WO1993006222A1

WO1993006222A1 - Immortalized cerebral endothelial cell lines, process for their preparation and applications thereof as a model for the study of cerebral physiopathology

Info

Publication number: WO1993006222A1
Application number: PCT/FR1992/000879
Authority: WO
Inventors: Michel Claire; Pierre-Olivier Couraud; Odile Durieu-Trautmann; Nathalie Foignant-Chaverot; Françoise ROUX; Arthur Donny Strosberg
Original assignee: Centre National De La Recherche Scientifique (Cnrs)
Priority date: 1991-09-24
Filing date: 1992-09-22
Publication date: 1993-04-01
Also published as: FR2681609A1; FR2681609B1

Abstract

Immortalized cerebral endothelial cell lines, process for their preparation and applications thereof as a model for the study of cerebral physiopathology and more especially, as a cellular model of the hemato-encephalic barrier. Said lines are obtained by transfection of cerebral endothelial cells by means of a nucelic acid fragment comprising at least one fragment immortalizing a viral or cellular oncogene, optionally associated with at least one marker gene. The lines thus transfected are immortalized and exhibit the non-transformed phenotype of differentiated cerebral endothelial cells.

Description

LINES OF IMMORTALIZED CEREBRAL ENDOTHELIAL CELLS, THEIR PREPARATION PROCESS AND THEIR APPLICATIONS AS A STUDY MODEL OF CEREBRAL PHYSIOPATHOLOGY.

The present invention relates to immortalized cerebral endothelial cell lines, to their preparation process and to their applications as a model for studying cerebral pathophysiology and more particularly as a cell model of the blood-brain barrier.

The blood-brain barrier controls the exchanges between the blood and the brain parenchyma. It essentially consists of the endothelial cells of the cerebral microvessels, associated with the perivascular astrocytes. These endothelial cells have a unique phenotype, essentially characterized by the presence of numerous tight intercellular junctions and the expression of specific enzymes, such as γ-glutamyltranspeptidase (γ-GT) and alkaline phosphatase.

It is known that the induction of this endothelial phenotype is controlled, in vivo, by perivascular astrocytes. The in vi tro study of the molecular mechanisms of this induction has the disadvantage of being dependent on the availability of primary cell cultures of endothelial cells from cerebral microvessels; moreover, in addition to the difficulty, at present, of having systems allowing the analysis of the endothelial cells of the blood-brain barrier, the limited lifespan of these cells in culture (of the order of twelve passages) leading, in addition, to considerable phenotypic changes concomitantly with cell aging (DURIEU-TRAUTMANN et al., J. Neurochem., 1991) is another major drawback associated with this study.

The inventors have therefore set themselves the goal of providing a model for studying endothelial cells of the blood-brain barrier, not pre not feeling the disadvantages of the cultures of these cells and allowing the study of the neuro- and immunochemical processes involved in controlling the activity of the blood-brain barrier.

The present invention relates to mammalian cerebral endothelial cell lines, characterized:

in that they are immortalized and exhibit at least one of the following characteristics of the differentiated cerebral endothelial cells, in a stable manner:

- the expression of endothelial markers such as the factor VIII related antigen, the angiotensin converting enzyme and specific enzymes (γ-GT, alkaline phosphatase),

- secretion of vasoactive substances (endothelin, NO, prostaglandins),

- the expression of molecules of the major histocompatibility complex (MHC),

- expression of hormone receptors

(neurotransmitter receptors such as β-adrenergic receptors), and

- the existence of tight junctions and

. in that they are capable of being obtained by transfection of cerebral endothelial cells with a nucleic acid fragment comprising at least one immortalizing fragment of a viral or cellular oncogene, optionally associated with at least one marker gene.

Such cell lines surprisingly exhibit at least one of the above properties of differentiated cerebral endothelial cells, and this in a stable manner.

According to the invention, said marker gene is advantageously a gene coding for resistance to an antibiotic.

According to an advantageous embodiment of the Said line, it is obtained by transfection of endothelial cells from bovine cerebral capillaries with the plasmid pSV3 neo containing the neo gene for resistance to neomycin and a fragment of the oncogene T of SV40.

The plasmid pSV3 neo is more particularly described in the article in the name of Southern and Berg, (J. Mol. Appl. Genêt., 1982, 1, 327-341).

This cell line was named SV-BEC by the inventors.

In accordance with the invention, said line was deposited under number I-1143 on September 19, 1991 with the National Collection of Cultures of Microorganisms held by the Institut Pasteur.

According to another advantageous embodiment of said line, it is obtained by transfection of endothelial cells of rat cerebral capillaries with a plasmid containing the region. early E1A of the adenovirus 2 genome and the neomycin resistance gene.

This cell line was named RBE-4 by the inventors.

In accordance with the invention, said line was deposited under number I-1142 on September 19, 1991 with the National Collection of Cultures of Microorganisms held by the Pasteur Institute.

Said viral or cellular oncogenic fragment and / or the marker gene are under the control of either a homologous promoter or a heterologous promoter, capable of allowing the expression of said immortalizing fragment.

Unexpectedly, said lines are immortal, have a stable and untransformed phenotype of differentiated cerebral endothelial cells, have unlimited proliferation activity and exhibit some of the properties of endothelated cells. brain links of the blood-brain barrier, allowing in particular their use as an in vitro cellular model of the blood-brain barrier.

The present invention also relates to a process for obtaining a cell line in accordance with the invention by a transfection method, which process is characterized in that:

. endothelial cells of cerebral microvessels are cultivated in a suitable culture medium, supplemented with serum and growth factor,

. they are transfected between the 2nd and the 6th passage with a nucleic acid fragment comprising at least one immortalizing fragment of a viral or cellular oncogene and optionally at least one marker gene, in particular a gene coding for resistance to an antibiotic and

. the transfected cells are selected on a selection medium suitable for said marker gene, if necessary.

According to an advantageous embodiment of said method, said nucleic fragment comprises a gene coding for resistance to neomycin and a fragment of a viral oncogene, such as a fragment of the SV40 T oncogene and a fragment containing the E1A early region of the genome of adenovirus 2 or of a cellular oncogene.

The present invention also relates to a model for studying and identifying the biochemical and cellular systems of the blood-brain barrier, characterized in that it comprises at least one cell line according to the invention.

In addition to the foregoing arrangements, the invention also comprises other arrangements, which will emerge from the description which follows, which refers to examples of implementation of the process which is the subject of the present invention, with reference to the accompanying drawings in which Figures 1 to 7 illustrate the properties of SV-BEC cells and Figures 8 to 14 illustrate the properties of RBE-4 cells.

More specifically:

FIGS. 1A and 1B illustrate the effect of FGFb on the proliferation of SV-BEC cells (FIG. 1A) and of CECB cells (FIG. 1B),

FIGS. 2A and 2B illustrate the effect of increasing amounts of FGFb on SV-BEC cells (FIG. 2A) and CECB cells (FIG. 2B),

FIGS. 3 show that the SV cells

BEC are differentiated endothelial cells (Figure 3A: presence of angiotensin converting enzyme; Figure 3B: presence of factor VIII related antigen; Figure 3C: immunostaining with agglutinin Griffonia simplicifolia,

FIG. 4 shows that the SV-BEC cells secrete endothelin-1,

FIG. 5 illustrates a vertical section of a confluent monolayer of SV-BEC cells, in electron microscopy,

FIG. 6 shows the number of β-adrenergic binding sites present on the surface of SV-BEC cells,

FIGS. 7A and 7B illustrate the effect of isoproterenol and of an antagonist on the amount of cAMP in SV-BEC cells,

FIG. 8 shows the structure of the plasmid used for the transfection of endothelial cells of rat cerebral microvessels, for obtaining the immortalized line RBE-4,

FIGS. 9A and 9B illustrate some of the characteristics of the differentiated endothelial phenotype of immortalized RBE-4 cells: expression of an antigen related to factor VIII related factor (FIG. 9A) and staining with the lectin Bandeiraea simplicifolia (FIG. 9B), FIGS. 9C to 9F show the histochemical localization of γ-GT, in the presence of FGFb in immortalized RBE-4 cells,

- Figures 10A-F show the histochemical localization of alkaline phosphatase, in the absence

(Figures 10A-B) or in the presence (Figures 10C-F) of FGFb,

- Figures 11 and 12 illustrate the synthesis of cAMP (Figure 11) and cGMP (Figure 12) by RBE-4 cells,

FIG. 13 illustrates the secretion of ET-1 by the RBE-4 cells, and

- Figure 14 illustrates the expression of MHC class I and class II molecules in RBE-4 cells.

It should be understood, however, that these examples are given only by way of illustration of the subject of the invention, of which they do not in any way constitute a limitation.

EXAMPLE 1 Preparation of a cell line in accordance with the invention: endothelial cells of bovine cerebral microvessels (line SV-BEC).

Endothelial cells of bovine cerebral microvessels are isolated from the cerebral cortex treated with collagenase (DURIEU-TRAUTMANN et al., J. Neurochem., 1991, 56, 3, 775-781); these cells are not contaminated with smooth muscle vascular cells or pericytes. They are cultured in boxes covered with 0.2% gelatin (w / v) containing a DMEM medium (Dulbecco's modified Eagle's Medium) supplemented with fetal bovine serum at 10% (v / v), in glutamine 2 mM and in FGFb at 1 ng / ml and glucose at 1 g / l.

They are subjected to a weekly passage carried out by resuspension in trypsin-EDTA [0.05% -0.02% (w / v)] at 1 to 10 dilutions; the medium is changed every 3 days.

The phosphate co-precipitation technique of calcium is used for the transfection of said cells, on the 5th passage, with the plasmid pSV3 neo (10 μg), containing a fragment of the oncogene SV40 and the gene for resistance to the drug called G418 (WHITLEY et al., Mol. Cell. Endocrinol., 1987, 52, 279-284).

After selection in a medium containing 800 μg / ml of G418, the cells present are cloned by limiting dilution.

The clone SV-BEC is thus obtained.

EXAMPLE 2: Characteristics of the SV-BEC line.

The cell line obtained in Example 1 has a certain number of characteristics of the cerebral endothelial cells.

A. Phenotype __ in differentiated dothelial:

a) FGFb and gelatin requirements of immortalized SV-BEC cells:

. When the cells are seeded at low density (5.10 ³ cells / cm ² ) in tissue culture dishes and cultured on a medium supplemented with serum, [DMEM supplemented with fetal bovine serum (10%) and glutamine (2 mM)] , bovine brain endothelial cells (CECB) proliferate at a low rate, while SV-BEC stop proliferating after 3-4 days in culture.

FIGS. 1A and 1B, which have the number of days on the abscissa and the number of cells / cm ² (x 10 ^-3 ) on the ordinate, illustrate the effect of FGFb on the proliferation of SV-BEC cells (FIG. 1A) and CECB cells (Figure 1B). The addition of FGFb (1 ng / ml) (FIG. 1A, curve - ■ - and FIG. 1B, curve - ▲ -), significantly improves the growth rate of the two cell types.

The confluence is reached after 5 days, after addition of FGFb each day.

. The maximum cell density obtained with SV-BEC cells, under these conditions, is only 65 to 70% of the density obtained with CECB cells. The growth rates of the two cell types are similar, with a population doubling time of approximately 12 hours, during the log phase.

. FIGS. 2A and 2B, which have the FGFb concentrations on the abscissa and the number of cells / cm ² (x 10 ^-3 ) on the ordinate illustrate the effect of increasing quantities of FGFb on SV-BEC cells (FIG. 2A) and on CECB cells (Figure 2B) in the presence or absence of gelatin. The concentration of FGFb necessary for optimal cell proliferation is 0.25 ng / ml for SV-BEC cells (FIG. 2A: culture dishes coated with gelatin: curve; dishes without gelatin: curve ❑) and 0.5 ng / ml for CECB cells (Figure 2B: culture dishes coated with gelatin: curve ▲; dishes without gelatin: curve ❑). In the test illustrated in FIGS. 2A and 2B, the FGFb is added on day 0 and on day 3.

The proliferative responses of the two cell types are significantly improved when the cells are seeded on culture dishes coated with gelatin. At confluence, SV-BEC cells form a monolayer of cells, inhibited by contact.

b) Immunochemistry:

By immunocytochemical analysis, it appears that SV-BEC cells express a factor VIII related antigen as well as the angiotensin converting enzyme, two specific markers of endothelial cells.

In addition, these cells are stained with the fluorescent agglutinin Griffonia simplicifolia (Sigma), which is also a specific marker for cerebral endothelial cells.

For these three tests, the cells were grown on coverslips coated with gelatin. They are fixed with a methanol: ethanol mixture (1: 1) at 22 ° C for 20 min, these cells are first incubated with antibodies directed against the factor VIII related antigen or the angiotensin converting enzyme, then with biotynilized antibodies and finally they are stained with fluorescent strep-tavidin. These results are observed using a standard microscope (Nikon) equipped with epifluorescence illumination.

The agglutinin Griffonia simplicifolia, labeled with fluorescein, is used at 1/400 in a PBS buffer.

All incubations are 60 minutes at room temperature.

SV-BEC cells are therefore real differentiated endothelial cells, as shown by the presence of factor VIII related antigen (Figure 3B), angiotensin converting enzyme (Figure 3A) and immunostaining with agglutinin Griffonia simplicifolia (Figure 3C).

c) Secretion of vasoactive substances:

SV-BEC cells are also tested for the synthesis and secretion of vasoactive substances, in comparison with CECB cells. Using a radioimmunoassay, immunoreactivity related to that of endothelin-1 is detected in a conditioned medium (Figure 4). The dilution curves generated by these conditioned media are parallel to the standard curve obtained with endothelin-1 (ET-1). In addition, the secretion observed is linear as a function of time and reaches a plateau after 24 hours. The RIA protocol is as follows:

a rabbit antiserum specific for endothelin (final dilution of 1/300) is incubated in DMEM supplemented with fetal bovine serum (10%), in the presence of different concentrations of synthetic unlabeled endothelin or conditioned medium, for 2 hours at 37ºC (final volume: 80 μl).

[ ¹²⁵ I] ET-1 (10,000 cpm in 40 μl) is then added and the incubation is 16 hours at 4ºC. Free and bound [ ¹²⁵ I] ET-1 are separated by the addition of Protein A-Sepharose ^® .

After 1 hour at room temperature, the tubes are centrifuged, the pellets washed with a buffer and the bound radioactivity is counted.

FIG. 4 illustrates the results of such a test and comprises on the abscissa the concentration of endothelin-1 (log fmol / tube) and on the ordinates the bound / free ratio (%). The conditioned media obtained from SV-BEC (curve -

-) and from CEBC (curve - ▲ -) are compared with the standard curve (curve -O-).

B. Absence of tumoriqenicity:

SV-BEC cells are not transformed: indeed, as specified above, they exhibit contact inhibition and their proliferation is dependent on exogenous FGFb.

C. Expression of markers of the blood-brain barrier:

- The protocol used is as follows:

The cells are rinsed with cold PBS buffer, pH 7.4, then fixed in 2% glutaraldehyde (v / v) in 0.1 M cacodylate buffer, for 60 min at 4ºC. After rapid rinsing in the same buffer, the cells are again fixed, either in ferro-osmium [OsO4 1%, K ₄ Fe (CN) ₆ 0.8%] in cacodylate buffer for 30 min, or in 1% aqueous IOSO _{4 and} 1% uranyl acetate in 50% ethanol. These cells are then dehydrated in ethanol and then removed from the culture dishes using a short treatment with butyl-2-3-epoxy-propyl-ether.

The monolayers were carefully collected and transferred to small capsules polyethylene and included in an EPON ^® mixture, using the epoxy 1-2 propane as an intermediate solvent.

Thin sections are stained conventionally with lead citrate and uranyl acetate and observed in a Philipps CM12 80 kv apparatus.

- Results:

The ultrastructural morphology of SV-BEC cells is observed under the electron microscope.

FIG. 5 illustrates a vertical section of a confluent monolayer; many tight intercellular junctions are visible (see arrows), characteristic of the blood-brain barrier.

In addition, a significant γ-glutamyl transferase activity is measured in the confluent SV-BEC cells (11.2 nmol.mg prot ^-1 .min ^-1 ), also specific for the blood-brain barrier, according to the measurement protocol for γ-glutamyl transpeptidase activity described in ORLOWSKI and MEISTER (Proc. Natl. Acad. Sci., 1970, 67, 1248-1255).

D. Coexpression of β1 and β2 adrenergic receptors:

* Connection to receptors.

- Protocol:

. For the measurement of the binding of β-adrenergic ligands, the cells are detached after a short treatment with trypsin-EDTA, washed and resuspended (approximately 10 ⁷ cells / ml) in a Hank's saline solution supplemented with HEPES 20 mM, pH 7.4 and bovine serum albumin (1 mg / ml).

The cells (10 ⁶ ) are incubated in the tam pon with increasing amounts of [ ³ H] CGP 12177, in a final volume of 300 μl at 37ºC for 60 min.

Nonspecific binding is measured in the presence of (±) alprenolol 3.10 ^-5 M. The reaction is stopped by adding 1 ml of cold buffer, and the samples are immediately filtered through GF / C fiberglass filters (Whatman ). After washing, the radioactivity retained on the filters is counted by liquid scintillation.

- Results:

Binding of [ ³ H] CGP 12177, a hydrophilic β-adrenergic antagonist, is used to assess the number of β-adrenergic binding sites present on the surface of SV-BEC cells. Scatchard analysis of saturation binding data indicates 3819 ± 229 sites / cell and a dissociation constant (K _D ) of 899 ± 113 pM (Figure 6).

Figure 6 has on the abscissa the concentration in [ ³ H] CGP 12177, in nM and on the ordinate the bound amount in fpmol / 10 ⁶ cells.

* Coupling of receptors to adenylate cyclase activity: accumulation of cellular cAMP.

- Protocol:

Aliquots of 10 ⁶ cells are incubated in a buffer (Hank's salt solution, 20 mM HEPES, pH 7.4,

IBMX (3-isobutyl-1-methyl-xanthine) 0.5 mM) with increasing amounts of (-) isoproterenol in a final volume of 200 μl at 37 º C for 10 min.

The inhibition of the accumulation of cellular cAMP is tested with (-) isoproterenol 3.10 ^-8 , in the presence of different concentrations of β1-selective adrenergic CGP 2071A ligand. The AMPc esc content measured as described in CHAPOT et al. (Hybridoma, 1989, 8, 535-543), using the Amersham [ ³ H] cAMP kit. - Results:

FIG. 7A shows that (-) isoproterenol induces an increase in the level of cAMP by a factor of 6.

These data indicate an IΕC ₅₀ value of 5 nM for (-) isoproterenol. A biphasic inhibition of the effect of (-) isoproterenol is observed in the presence of CGP 20712A, suggesting the existence of two populations of β-adrenergic receptors as in the original endothelial cells (FIG. 7B).

A computer analysis of these data reveals that 36% of the receptors have a high affinity for CGP 20712A (CI ₅₀ ≈ 29 pM) and that 65% have a low affinity (CI ₅₀ ≈ 1 nM). These values are in agreement with the known affinities of CGP 20712A for the β1 and β2 adrenergic receptors (MARULLO et al., Bio / Technology, 1989, 7, 923-927).

FIG. 7A, which has the (-) isoproterenol (log M) concentration on the abscissa and the amount of pmol of cAMP / 10 ⁶ cells on the ordinate, illustrates the effect of isoproterenol on the amount of cAMP in SV-BEC cells and FIG. 7B, which has the concentration of CGP 20712A (log M) on the abscissa and the percentage of accumulated cAMP on the ordinate, illustrates the inhibition of this effect by CGP 20712A.

EXAMPLE 3 Preparation of a cell line in accordance with the invention: endothelial cells of Lewis rat cerebral microvessels (RBE-4 line).

- Endothelial cells of Lewis rat cerebral microvessels are immortalized by transfection with a plasmid containing the early E1A region of the adenovirus 2 genome and the neomycin resistance gene under the control of the SV40 promoter (FIG. 8) as in Example 1, after culturing these cells in boxes covered with collagen, containing an α-MEM / F10 medium (2/3; 1/3), supplemented with 10% fetal calf serum , in FGFb 1 ng / ml, glutamine and penicillin / streptomycin. EXAMPLE 4: Characteristics of the RBE-4 line.

The cell line obtained in Example 3 has some of the characteristics of cerebral endothelial cells, studied according to the same protocols as those of Example 2; in particular, it has an untransformed phenotype: contact inhibition, proliferation dependent on growth and adhesion factors, expression of markers of endothelial differentiation and non-tumorigenicity.

A. Differentiated endothelial phenotype:

RBE-4 cells express an antigen related to factor VIII related factor (Figure 9A); moreover, these cells are stained with the lectin Bandeiraea simplicifolia (FIG. 9B); the protocol used is the same as that of example 2 and the results clearly show that the RBE-4 cells are differentiated endothelial cells.

B. Expression of markers of the blood-brain barrier:

The activities of two enzymes, which are considered to be markers of the differentiated endothelial cells of the cerebral microvessels, γ-glutamyl transferase (γ-GT) and alkaline phosphatase, are demonstrated in RBE-4 cells at using histochemical methods.

RBE-4 cells are seeded in tissue culture dishes and cultured in a medium containing FGFb [α Medium / Ham's F10 (1: 1 Seromed, France), supplemented with 2 mM glutamine, fetal calf serum heat inactivated at 10% and FGFb 1 ng / ml; then they are spread at a density of 10 ⁴ cells / cm ² on boxes covered with collagen]. The confluent cultures develop, after several days, ramifications which extend beyond the monolayer and form a network of tubular structures, recalling capillaries (Figure 9C). Γ-GT (Figure 9C) and alkaline phosphatase activities can be detected in some of these tubular structures, but are not observed in the cells of the surrounding monolayer.

The RBE-4 cells are cocultivated with conditioned medium or plasma membranes either of rat primary astroglial cells (FIG. 9D) or of C6 gliomal cells (FIG. 9E). In monolayers treated with plasma membranes of the two aforementioned types, whether or not in the presence of FGFb, the tubular structures develop rapidly and form important shapeless aggregates; such proliferation appears less evident in a conditioned environment. The γ-GT (Figure 9F) and alkaline phosphatase (Figure 10A-F) activities are found in many cells of these three-dimensional structures, causing an overall increase in these two BBB markers in cocultures. The addition of a cAMP analog, 8-bromo-cAMP, reduces this angiogenic process and induces the expression of alkaline phosphatase activity in each of the cells forming the residual tubular structures or the aggregates and in some cells of the monolayer. The effects of this substance on γ-GT are less pronounced.

Figures 9C to 9F show the histochemical localization of γ-GT, in the presence of FGFb (a line corresponds to 60 μm): 9C: RBE-4 cells at confluence, with a capillary type structure and the presence of a few γ- cells GT +; 9D: RBE-4 cells treated for 6 days with plasma membranes of primary astrocytes; 9E RBE-4 cells treated for 6 days with plasma membranes of C6 gliomal cells; 9F: RBE-4 cells treated for 6 days with plasma membranes of C6 gliomal cells and 0.1 mM c-8-bromo-cAMP: most of the cells pre sense γ-GT activity.

FIGS. 10A-F show the histochemical localization of alkaline phosphatase, in the absence (FIGS. 10A-B) or in the presence (FIGS. 10C-F) of FGFb: FIGS. 10A and C: RBE-4 cells at confluence; FIGS. 10B and D: RBE-4 cells treated for 6 days with plasma membranes of C6 gliomal cells; FIG. 10E: RBE-4 cells treated for 10 days with a medium conditioned for gliomal cells C6: FIG. 10F: cells further treated with 0.1 mM 8-bromo-cAMP. These various figures show that the expression of the alkaline phosphatase activity exhibits the same evolution in ec coculture in the presence of 8-bromo-AMPc as the γ-GT.

These various results show that in the monolayers of endothelial cells of the clone RBE-4, as in the primary cultures of microvascular endothelial cells, the formation of capillary tubes can occur, in the presence of a soluble angiogenic mitogen; moreover, the formation of such a tubular structure can be stimulated by astroglial factors (membrane or soluble).

C - Expression of hormone receptors.

. cAMP production:

Aliquots of 4.10 ⁶ cells are incubated in buffer A [Hank's saline solution, Hepes 20 mM pH 7.4, IBMX 0.5 mM} in the presence of increasing amounts of (-) isoproterenol, in a final volume of 250 μl , at 37ºC and for 10 minutes; cAMP is measured as described in DURIEU-TRAUTMANN O. et al. (J. Neurochem., 1991, 56, 775-781), using an Amersham cAMP kit [ ³ H].

The RBE-4 cells were tested for their capacity to produce cAMP under the influence of an extracellular regulation signal; FIG. 11, which has the concentration of isoproterenol on the abscissa and ordered the cAMP concentration (pmol / 10 ⁵ cells), shows that isoproterenol, a β-adrenergic agonist, stimulates the accumulation of cAMP in RBE-4 cells, this stimulation being blocked by propranolol.

. cGMP production:

Aliquots of 2.10 ⁵ cells are incubated in the same buffer A as above, in the presence of increasing amounts of natriuretic peptides (ANP) in a final volume of 500 μl at 37ºC, for 10 minutes.

When testing the soluble guanylyl cyclase activity, aliquots of 10 ⁶ cells are incubated in buffer A containing nitroprusside at 37ºC, for 5 minutes.

The production of cGMP, from GTP, is catalyzed by different enzymes: FIG. 12, which has the ANP concentration on the abscissa and the cGMP concentration (pmol / 10 ⁵ cells) on the ordinate, shows that the atrial natriuretic factor ( FAN or ANP for atrial natriuretic peptide) stimulates the accumulation of cGMP in RBE-4 cells.

This shows that the immortalized cells according to the invention, RBE-4 have β-adrenergic receptors and FAN receptors.

D - Secretion of vasoactive substances.

. secretion of endothelin-1 (ET-1):

RBE-4 cells are grown in dishes containing 24 wells for 3 days.

The medium is then changed and replaced with 500 μl of medium devoid of serum containing 0.15% BSA and various effectors at the concentrations indicated below.

The incubation lasts from 30 min to 6 h. The levels of ET-1 in the culture supernatants are determined by EIA, based on the use of a tandem of monoclonal antibodies directed against two different epitopes of ET-1 (sensitivity of the test, of the order of pg / ml); the accumulation of ET-1 in the extracellular medium is linear over a period of 6-8 hours (110 ± 4 pg / 10 ⁶ cells / h, at confluence) (Figure 13), then reaches a plateau around the 16th hour . This secretion is stimulated by serum and thrombin; in addition, 8-bromo-cAMP (0.5mM) induces a strong release of ET-1, while 8-bromo-cGMP (0.5mM) and FAN (100 nM) inhibit this secretion.

Figure 13 highlights these results, obtained under the aforementioned conditions:

in the presence of: 1: nothing; 2: 8-bromo-cAMP 0.5 mM; 3: IBMX 0.5 mM and 8-bromo-AMPc 0.5 mM; 4: isoproterenol 100 μM; 5: 8-bromo-GMPc 0.5 mM; 6: FAN 100 nM and 7: IBMX 0.5 mM. After these treatments, the ET-1 immunoreactivity is measured in the supernatants. The results are expressed as the mean ± SEM of 8 to 16 determinations (p <0.01 for columns 2 to 6).

. NO secretion:

NO, identified as an endothelium-relaxing factor, is synthesized from L-arginine by at least two different NO synthases (the first: dependent on calcium and calmodulin and expressed, constitutively, only in a small number cell types, including some neurons; the second: inducible by cytokines).

RBE-4 cells are grown in 24-well dishes for 3 days.

They are then stimulated by the addition of 100 U / ml of rat IFN-γ and / or of 50 U / ml of human TFNα, in the presence or in the absence of the various effectors mentioned below: N-methylarginine (NMA), nitroarginine (NOA), cycloheximide (CHX), 8-bromo-AMPc (BrAMPc) or isoproterenol (ISO).

Table I below shows the regulation of inducible NO synthase activity in RBE- cells

The release of NO by the RBE-4 cells is detected by colorimetric determination of the accumulated nitrites, after 48 h, by adding 500 μl of cellular supernatant to 500 μl of Greiss reagent (sulfanilamide 0.5% and naphthylethylenediamine dihydrochloride 0.05 %, in 5% phosphoric acid), then incubating for 10 min at room temperature. The DO540 is measured with a spectrophotometer. As shown in Table I, only inducible NO synthase is detectable in RBE-4 cells. The production of nitrites by RBE-4 cells is induced by treatment with IFN-γ and TNFα potentiates this effect. This activity is blocked by N-methyl-arginine and to a lesser extent by nitro-arginine. This shows the absence of expression of the constitutive NO synthase by these cells, which would constitute a particular characteristic of the endothelial cells of the blood-brain barrier.

Table I also shows that BrAMPc, although having no effect alone, potentiates the inducing effect of cytokines. Isoproterenol stimulates the inducing effect of IFN-γ and TNFα, although without effect alone.

The activity of isoproterenol is dose dependent and simulates that of 8-Br-cAMP.

The activation of NO synthesis is preceded by a lag phase of 7-8 h.

. secretion of prostaglandins:

RBE-4 cells constitutively secrete significant quantities of PGE2 (> 1000 pg / ml), but practically no 6-keto-PGF- _1α , the stable derivative of PGI ₂ (<50 pg / ml).

E - Expression of molecules of the major histocompatibility complex.

The expression of class I and class II molecules in RBE-4 cells has been studied.

By flow cytometry analysis under standard conditions, RBE-4 cells constitutively express class I molecules only. Analysis after treatment with IFN-γ reveals that the expression of class I molecules is increased; it also reveals a significant induction of the expression of class II molecules (Figure 14: intensity of fluorescence on the abscissa, number of cells, on the ordinate): the maximum expression of class I molecules is observed after 16 hours and remains stable for several hours, while 24 hours of treatment are necessary to obtain optimal expression of class II molecules.

As is apparent from the above, the invention is in no way limited to those of its modes of implementation, embodiment and application which have just been described more explicitly; on the contrary, it embraces all the variants which may come to the mind of the technician in the matter, without departing from the scope or the scope of the present invention.

Claims

1º) Lines of mammalian cerebral endothelial cells, characterized:

- expression of endothelial markers,

- the secretion of vasoactive substances, - the expression of molecules of the major histocompatibility complex (MHC),

- the expression of hormone receptors, and

- the existence of tight junctions and

2º) Cell line according to claim 1, characterized in that it is capable of being obtained by transfection of endothelial cells of bovine cerebral capillaries with the plasmid pSV3 neo containing the neo gene for resistance to neomycin and a fragment of l SV40 oncogene T.

3º) Cell line according to claim

2, characterized in that it was deposited under No. I-1143 dated September 19, 1991 with the National Collection of Cultures of Microorganisms held by the Pasteur Institute.

4º) Cell line according to claim

1, characterized in that it is capable of being obtained by transfection of endothelial cells of rat cerebral capillaries with a plasmid containing the early region ElA of the genome of adenovirus 2 and the gene for resistance to neomycin.

5º) Cell line according to claim 4, characterized in that it was deposited under the nº I-1142 dated September 19, 1991 with the National Collection of Cultures of Microorganisit.es held by the Institut Pasteur.

6º) Method for obtaining a cell line according to any one of Claims 1 to 5, by a transfection method, which method is characterized in that:

7º) Method according to claim 6, characterized in that said nucleic fragment comprises a gene coding for resistance to neomycin and a fragment of a viral or cellular oncogene.

8º) Method according to claim 7, characterized in that the fragment of viral oncogene is selected from the group which comprises SV40 dOncogene fragments and fragments containing the early ElA region of the genome of adenovirus 2.

9º) Model for studying and identifying biochemical and cellular systems of the blood-brain barrier, characterized in that it comprises at least one cell line according to any one of claims 1 to 5.