb;11(2):323-36
references.pmid: 19046337
references.citation: Hum Cell. 2005 Mar;18(1):67-72
references.pmid: 16130902
references.citation: Front Cell Neurosci. 2012 Aug 09;6:33
references.pmid: 22907989
references.citation: Semin Cancer Biol. 1994 Apr;5(2):137-45
references.pmid: 7520302
references.citation: J Pharmacol Exp Ther. 1982 Jul;222(1):1-6
references.pmid: 7086691
references.citation: Fluids Barriers CNS. 2013 Mar 26;10(1):16
references.pmid: 23531482
references.citation: Proc Natl Acad Sci U S A. 1991 Jun 15;88(12):5096-100
references.pmid: 1711218
references.citation: Breast Cancer Res. 2010;12(5):R83
references.pmid: 20942910
references.citation: Endocrinology. 1993 Nov;133(5):2116-26
references.pmid: 8404661
references.citation: Trends Immunol. 2012 Dec;33(12):579-89
references.pmid: 22926201
references.citation: Nat Immunol. 2009 May;10(5):514-23
references.pmid: 19305396
references.citation: Methods Mol Biol. 2013;945:13-29
references.pmid: 23097098
references.citation: PLoS One. 2014;9(6):e99733
references.pmid: 24936790
references.citation: Methods Mol Biol. 2010;633:207-20
references.pmid: 20204630