Blood-brain barrier-specific properties of a human adult ...loyce2008.free.fr/MENINGO/Trucs%20en%20g%E9n%E9ral/Weksler,%20BBB%20... · cells expressed chemokine receptors, up-regulated

©2005 FASEB The FASEB Journal express article 10.1096/fj.04-3458fje. Published online September 1, 2005.

Blood-brain barrier-specific properties of a human adult brain endothelial cell line B. B. Weksler,* E. A. Subileau,† N. Perrière,‡ P. Charneau, K. Holloway,† M. Leveque,* H. Tricoire-Leignel,* A. Nicotra,§,1 S. Bourdoulous,* P. Turowski,§ D. K. Male,† F. Roux,‡ J. Greenwood,§ I. A. Romero,†,2 and P. O. Couraud*,2 *Institut Cochin, Centre National de la Recherche Scientifique UMR 8104, Institut National de la Santé et de la Recherche Médicale (INSERM) U567, Université René Descartes, Paris, France; †Department of Biological Sciences, The Open University, Walton Hall, Milton Keynes United Kingdom; ‡INSERM, Hôpital Fernand Widal, Paris, France; §Division of Cellular Therapy, Institute of Ophthalmology, University College London, London, United Kingdom; and Institut Pasteur, Paris, France

1Present address of A Nicotra: Department of Biochemistry, Faculty of Medicine, University of Catania, Italy 2These authors contributed equally to this work.

Corresponding authors: B. B. Weksler, Weill Medical College of Cornell University, New York, NY, 10021, USA. E-mail: [email protected], and P. O. Couraud, Institut Cochin, Departement de Biologie cellulaire, 22 rue Mechain 75014 Paris, France. E-mail: [email protected]

ABSTRACT

Establishment of a human model of the blood-brain barrier has proven to be a difficult goal. To accomplish this, normal human brain endothelial cells were transduced by lentiviral vectors incorporating human telomerase or SV40 T antigen. Among the many stable immortalized clones obtained by sequential limiting dilution cloning of the transduced cells, one was selected for expression of normal endothelial markers, including CD31, VE cadherin, and von Willebrand factor. This cell line, termed hCMEC/D3, showed a stable normal karyotype, maintained contact-inhibited monolayers in tissue culture, exhibited robust proliferation in response to endothelial growth factors, and formed capillary tubes in matrix but no colonies in soft agar. hCMEC/D3 cells expressed telomerase and grew indefinitely without phenotypic dedifferentiation. These cells expressed chemokine receptors, up-regulated adhesion molecules in response to inflammatory cytokines, and demonstrated blood-brain barrier characteristics, including tight junctional proteins and the capacity to actively exclude drugs. hCMEC/D3 are excellent candidates for studies of blood-brain barrier function, the responses of brain endothelium to inflammatory and infectious stimuli, and the interaction of brain endothelium with lymphocytes or tumor cells. Thus, hCMEC/D3 represents the first stable, fully characterized, well-differentiated human brain endothelial cell line and should serve as a widely usable research tool.

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Key words: drug permeability • cellular junctions • multidrug resistance • in vitro model

ndothelial cells that line the microvasculature of the central nervous system (CNS) differ fundamentally from other vascular endothelia in their capacity to regulate the passage of molecules and cells to and from the neural parenchyma and constitute the blood-brain

barrier (BBB). This selectivity resides in specialized features unique to CNS endothelia (1), including expression of tight intercellular junctions that markedly limit paracellular permeability plus a unique pattern of receptors, transporters, and nonselective drug export pumps that protect the CNS from a very large variety of potentially harmful hydrophobic compounds (2, 3). Although much has been learned about the unique properties of brain endothelium from in vivo studies, the limitations imposed by whole animal experiments has led to the development of techniques to isolate and culture CNS-derived endothelial cells from various species (4). Unfortunately, these cultured endothelial cells usually fail to develop functional tight junctions. To address this limitation, coculture systems have been developed in which brain endothelial cells, especially of bovine origin (5, 6) are grown on microporous filter inserts in the presence of primary cultures of astrocytes in order to mimic the anatomical and functional relationship between brain endothelium and surrounding astrocyte foot processes. These coculture systems exhibit a good correlation between in vitro permeability of standard molecules, over a wide range of hydrophilicity and the in vivo cerebral bioavailability of the same molecules (7). Alternatively, a model based on monocultures of porcine brain endothelial cells in serum-free conditions, in the presence of hydrocortisone, was also shown to favor BBB differentiation (8). Although primary cultures of human brain endothelial cells have been shown to retain some phenotypic characteristics of brain endothelium (9), they rapidly undergo dedifferentiation and senescence even upon limited passaging, thus hampering usefulness as in vitro models of the human BBB. Recently, transgenic expression of the catalytic unit of telomerase (hTERT), alone or in combination with an oncogene, has been shown to prevent telomere shortening, to extend cellular lifespan and in some cases to immortalize human endothelial cells of different peripheral organs in culture (10, 11). In this report, we describe the production, characterization and properties of an immortalized human brain endothelial cell line, hCMEC/D3, derived from a primary cell culture through coexpression of hTERT and the SV40 large T antigen via a highly efficient lentiviral vector system (12). This cell line retains most of the morphological and functional characteristics of brain endothelial cells, even without coculture with glial cells and may thus constitute a reliable in vitro model of the human BBB.

MATERIALS AND METHODS

Materials

EBM-2 medium was from Clonetics (Cambrex BioScience, Wokingham, UK) and was supplemented with VEGF, IGF-1, EGF, basic FGF, hydrocortisone, ascorbate, gentamycin and 2.5% fetal bovine serum (FBS) as recommended by the manufacturer: this fully supplemented medium is designated Microvascular Endothelial Cell Medium-2 (EGM-2 MV, herein referred to as EGM-2 medium). Collagen type I was obtained from BD Biosciences PharMingen (Le Pont de Claix, France).

Human recombinant IFN-γ was obtained from Genzyme (Cambridge, MA). Human TNF-α and anti-human platelet endothelial cell adhesion molecule-1 (PECAM-1, clone 9G11) monoclonal

E


antibody were obtained from R&D systems (Abingdon, Oxon, UK). Mouse anti-intercellular adhesion molecule-1 (ICAM-1, clone HA-58), anti-vascular cell adhesion molecule 1 (VCAM-1, clone 51-10C9) monoclonal antibodies were from BD Biosciences. Mouse anti-ICAM-2 (CBR-IC2/2) monoclonal antibody and rabbit anti-VE-cadherin polyclonal antibody were obtained from Dako (Oxford, UK). Polyclonal anti-ZO-1, anti-occludin, anti-claudin-1, anti-claudin-3, and anti-claudin-5 antibodies and monoclonal anti-occludin antibodies were obtained from Zymed (San Fransisco, CA). Monoclonal antibodies against β- and γ-catenins were obtained from Transduction Laboratories (Lexington, KY). Monoclonal anti-CD40 (EA-5) antibody, anti-human MRP1 (QCRL-1), anti-human BCRP, anti-human P-glycoprotein (C219) antibodies were all obtained from Calbiochem Merck Biosciences (Nottingham, UK); monoclonal anti-SV40-T antigen and anti-h-TERT antibodies were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA). Monoclonal anti-JAM-A antibody was kindly provided by Dr E. Dejana (FIRC Institute of Molecular Oncology, Milano, Italy). Monoclonal anti-MHC-II and Cy-2, Cy-3, TRITC, or FITC-conjugated secondary antibodies were obtained from Jackson Immunoresearch (West Grove, PA). [14C]- or [3H]-radiolabeled sucrose, inulin, imipramine, diazepam, prazosin, colchicine, vincristine, morphine-6-glucuronide, and daunorubicin were obtained from Amersham (Les Ulis, France). Calcein-acetoxymethylester (calcein-AM) was from Molecular Probes (Eugene OR). Rhodamine 123, recombinant human bFGF, FITC, or TRITC-conjugated phalloidin and all other reagents were obtained from Sigma (St. Quentin, Fallavier, France), unless otherwise specified.

Isolation of human brain endothelial cells

Human brain tissue was obtained following surgical excision of an area from the temporal lobe of an adult female with epilepsy, carried out at Kings College Hospital, London, in accordance with the guidelines of the Local Ethics Committee and research governance guidelines. The isolation procedure closely follows that of Abbott et al. (13) for primary rat brain endothelial cells with minor modifications. Brain tissue was freed of meninges and surface vessels and was finely chopped and dispersed by enzymatic digestion in collagenase/dispase (1 mg/ml), DNase I (20 U/ml) and tosyl-lysyl-chloromethylketone (0.15 μg/ml) solution in Ca- and Mg-free HBSS for 1 h at 37°C. Microvessel fragments were separated from other material and single cells by density-dependent centrifugation on 25% bovine serum albumin (BSA). The floating single cell layer was used to isolate primary astrocytes, as described previously (14). The pelleted microvascular fraction was subjected to a second digestion in collagenase/dispase solution at 37°C for 2 h. The microvessel fragment suspension was then centrifuged over a preformed 50% Percoll gradient, and the microvessel fragments were washed and plated onto Type I collagen-coated plastic tissue culture flasks in EGM-2 growth medium containing FBS and growth supplements, as indicated above. To eliminate nonendothelial cells with low levels of P-glycoprotein (P-gp) expression that are sensitive to puromycin cytotoxicity, puromycin (2 μg/ml) was added to the culture medium for the first three days following isolation (15). This procedure resulted in more than 95% pure endothelial cultures, as assessed by morphological criteria and by expression of endothelial-specific markers (see Results section). The cultures were maintained at 37°C in 5% CO2 and EGM-2 medium was replaced every 3 days until the cells reached 50% confluence when they were used for lentiviral transduction or RT-PCR analysis.


Immortalization and selection of human brain endothelial cells

The immortalization of human brain endothelial cells was performed via sequential lentiviral transduction of hTERT and SV40 large T antigen transduction of hTERT and SV40 large T antigen into a primary culture of adult brain endothelial cells. Subconfluent cells at passage 0 were first incubated overnight with a low concentration of a highly efficient DNA flap lentiviral vector (12), encoding hTERT (final concentration 100 ng p24 units/ml) in EGM-2 medium. Cells were then washed and left to grow for 1−2 days. This was followed by another overnight incubation with a low concentration of a DNA flap lentiviral vector encoding SV40-T antigen (20 ng p24 units/ml). Individual transduced cells were then isolated in the first subsequent passage by two rounds of limiting dilution cloning, which produced several hundred proliferating cell lines. One clonal population, designated hCMEC/D3, was selected from these clonal proliferating cell lines on the basis of its endothelial morphology, growth capacity, contact inhibition at confluence, and stable expression of markers of a normal endothelial phenotype: von Willebrand factor and PECAM-1, in addition to β-catenin and ZO-1. This cell line was routinely grown on Type I collagen-coated (0.15%) plates in EGM-2 medium. Differentiation and expression of junction-related proteins were favored at confluence by further culture in EBM-2 medium lacking added growth factors and containing 0.25% FBS and 1 μM dexamethasone. For safety reasons, transduced cells were tested for any production of retroviral particles, using a sensitive enzyme assay for p24 (not shown) in the first and second passage, and both cells and conditioned media were negative.

Immunocytochemistry and flow cytometry

Immunocytochemical studies were performed on both immortalized cells and the primary cultures, from which they were derived. Cells were seeded onto Type I collagen-coated Thermanox chamber slides, Thermanox coverslips (Nunc LabTek, Gibco, Paisley, UK) or the center of 35 mm plates and grown to confluence. For characterization of junctional proteins, confluent cells were washed, fixed for 10 min in 4% paraformaldehyde, washed 3 times in PBS, permeabilized for 10 min in 0.2% Triton X-100, and rewashed and blocked in 3% BSA for 30 min. Alternatively, cells were fixed in 3.7% formaldehyde in PBS, permeabilized with ice-cold acetone or methanol, washed in PBS, and blocked in 0.5% BSA before staining. In all cases, cells were then incubated for 2 h with relevant antibodies, washed extensively in PBS and then incubated with fluorophore-conjugated secondary antibody for 1 h, followed by extensive rinsing before mounting in Mowiol (10% Mowiol 4-88, Calbiochem). Omission of the primary antibody served as negative control. After mounting, the cells were observed and photographed using an epifluorescence or confocal fluorescence microscope (Leica or Zeiss).

Flow cytometry analysis of endothelial cells was performed on a FACScan (Becton Dickinson, Oxford, UK), as described previously (14).

Matrigel and soft agar assays

Confluent hCMEC/D3 cells were resuspended in fresh EGM-2 medium at 50–100,000 cells/ml, and 1 ml aliquots were layered over a preformed gelled layer of 250 μl reconstituted extracellular matrix (Matrigel, BD Biosciences) in wells of a 48-well culture plate and incubated at 37°C for


6–48 h. Development of capillary-like networks and tubular structures was observed and photographed under a phase-contrast microscope.

For evaluation of the capacity of cells for anchorage-independent growth in soft agar, hCMEC/D3 cells at 200–2,000 cells/ml of EGM-2 medium were suspended in a final concentration of 0.37% agar in the same medium and layered over a coating of 0.5% solid agar in wells of 24-well tissue culture dishes using triplicate wells for each concentration. The plates were incubated in a 5% CO2 incubator at 37°C for up to 4 wk. Highly malignant human glioblastoma U87 cells were used as positive controls, as described (16). Colonies forming in the soft agar were counted at weekly intervals in 5 microscopic fields per well.

RNA extraction and RT-PCR

Total RNA was extracted from hCMEC/D3, and primary human brain endothelial cells using 2 ml Trizol reagent (Invitrogen, Paisley, UK), according to the manufacturer’s instructions. For reverse transcription (RT) a 20 μl reaction containing 1 μg of RNA was carried out using random hexamers and ImProm-II Reverse Transcriptase (Promega) in the presence of 20 U RNasin (Promega). Primer annealing was performed at 25ºC for 5 min and transcription was run at 40ºC for 1 h, followed by inactivation of the enzyme at 70ºC for 15 min. Four-microliter aliquots of the RT reaction product were used for PCR analyses in a reaction mixture containing 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM dNTPs, 1 (M of sense and anti-sense primers and 1 U Taq DNA polymerase in a total volume of 20 μl. Amplification was performed in an iCycler (Biorad) with an initial denaturation step at 94ºC for 5 min; 35 cycles of 94ºC for 45 s, 55ºC for 45 s and 72ºC for 1 min, and a final elongation step at 72ºC for 7 min. The annealing temperature was reduced to 53ºC for MRP5. For semiquantitative expression analysis, a cyclophilin fragment was amplified simultaneously. The following primer pairs were used: MRP-1 forward: ACC AAG ACG TAT CAG GTG GCC, reverse: CTG TCT GGG CAT CCA GGA T (fragment size 286 bp); MRP-5 forward: AGT GGC ACT GTC AGA TCA AAT T, reverse: TTG TTC TCT GCA GCA GCA AAC (fragment size 455 bp); BCRP forward: TGG CTG TCA TGG CTT CAG TA, reverse: GCC ACG TGA TTC TTC CAC AA (fragment size 235 bp); MDR-1 forward: GCC TGG CAG CTG GAA GAC AAA TAC ACA AAA TT, reverse: CAG ACA GCA GCT GAC AGT CCA AGA ACA GGA CT (fragment size 286 bp); cyclophilin forward AGC ACT GGA GAG AAA GGA TT, reverse GGA GGG AAC AAG GAA AAC AT (fragment size 527 bp). Western blot analysis The expression of MRP1, P-gp, and MDR1 in hCMEC/D3 was evaluated by immunoblot analysis. Following incubation with specific antibodies and extensive washing in PBS containing 0.05% Tween-20, membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (Pierce, Chester, UK) for 1 h at room temperature. Membranes were extensively washed in PBS containing 0.05% Tween-20, and immunoblots were visualized by enhanced chemiluminescence detection (ECL, Amersham, Bucks, UK).


Permeability studies

The permeability of hCMEC/D3 cell monolayers to molecules of different sizes was measured as described (17, 18) on Transwell polycarbonate insert filters (pore size 3 μm, Corning, Brumath, France). HCMEC/D3 cells were seeded on the filters at a confluent density of 2 × 105 cells/cm2 in EGM-2 medium. After 48 h, [14C]-sucrose, -diazepam, -morphine-6-glucuronide (M6G) or [3H]-inulin, -imipramine, -prazosin, -colchicine, -vincristine (0.8 (Ci/ml), or FITC-labeled dextrans of molecular weight 4, 40, and 70 kDa (1–2 mg/ml) were then added to the upper chamber, the lower chamber was sampled at 10-min intervals and the radioactivity or fluorescence that passed through the cell-covered inserts was determined using a β-scintillation counter (Pharmacia Wallac 1410) or a fluorescence multiwell plate reader (Wallac Victor 1420), respectively. The volume cleared was plotted against time, and the slopes of the curves were used to calculate the permeability coefficients (Pe) of the endothelial monolayer, as described (17): 1/PS = 1/me – 1/mf and Pe = PS/s, where me and mf are the slopes of the curves corresponding to endothelial cells on filters and to filters only, respectively. PS (clearance) is the permeability surface area product and s is the surface area of the filter (1 cm2). Permeability coefficients were correlated to recently reported rat or mouse in situ brain perfusion values (19, 20) expressed as transport coefficients (Kin, μl•s−1•g−1): PS = –F Ln(1-E), with E = Kin/F, where E is the brain extraction and F is the blood flow (μl•s−1•g−1).

Transendothelial electrical resistance (TEER) of hCMEC/D3 monolayers on Transwell-Clear filters was measured using an Endohm 12 chamber and an Endohmeter (World Precision Instruments, Sarasota, FL). The resistance of ECM-coated inserts was always subtracted from the resistance obtained in the presence of the endothelial cultures.

Drug accumulation studies

Confluent hCMEC/D3 cells, grown in 24-well plates, were washed 3 times with PBS and preincubated for 30 min at 37°C in a shaking waterbath with culture medium with or without the efflux transporter inhibitor PSC-833 (10 µM, a gift from Novartis) (21), MK-571 (10 µM; a gift from Merck) (22), or KO-143 (1 µM; a gift from Dr. Schinkel, University of Amsterdam, NL) (23). Cellular uptake of the fluorescent substrates rhodamine 123 (10 µM), calcein-acetoxymethylester (calcein-AM) (10 µM), or [3H] daunorubicin (0.2 μCi/well) was measured for 1 h, as described previously (24), with minor modifications. The cells were then rapidly washed 3 times with ice-cold PBS and lysed in 1% Triton-X100. The amount of fluorescent substrates retained in the cells was determined in a Wallac F-Lite 2001 fluorescence plate reader with a 485-nm bandpass excitation/emission filter, whereas the radioactivity retained in the cells was measured using a β-scintillation counter (Pharmacia Wallac 1410).

Statistical analysis

Results are expressed as the means ± SEM, and significant differences between groups were determined by Student's t test or ANOVA as appropriate. P < 0.01 was considered significant.


RESULTS

Nontransformed phenotype of immortalized human brain endothelial cells

Endothelial cell colonies emerged from human brain cortical microvessel fragments by days 5−10 following isolation and seeding and reached confluence from days 25 to 35. At confluence, primary cultures of human brain endothelia exhibited spindle-shaped morphology previously documented in brain endothelial cells derived from human (9) and other species (13) (Fig. 1A).

Following lentiviral infection of preconfluent primary brain endothelial cell cultures, several hundred parent cell lines of immortalized human brain microvessel endothelial cells were obtained by two subsequent rounds of limiting dilution cloning, out of which one clonal population, named hCMEC/D3, was selected. The hCMEC/D3 cell line showed a morphology similar to primary cultures of brain endothelial cells, with monolayers of tightly packed elongated cells that exhibited contact inhibition at confluence (Fig. 1A, 1B). The hCMEC/D3 cell population displays the presence of nuclear SV40-T antigen protein (Fig. 1C) and a high expression level of nuclear and perinuclear hTERT protein, as shown by confocal microscopy (Fig. 1D). The hCMEC/D3 cell line maintained a nontransformed phenotype over more than 100 population doublings (35 passages), without any sign of senescence or dedifferentiation, as supported by the following observations: 1) a diploid karyotype, without any major chromosomal rearrangements, both at passage 22 (Fig. 2A) and passage 33 (not shown); 2) hCMEC/D3 cells did not form any colonies in soft agar, indicating that their proliferation is adhesion-dependent, in contrast with the malignant human glioblastoma cell line U87 used as a positive control (16) (data not shown); and 3) when hCMEC/D3 cells were seeded on reconstituted extracellular matrix (Matrigel), they rapidly formed a branched network of capillary-like cords, a characteristic typical of primary endothelial cells, suggesting a normal capacity for angiogenesis (Fig. 2B).

Expression of endothelial and BBB-specific markers

The hCMEC/D3 cell line constitutively expresses many endothelial markers with the appropriate subcellular localization pattern: PECAM-1 and VE-cadherin, appearing as cellular junction-associated proteins (Fig. 3) and cytoplasmic granules of von Willebrand factor (not shown). In addition, β- and γ-catenins, together with F-actin, were mostly localized to the cortical region, as expected from cells with properly organized multiprotein complexes at adherens junctions (Fig. 3). All together, these observations strongly suggest that the hCMEC/D3 cell line displays a phenotype close to that of normal primary endothelial cells. No change was detected in the junctional expression of VE-cadherin, β-catenin (Fig. 3) or any other tested endothelial marker (not shown) from early passages to passage 33, the highest passage investigated so far.

We further investigated whether the hCMEC/D3 cell line also expresses several discriminatory proteins. The submembranous tight junction-associated protein ZO-1 and the integral membrane protein JAM-A are components of the BBB endothelium in vivo, together with the transmembrane proteins occludin and claudin-3 and -5. Interestingly, immunodetection of ZO-1 and JAM-A in hCMEC/D3 cells revealed the continuous expression of both markers at cell-cell contacts (Fig. 3). In addition, although claudin-3 and occludin were not consistently detected at the cell-cell contacts, junctional expression of claudin-5 was clearly observed (Fig. 3). As a


negative control, the epithelium-selective claudin-1, which is not expressed by brain endothelium in situ, was not detected in hCMEC/D3 cells (data not shown).

Expression of adhesion molecules

A number of adhesion molecules known to be expressed by brain endothelium are involved, under inflammatory conditions, in migration of activated leukocytes across the BBB. Flow cytometric analysis of hCMEC/D3 cells not only confirmed the strong expression of PECAM-1 (Fig. 3), but also revealed the constitutive expression of the adhesion molecules ICAM-2 and CD44 (Fig. 4A), as reported for human brain endothelial cells in situ (24a). In addition, basal expression of ICAM-1 and CD40 was low in confluent, unstimulated cells and became markedly up-regulated following a 24 h treatment with 100 ng/ml IFN-γ and 100 U/ml TNF-α, whereas VCAM-1 and MHC-II expression was undetectable in nontreated cells, and VCAM-1 and MHC-II were strongly induced by these inflammatory cytokines (Fig. 4), as previously reported with primary cultures of human brain endothelial cells (25, 26). By contrast, hCMEC/D3 cells were not stained by markers specific for other cell types: GFAP (for astrocytes), CD69 (for monocytes), DC-SIGN-1 (for dendritic cells), or DC-SIGN-2, which has been reported to be expressed only by some nonbrain endothelial cells (data not shown).

Expression of chemokine receptors

Multiple chemokine receptors stimulate signal transduction in brain endothelial cells that favor migration of activated leukocytes across the BBB (27). The flow cytometry analysis presented in Fig. 5 revealed that hCMEC/D3 cells, in a manner similar to human brain microvessels or primary cultures of human brain endothelial cells (27, 28), express a variety of chemokine receptors: CXCR-1 to CXCR-5 and CCR-3 to -CCR-6 (Fig. 5). Only CCR-1 and -2 could not be detected either in hCMEC/D3 cells (Fig. 5) or in primary human brain endothelial cells (data not shown).

Drug permeability: correlation with in situ brain perfusion

Monolayers of the hCMEC/D3 cells line, grown to confluence on semipermeable Transwell filters, were tested for their permeability to hydrophilic molecules that are used as paracellular permeability markers. As shown in Table 1 (upper panel), permeability assays using fluorescent dextrans of increasing molecular size (4–70 kDa) revealed that hCMEC/D3 monolayers exert a better restriction than primary cultures of bovine brain endothelial cells (29, 30) and a much more stringent restriction than do GPNT cells (31) on the transendothelial passage of both low-molecular-weight and high-molecular-weight dextran molecules. In addition, compared with permeability values previously reported for the reference bovine BBB coculture model (6), the permeability coefficient for [14C]-sucrose (MW=340 Da) was higher for hCMEC/D3 cells (1.65 vs. 0.75×10−3 cm/min), but in the case of [3H]-inulin (MW=4,000 Da), the permeability for both models was of the same magnitude (0.36 vs. 0.37×10−3 cm/min (Table 1, bottom). It should be mentioned that transendothelial electric resistance resistance (TEER) was found to remain constantly at low levels (<40 Ω.cm2), reflecting a high ionic permeability. Preliminary data indicate that treatments with a cAMP-elevating agent (1 μM of chlorophenylthio-cAMP) and culture medium conditioned by primary human astrocytes (50%), described previously for their


ability to decrease endothelial permeability failed to significantly increase the TEER value (not shown).

To evaluate further the permeability characteristics of the hCMEC/D3 cell line, we carried out in vitro/in vivo comparisons for several standard compounds with different physico-chemical properties and increasing hydrophobicity: sucrose, morphine-6-glucuronide, inulin, vincristine, colchicine, prazosin, diazepam, and imipramine. Permeability coefficients (Pe in cm/min) calculated with hCMEC/D3 cells grown to confluence on polycarbonate filters were correlated with in vivo permeability data (expressed as Kin transport coefficient values) obtained by brain perfusion of adult rats or mice (19, 20), which are considered quantitatively similar to human values. It must be noted that because imipramine was partly retained on the filters by hydrophobic interactions, as previously reported (6), the corresponding permeability value (2.53×10−3 cm/min) was not included in the regression analysis. As shown in Fig. 6A, our results demonstrate a good correlation (R=0.938) between in vitro and in vivo data. For comparison, similar experiments were performed with human umbilical vein endothelial cells (HUVECs), which are not expected to form a tight diffusion barrier and with the rat brain endothelial cell line GPNT (31). Permeability data for vincristine, colchicine, prazosin, and diazepam showed a poor correlation between in vivo brain perfusion values and in vitro data (R=0.881) with HUVECs, whereas a good correlation (R=0.965) was observed with GPNT cells. Figure 6B and C reveal a good correlation between hCMEC/D3 and GPNT permeability data (R=0.990), but no correlation between hCMEC/D3 and HUVEC permeability data (R=0.628). Although this study will need to be extended to a larger number of compounds, these observations clearly indicate that a good correlation between in vitro and in vivo BBB permeability data could only be achieved with monolayers of brain endothelial cells; moreover, they point to the hCMEC/D3 cell line as a potential in vitro model of the human BBB both for hydrophobic drugs and for hydrophilic molecules of molecular weight higher than sucrose.

Expression and functionality of multidrug resistance proteins

In conjunction with the presence of interendothelial tight junctions, BBB endothelia are characterized by a highly restrictive permeability to hydrophobic molecules, resulting from the expression of several multidrug resistance proteins of the ATP binding cassette (ABC) transporter family that function as nonselective drug export pumps and protect the CNS from a very large panel of potentially harmful hydrophobic compounds. Among these efflux pumps are P-gp (also called MDR1 or ABCB1) (2, 3), the multidrug resistance-associated protein-1 (MRP1 or ABCC1) (3); and the more recently described breast cancer resistance protein (BCRP or ABCG2) (32, 33), all of which exhibit distinct, but overlapping substrate preferences. As shown by the RT-PCR results presented in Fig. 7A, all three transcripts were detected in hCMEC/D3 cells, as well as in primary cultures of human brain endothelial cells. In addition, we also detected the MRP5-encoding transcript both in hCMEC/D3 cells and in primary human brain endothelial cells (Fig. 7A), as previously detected in bovine brain endothelial cells (34). Protein expression of P-gp, MRP1, and BCRP was further confirmed by Western blot analysis (Fig. 7B); these efflux pumps were detected as 170 kDa, 190 kDa, and 70 kDa proteins, respectively, as expected from previous reports (24, 33). As control, primary cultures of human astrocytes were shown not to express MRP1 or BCRP, but to express low levels of P-gp, as described (35).


The functionality of these pumps was then evaluated by assessing the uptake by hCMEC/D3 monolayers of drugs that are known as substrates of at least two ABC transporters: calcein-AM, a P-gp and MRP1 substrate (Fig. 8A) (36); rhodamine 123, a P-gp and BCRP substrate (Fig. 8B), [3H] daunorubicin, a shared substrate of all three P-gp, MRP1, and BCRP transporters (Fig. 8C), in the absence or presence of specific inhibitors: PSC-833 (10 µM), MK-571 (10 µM) or KO-143 (1 µM) blocking P-gp, MRP1 and BCRP, respectively (21–23). As shown in Fig. 8, PSC-833 markedly increased calcein-AM, rhodamine 123, and daunorubicin uptake by hCMEC/D3 cells, indicating that P-gp was highly active in these cells. In addition, MK-571 significantly increased calcein-AM and daunorubicin uptake, but not rhodamine 123 uptake: this result is in good agreement with the reported selectivity of these substrates and suggests that MRP1 is functional in hCMEC/D3 cells. Finally, KO-143 significantly increased [3H]daunorubicin uptake, albeit to a limited extent, showing that this ABC transporter is moderately expressed and active in hCMEC/D3 cells. This finding is in agreement with a recent report on primary cultures of human brain endothelial cells (33), where KO-143 did not significantly affect rhodamine 123 uptake, suggesting, as recently proposed (37), that rhodamine 123 uptake is not sensitive enough to detect BCRP activity. Taken together, these results demonstrate that hCMEC/D3 cells express three ABC efflux transporters (P-gp, MRP1 and BCRP) known to be expressed by brain endothelium and that all three are functional.

DISCUSSION

The present results constitute an extensive phenotypic characterization of an immortalized human brain microvascular endothelial cell line, which stably maintains in culture most of the unique structural and biochemical properties of brain endothelium in vivo. The restricted permeability of monolayers of these endothelial cells to macromolecules demonstrates functional retention in vitro of physiological properties of the BBB. Unlike primary cultures of CNS-derived endothelia that are difficult to obtain pure, are phenotypically unstable, and rapidly undergo cellular senescence after a limited number of divisions, this immortalized clonal cell line showed no indications of phenotypic drift or decrease in the rate of population doubling over the course of 35 passages (>100 population doublings). The hCMEC/D3 cell line maintains a stable and physiologically normal endothelial phenotype, including the capacity rapidly to form networks of capillary-like vascular cords within three-dimensional extracellular matrix and the constitutive or regulated expression of a number of endothelial markers, adhesion molecules, and chemokine receptors. We have previously produced immortalized rat brain endothelial cell lines (38, 39), which proved to be a valuable resource for studying biochemical (40), toxicological (41), and immunological properties (42) of cerebral endothelia. The strategy used here to produce the hCMEC/D3 cell line was designed on the basis of 1) using a new generation of lentiviral vectors that have been shown to support highly efficient gene transduction in postmitotic or slowly proliferating cells, including various primary neural cells (12) and 2) inserting hTERT to prolong the stable life span of differentiated cells in culture, as well as SV40 T antigen to foster proliferation. This combination has been shown to permit long-term culture of various cell types, particularly fully differentiated cells, with maintenance of their differentiated properties and without cellular transformation, including human endothelial cells (10). The tight junctions of the BBB are the major contributor to the extremely low permeability to both solutes and cells. Junctional expression of certain claudins and occludin, the integral membrane components of tight junctions, as well as continuous junctional expression of JAM-A and ZO-1, all demonstrated in hCMEC/D3 cells, correlate in situ with functional tight junctions (1). In


terms of the relative contribution of these proteins to tight junction formation, it is of interest to note that, although occludin unexpectedly appeared to be dispensable (43), loosening of the BBB was reported in claudin-5-deficient mice (44), highlighting the key contribution of this adhesion molecule to BBB integrity. We present evidence here that hCMEC/D3 cells display a junctional expression of JAM-A, claudin-5, and ZO-1; occludin, however, was expressed but not consistently detected at cell-cell contacts. These results support the conclusion that hCMEC/D3 cells maintain the overall tight junction organization known to be present in brain endothelium.

The suitability of the hCMEC/D3 model for transendothelial permeability studies is supported by our results, which constitute one of the key observations of the present study: we observed that hCMEC/D3 cell monolayers display a highly restricted permeability to standard drugs over a wide range of hydrophobicity, in good correlation with in vivo permeability values from brain perfusion studies in adult rats or mice (19, 20). These data clearly indicate that the hCMEC/D3 cell monolayers, even in the absence of coculture with glial cells, possess functional intercellular junctions with highly restrictive permeability properties. Restriction was found to be much more stringent than in previously immortalized rat brain endothelial cell lines (RBE4, GPNT cell lines) (18, 31) and quite similar to the reference bovine coculture model (6), although sucrose permeability was slightly higher and TEER values were consistently low. Prolonged coculture with glial cells in the presence or absence of cAMP-inducing agents or with dexamethasone, conditions which have been extensively documented to reduce the passive diffusion permeability of cultured brain endothelial cells by inducing the appropriate organization of tight junction molecular complexes, will need to be explored with hCMEC/D3 cell monolayers. Preliminary experiments indicated that a 24 h treatment of hCMEC/D3 cells with a cAMP-inducing agent and 50% culture medium conditioned by primary human astrocytes increased the expression of claudin-5 and occludin, as assessed by Western blot analysis (data not shown), suggesting that hCMEC/D3 cells can respond to astrocytic influences and/or differentiating factors. However, under those conditions, no significant increase of TEER values was observed. Further experiments are required for optimization of the coculture conditions. Taken together, our results demonstrate that hCMEC/D3 cell monolayers, even in absence of coculture with glial cells, constitute a valuable in vitro model of human BBB, which mimics brain endothelium restricted permeability for a wide variety of compounds, except for small hydrophilic molecules (<300 Da) and ions.

The transendothelial permeability of many hydrophobic compounds at the BBB is known to be strictly limited by the expression of multiple efflux transporters of the ABC cassette superfamily. Among the seven identified subfamilies of these transporters in humans, consisting of more than 40 known members, three (B, C, and G) contain efflux transporters that are expressed and active at the BBB. P-gp (or ABCB1) and the recently identified BCRP (or ABCG2) are inserted in the luminal cell membrane of brain endothelial cells and thus constitute a first line of defense against substrate penetration into brain (3). Of the seven members of the MRP family, MRP1 is reported to be expressed at a very low level at the abluminal membrane of brain endothelium, both in situ or in freshly isolated brain endothelial cells, but at a much higher level in cultured rat and human brain endothelial cells (22, 24, 45). In addition, other members, MRP2, MRP5, with some species variations, were also shown to be expressed by brain endothelial cells (3, 34). All together, these ABC efflux transporters contribute to the multidrug resistance phenomenon at the BBB. In the present study, we show the expression of transcripts encoding P-gp, MRP1, MRP5, and BCRP and, focusing on P-gp, MRP1 and BCRP, we clearly demonstrate that all three


transporters are expressed and functional in hCMEC/D3 cells. Considering the previously reported species difference in P-gp substrate specificity (46), availability of our hCMEC/D3 cell line might significantly increase the predictability of in vivo results in humans regarding BBB permeability to lipid-soluble molecules. In previously described in vitro models of the BBB, coculture of brain endothelial cells with glial cells was reported to increase the functional expression of P-gp and other ABC efflux transporters, in parallel with the above mentioned decrease in monolayer permeability. Accordingly, future optimization of the hCMEC/D3 model might include coculture with human glial cells.

Although the brain has been often described as an "immune-privileged site" due to the presence of the BBB, it is now well established that, during inflammatory situations, such as multiple sclerosis, massive infiltration of leukocytes can be observed within the brain tissue. This leukocyte recruitment coincides with the up-regulated expression of a number of adhesion molecules on the surface of cerebral vascular endothelium (47). Extending previous observations on primary cultures of human brain endothelial cells (26), we report here that hCMEC/D3 cells constitutively express ICAM-1, ICAM-2, PECAM-1, and CD40 and that, upon cytokine activation, ICAM-1 and CD40 are up-regulated, while expression of VCAM-1 is induced. Leukocyte migration across the BBB is also known to be controlled by brain-derived chemokines, which can either reach their cellular targets following receptor-mediated transcytosis through brain endothelium or can activate intracellular signaling pathways in brain endothelial cells that favor leukocyte migration. In this context, our observations that hCMEC/D3 cells express a large number of chemokine receptors confirm and extend recent observations on human brain microvessels (27) and pave the way to further investigations on the mechanisms of human leukocyte infiltration into the CNS.

An immortalized human brain endothelial cell line had been previously reported, derived by transfection of primary cultures with a plasmid encoding SV40 T antigen (48); this cell line was shown to express certain endothelial markers (von Willebrand factor, occludin), but its phenotype was unstable and contact inhibition did not occur (49). Also, brain endothelial cells with extended life span have been produced by hTERT expression (50): the phenotype of these cells was described only in terms of von Willebrand factor and PECAM-1 expression, limiting any conclusion regarding their potential use as a BBB in vitro model. An additional human brain endothelial cell line termed SV-HCEC was described several years ago in terms of expression of endothelial markers, BBB-specific enzymes and restricted permeability (51). However, to our knowledge, this cell line has not since been extensively characterized, probably indicating that the SV-HCEC cell line may be unstable and/or may have lost its barrier characteristics over time.

In conclusion, we believe that the hCMEC/D3 cell line constitutes the first example of an extensively characterized human brain endothelial cell line, which recapitulates most of the unique properties of the BBB, even without coculture with glial cells. This cell line will be a useful model for studying the biology of human brain endothelium in the context of neuroinflammatory or infectious diseases and for large-scale screening of CNS drug candidates.

ACKNOWLEDGMENTS

We are grateful to Dr. Schinkel (Amsterdam, NL) for kindly providing us with KO 143, to Dr. R. Selway for surgical isolation of the brain tissue material and to Dr. S. Cisternino (INSERM U26,


Paris) for helpful discussions. This work was supported by SIDACTION, the Association de Recherche sur la sclérose en plaques (ARSEP), the Centre National de la Recherche Scientifique, the Institut National de la Santé et de la Recherche Médicale, the Université Paris-5 René Descartes, the Wellcome Trust, the Leverhulme Trust, the Multiple Sclerosis Society, the Open Society Development Research Fund, and the National Heart Lung and Blood Institute. A. Nicotra's salary was supported by a traveling fellowship from the University of Catania, Italy.

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Received December 21, 2004; accepted July 12, 2005


Table 1 Paracellular permeability of hCMEC/D3 monolayers to hydrophilic molecules

FITC-Dextrans, kDa

Permeability coefficients (× 10-3 cm/min)

HCMEC/D3 GPNT rat cell line5

bovine brain endothelial cells (monocultures)

4 0.325 0.975 0.434a 0.370b

40 0.027 0.114 0.046a n.d.

70 0.013 0.053 0.008a 0.126b

Permeability coefficients (× 10-3 cm/min)

HCMEC/D3 bovine coculture modelc

RBE4 rat cell lined

GPNT rat cell linee

[14C]-sucrose 1.65 ± 0.18 0.75 4.8 7.37

[3H]-inulin 0.36 ± 0.03 0.37 1.3 n.d.

TEER (Ω.cm2) 30−40 400−600 < 30 < 30

HCMEC/D3 cells were grown to confluence on polyester Transwell filters; then permeability to the indicated molecules and transendothelial electrical resistance (TEER) was measured as indicated in Materials and Methods. Experimental permeability data (in bold) were compared with published values of permeability coefficients for the same compounds (avan Bree et al., 1988; bMark and Miller, 1999; cCecchelli et al., 1999; dRist et al., 1997; eRomero et al., 2000); (n. d., not done).


Fig. 1

Figure 1. Morphological characteristics of hCMEC/D3 cells and expression of the immortalizing genes. Phase contrast microscopic view of the primary culture of human brain endothelial cells showing elongated, tightly packed, contact-inhibited morphology (A) and of the immortalized hCMEC/D3 clonal cell line (B), with a similar morphology. C) Confocal microscopic photograph of hCMEC/D3 cells showing a nuclear staining for SV40 T antigen and (D) perinuclear expression of hTERT.


Fig. 2

Figure 2. Nontransformed phenotype of hCMEC/D3 cells. A) hCMEC/D3 cells display a normal, nontransformed human female karyotype: results are presented at passage 22, and similar results were obtained at passage 33. B) Phase contrast microscopic view of a branched network of capillary-like cords formed by hCMEC/D3 cells in Matrigel extracellular matrix after 5 days in EGM-2 medium (×40).


Fig. 3

Figure 3. Expression of endothelial and BBB markers by hCMEC/D3 cells. Confluent monolayers of hCMEC/D3 cells (passage 22) were stained for the endothelial junctional markers PECAM-1 and VE-cadherin and for the junction-associated proteins β-catenin, γ-catenin, ZO-1, JAM-A, and claudin-5 as indicated. Cy2-phalloidin staining of F-actin (blue) is essentially localized at the cell cortex (counterstained nuclei in red). No change was detected in the junctional expression of VE-cadherin or β-catenin at passage 33 (p33).


Fig. 4

Figure 4. Adhesion molecule expression by hCMEC/D3 cells. Membrane expression of various adhesion molecules was assessed by flow cytometry analysis of hCMEC/D3 cells, untreated (–) or treated (+) by TNF-α (100 U/ml) and IFN-γ (100 ng/ml) for 24 h. Fluorescence intensity is presented on x-axis. Gray tracks are negative controls, corresponding to staining in the absence of primary antibodies; white tracks are specific stainings for the indicated adhesion molecules.


Fig. 5

Figure 5. Chemokine receptor expression on hCMEC/D3 cells. Membrane expression of chemokine receptors was assessed by flow cytometry analysis of hCMEC/D3 cells. Fluorescence intensity is presented on x-axis. Gray tracks are negative controls, corresponding to staining in the absence of primary antibodies; white tracks are specific stainings for the indicated chemokine receptors.


Fig. 6

Figure 6. A) Correlation between in vitro permeability of hCMEC/D3 cells and reported in vivo BBB permeability for a variety of chemical compounds. In vitro permeability of indicated drugs across confluent monolayers of hCMEC/D3 cells on polycarbonate Transwell filters is expressed as permeability coefficients (Pe, 10-3 cm/min). Results of in vivo BBB permeability for the same compounds (expressed as transport coefficients: Kin, 10-3 µl.s−1.g−1 ) were assessed by the brain perfusion technique in adult rats or mice and were previously reported (19, 20). Tested compounds are [14C]-diazepam and -morphine-6-glucuronide (M6G), [3H]-imipramine, -prazosin, -colchicine and -vincristine. Correlations between the in vitro permeability data for [14C]-diazepam, [3H]-prazosin, -colchicine and -vincristine obtained with hCMEC/D3 cells (y-axis) and rat brain GPNT cells (B) or with hCMEC/D3 cells (y-axis) and HUVECs (C) are presented for comparison.


Fig. 7

Figure 7. Expression of ABC transporters by hCMEC/D3 cells. A) RT-PCR analysis of the expression of mdr1, mrp1, mrp5, and bcrp gene transcripts by hCMEC/D3 cells (D3) and primary human brain endothelial cells (Pr); cyclophilin was used as control. The RT-PCR analysis was performed in the absence (–) or presence (+) of reverse transcriptase (RT) in order to exclude any amplification of genomic sequences. B) Western blot analysis of hCMEC/D3 cells (D3) and human primary astrocytes (A) for the expression of P-gp (MDR1, 170 kDa), MRP-1 (190 kDa) and BRCP (70 kDa) proteins (arrows).


Fig. 8

Figure 8. Functionality of ABC transporters expressed by hCMEC/D3 cells. Cellular uptake of calcein-AM (A), a fluorescent substrate of P-gp and MRP1, rhodamine 123 (B), a fluorescent substrate of P-gp and BCRP, or [3H] daunorubicin (C), a shared substrate of P-gp, MRP1 and BCRP, was measured after 1 h, in the absence (–) or presence of specific P-gp, MRP1 or BCRP inhibitor, respectively PSC-833 (10 µM), MK-571 (10 µM) or KO-143 (1 µM). Fluorescence intensity (A, B) or radioactivity (C) remaining associated with the hCMEC/D3 cells after extensive washing was measured: results are expressed as a percentage of the corresponding value in the absence of inhibitor (–). Three independent experiments were performed; results from one representative experiment in triplicates are presented; *Significant difference (P<0.01) from control (–).


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