Brain Research, 566 (1991) 325-328
325
Elsevier
BRES 24946
Class II MHC antigen expression by cultured human cerebral vascular endothelial cells* R i c h a r d M . M c C a r r o n 1, L a n W a n g 1, E l l i o t P. C o w a n 2 a n d M a r i a S p a t z 1 SStroke Branch and 2Neuroimmunology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892 (U.S.A.)
(Accepted 27 August 1991) Key words: Endothelium; Brain; Class II MHC; Blood-brain barrier; HLA-DR
Cerebral vascular endothelial cells (EC) isolated from human brain do not constitutivelyexpress class II MHC antigens. However, incubation in the presence of human interferon-gamma(IFNy) resulted in the expression of both HLA-DR and -DP antigens. FACS analysis revealed that approximately40% of the EC population expressed HLA-DR antigen. Ouantitation by ELISA demonstrated that maximum expression was observed with 100 Ulml IFN~ for 4 days. Treatment with IFN~ also increased class II mRNA levels in all EC cultures tested.
The cerebral capillary endothelial cell (EC) is strategically located to play a critical role(s) in regulating peripheral blood cell trafficking into the central nervous system (CNS). The cerebral microvascular endothelium differs from other endothelium in several respects, e.g., they form tight continuous junctions, have fewer vesicles, and also possess a variety of highly specific transport mechanisms. These characteristics are at least partially responsible for the lack or limited amount of lymphocytes trafficking into the CNS, and account for the term blood-brain barrier (BBB). Numerous studies have identified alterations in the BBB permeability in CNS diseases such as multiple sclerosis (MS) and the model disease experimental allergic encephalomyelitis (EAE) s'7,t2'17'19. The mechanism(s) by which these alterations in the BBB occur allowing the entry (extravasation) of inflammatory cells into the brain parenchyma are not known. One factor which plays a well.documented role in interactions with immune cells is the class II MHC (la) molecule. The expression of these molecules on EC has implicated these cells in a variety of phenomena including antigen presentation 9'11'24, lymphocyte adhesion 3's and cytolytic T cell interactions I°'13'15. It is shown here that cerebral capillary EC isolated from human brain do not constitutively express class II MHC (HLA-DR, -DP, -DO) antigens. However, these cells can be induced to express both HLA-DR and HLA-DP antigens by in vitro culture with recombinant human interferon-gamma
(IFNy). This is the first demonstration of class II MHC antigen expression by cultured human cerebral vascular EC, and indicates a possible mechanism by which these cells interact with immune cells at the site of the BBB. E C culture. EC were prepared from small samples of human brain tissue surgically removed for treatment of idiopathic epilepsy. Microvessels were isolated by mechanical dispersion (homogenization) and filtration techniques similar to the method by Gerhard et al. 4. Cells were passaged by trypsinization and were utilized between passages 4 and 12. EC purity was assessed with antibody to human factor VIII-related antigen, and contamination by astrocytes measured with antibody to glial fibrillary acidic protein (GFAP). EC were cultured on gelatin-coated coverslips, fixed (3.7% formalin at room temperature for I0 min), permeabilized (0.1% Triton X.100, room temperature for 5 rain), and stained with rabbit anti-human factor VIII-related antigen (I :50) (Accurate Chemical and Scientific Corporation, Burlingame, CA) or rabbit anti-GFAP (Accurate) (1:200) in a humidified chamber for 45-60 rain at room temperature. After washing, secondary antibody [rhodamine-conjugated goat F(ab')2anti-rabbit IgG (H + L) (1:200)] was added at room temperature for 30-45 min. Staining for HLA-DR, -DP and -DQ antigens was performed with mouse antibodies to HLA-DR antigen (clone L243; 1:50), HLA-DP antigen (clone B7/21, 1:50) and HLA-DQ antigen (clone SK10, 1:25) (Becton Dickinson; Mountain View, CA). Cells were fixed as described above, but were not per-
* Presented in part at the American Associationof Immunologists/AmericanSociety for Biochemistryand Molecular BiologyJoint Meeting, New Orleans, LA, June 7, 1990. Correspondence: R.M. McCarron, National Institutes of Health, 9000 RockvillePike, Building36, Room 4D04 Bethesda, MD 20892, U.S.A.
326 meabilized. Single cell suspensions of EC populations were stained as described above and analyzed by flow cytometry on a FACS IV analyzer (Becton-Dickinson FACS Systems). ELISA. Cell surface expression of HLA-DR molecules was measured by ELISA essentially as previously described 2°. EC (2 × 104 cells/0.1 ml/well in 0.3 cm2 well (pre-coated with type I collagen; Collaborative Research, Bedford, MA) were incubated for 24-96 h at 37 °C in a 5.0% CO2 atmosphere in the absence or presence of indicated concentrations of recombinant human IFN7 (Interferon Sciences, Rockviile, MD). Nonspecific binding sites of cells were blocked by the addition of ~00/~1 phosphate buffered saline (PBS) containing 2% bovine serum albumin and 50/~g aggregated rabbit IgO, prepared by heating monomeric IgO at 63 °C for 20 min21. After incubation with anti-HLA-DR or anti-HLA-DP antibody (1:1000), cells were treated with biotin-conjugated antimurine IgO (Tago, Burlingame, CA) (1:100; not crossreactive with rabbit IgO) followed by avidin horseradish peroxidase (5 mg/ml; Sigma Co., St. Louis, MO) diluted
1:3000. Following development by addition of phosphate citrate buffer containing 0.04% O-phenylenediamine and 0.012% H202, pH 5.0, the reaction was stopped with 4 N HCI, and the optical density (OD) was read at 490 nm (reference, 3 nm). Six wells of each experimental condition were routinely used. Northern blot analysis. Cultured cells were washed twice with PBS, and total cellular RNA was isolated by the acid guanidine:phenol:chloroform method I. RNA was separated on agarose-formaldehyde gels, transferred to nylon membranes (Nytran, Schleicher and Schuell, Keene, NH) and hybridized with probes for HLA-DRa and human p-actin sequentially as previously described 2. The DRa probe was a 600 bp 3" Pst I fragment of cDNA clone DRa-10 x6 (generously provided by Dr. E. Long, NIH). The actin probe was a 1.9 kb Eco RI-Hind III fragment of human/~-actin (a gift from Dr. Herbert Cooper, NIH). The blots were analyzed by either auto-radiography or signal quantitation on an Ambis Radioanalytic Imaging System (Ambis, San Diego, CA) using a 1.6 x 3.2 x 0.5 mm resolution plate. All DRa signals
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10 0 101 102 10 3 10 4 10 s Fluorescence Intensity Fig. I. Fluorescence microscopy of cultured human cerebral vascular endothelial cells(EC) incubated in the presence of I00 U/ml human interferon-gamma (IFN},) and stained with antibody to Factor Vlll-relatedantigen (A) or antibody to H L A - D R antigen (B); Bar = 2 ~m, C: fluorescenceflow cytometric analysisof the H L A - D R antibody binding to EC, The binding of H L A - D Q (top panel) and H L A - D R (bottom panel) is demonstrated as histograms plottingcell number (X-axis) vs fluorescence intensity(Y-axis), Identical cultures treated with
secondary antibody alone are indicated by dotted lines.
327 quantitated on the Imaging System were corrected for lane to lane differences in the amounts of R N A present by determining the relative intensities of the signal obLained with the actin probe and dividing this factor into the number of counts obtained using the D R a probe. Confluent cultures examined by light microscopy exhibited the 'cobblestone' appearance characteristic of EC cultures. Greater than 95% of the cells in culture were positively stained for yon WiUebrand Factor VIII antigen (Fig. 1A). Less than 2% of initially passaged cells expressed the astrocyte-specific marker, GFAP. In latepassaged cell cultures, no GFAP-positive cells were detected. These cells were obtained from the fifth passage of cultured cells obtained from one brain tissue specimen. In all passages tested and in identical cultures obtained from other brain biopsy tissue, no constitutive expression of class II MHC antigen was observed. Incubation of EC cultures for 4 days in the presence of human IFN), resulted in expression of class II MHC antigens in all human cerebral EC cultures (Fig. 1B). In addition to the positive staining observed with HLADR-specific antibody, all cultures also stained positively for HLA-DP antigen. Little to no staining was observed with antibody to H L A - D Q antigen, which positively stained B cells (results not shown). Analysis of IFNy-treated (100 U/ml, 4 days) cells with FACS revealed that as much as 40% of the EC population expressed H L A - D R antigen (Fig. 1C). In FACS analysis of 3 separate EC populations, an average of 40.7% .4. 4.5% of the EC expressed HLA-DR antigen. The mean fluorescence intensity of the H L A - D R antigen staining in these experiments was 139.7 ± 36.5.
To quantitate the degree of class II MHC antigen staining by EC, an ELISA was performed using HLADR, -DP and -DQ specific antibodies (Fig. 2). In all experiments, the magnitude of staining was greatest for H L A - D R > HLA-DP > > HLA-DQ. Very little HLA-DQ antigen was detected in any of the EC cultures, confirming the results obtained by examination with fluorescent microscopy (results not shown). The extent of antigen expression was proportional to the concentration of exogenous IFN), with maximum staining at 100 U/ml IFN),. No class II MHC antigen expression was detected on EC incubated in the presence of < 10 U/ml of IFN~, (data not shown). Although class II MHC antigen could be detected after culture with IFN), for 48 or 72 h, maximal expression was seen after 4 days in culture. IFNT-induced expression of HLA-DR antigen on the surfaces of cerebral vascular EC was also reflected at the RNA level. Total cellular R N A extracted from EC incubated in the absence or presence of IFN7 for two days was examined by Northern blot analysis using probes for H L A - D R a and actin (Fig. 3). In all experiments, signals were quantitated using a radioanalytic imaging system and normalized to the actin signal. The results indicate that IFNT-treatment effectively increased class II mRNA levels in all EC cultures tested. The results described here provide the first demonstration of class II MHC antigen on pure cultures of cerebral vascular EC isolated from human brain. The ability to express class II MHC antigens implicates these cells as potential participants in diseases characterized by immune-mediated reactions which ultimately alter the
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Fig. 2. Cell surface expression of HLA-DR and DP molecules as measured by ELISA. EC were cultured in the presence of indicated concentrations of recombinant human IFNy for 4 days at 37 °C. The data represent the HLA-DR or -DP specific OD value of each experimental condition minus the HLA-DR or -DP specific OD value obtained in the absence of IFNT. Background OD values (no primary antibody) were similar in all experiments (0.113 "" 0.24) and were subtracted from all data.
Fig. 3. Effect of IFNy on levels of HLA.DRa transcripts. A: total cellular RNA was isolated from microvessel EC that were either untreated (UnRx) or treated with IFNT,(100 U/ml) (IFN) for 2 days in two separate experiments, and analyzed by Northern blotting with a probe for HLA-DRa (top) and fl-actin (bottom). Results from two separate experiments are shown. B: results from A. quantitated by means of an Ambis Radioanalytic Imaging System, and normalized to actin counts as described in the text.
328 permeability of the BBB (i.e., autoimmune disorders such as MS). Class II MHC-positive EC could function as accessory cells and present antigen at the site of the BBB to antigen-specific T lymphocytes. This interaction could lead to the elaboration and release of both lymphokines and endothelium-derived factors (endokines) which may affect BBB permeability by either opening tight junctions or increasing endothelial transport mechanisms. The fact that encephalitogenic T cells induce permeability changes in la.positive but not la-negative EC provides additional evidence for the potential role of class II MHC-positive EC in pathogenic mechanisms t°. T cell-mediated alterations in permeability of cerebral vascular EC at the BBB could permit the localized infiltration of peripheral blood components into the CNS. Further evidence implicating class II MHC antigens in
pathogenic mechanisms includes observations in the E A E model system. Cerebral capillary EC from mice with E A E express la antigen, but EC from normal mice are Ia-negative 11'22. There is also evidence that la antigen expression in E A E precedes extravasation of lymphocytes into the CNS 14'm. Interestingly, H L A - D R antigen expression has been shown in situ on human EC and is believed to be associated with the development of MS lesions 23. Understanding the mechanism of class II MHC antigen expression on EC and its modulation may enlighten our knowledge on the influence of this antigen and these cells on BBB permeability and their role in neuroimmunological disorders.
1 Chomczynski, P. and Sacchi, N., Single step method of RNA isolation by acid 8uanidinium thiocyanate-phenol-chloroform extraction, Anal. Biochem., 162 (1987) 156-159. 2 Cowan, E.P., Pierce, M.L. and Dhib-Jalbut, S., Interleukin-1/~ decreases HLA class II expression on a glioblastoma multiform cell line, ]. Neuroimmunol., 33 (1991) 17-28. 3 Doyle, C. and Strominger, .I., Interaction between CD4 and Class 11 MHC molecules mediates cell adhesion, Nature, 330 (1987) 256-.259. 4 Oerhart, D.Z., Broderius, M.A. and Drewes, L.R., Cultured human and canine endothelial cells from brain microvessels, Brain Res. Bull., 21 (1988) 785-793. 5 Kato, S. and Nakamura, H., Ultrastructural and ultrachemical studies on the blood.brain barrier in chronic relapsing experi. mental allergic encephalomyelitis, Acta Neuropathoi., 77 (1989) 4~$.-464, 6 Krlstensson, K, and Wlsniewski, H,M,, Chronic relapsing experimental allergic encephalomyelitis: studies in vascular per. meablllty changes, Acta Neuroparhol., 39 (1977) 189-194. 7 Louinsky, A,S., Badmajew, V,, Robson, J.A,, Moretz, R.C, and Wisniewski, H.M., Sites of egress of inflammatorycells and horseradish peroxidase transport across the blood-brain barrier in a murine model of chronic relapsing experimental allergic encephalomyelitis, Acta Neuropathol., 78 (1989) 359-371. 8 Masuyama,J.-l., Minato, N. and Kano, S., Mechanisms of lymphocyte adhesion to human vascular endothelial cells in culture. T Lymphocyte adhesion to endothelial cells through endothelial HLA.DR antigens induced by gamma interferon, 3. Clin. Invest., 77 (1986) 1596--1605. 9 McCarron, R.M., Kempski, O., Spatz, M. and McFarlin, D.E., Presentation of myelin basic protein by murine cerebral vascular endothelial cells, 3. lmmunol., 134 (1985) 3100-3103. 10 McCarron, R.M., Racke, M., Spatz, M. and McFarlin, D.E., Cerebral vascular endothelial cells are effective targets for in vitro lysis by encephalitogenic T lymphocytes,3. immunol., 147 (1991) 503-508. II McCarron, R.M., Spatz, M., Kempski, O., Hogan, R.N., Muehl, L. and McFarlin, D.E., Interaction between myelin basic protein-sensitized T lymphocytes and murine cerebral vascular endothelial cells, 3. lmmunol., 137 (1986) 3428-3435. 12 McDonald, W.I. and Barnes, D., Lessons from magnetic resonance imaging in multiple sclerosis, Trends Neurosci., 12 (1989) 376-379. 13 Risau, W., Engelhardt, B. and Wekerle, H., Immune function of the blood-brain barrier: incomplete presentation of protein (auto-)antigens by rat brain microvaseularendothelium in vitro,
J. Cell. Biol., 110 (1990) 1757-1766. 14 Sakai, T., Tabira, T., Endoh, M. and Steinman, L., la expression in chronic relapsing experimental allergic encephalomyelitis induced by long-term T cell lines in mice, Lab. Invest., 54 (1986) 345-352. 15 Sedgwick, J.D., Hughes, C.C., Male, D.K., MacPhee, I.A.M. and ter Meulen, V., Antigen-specific damage to brain vascular endothelial cells mediated by encephalitogenic and nonencephalitogenic CD4 + T cell lines in vitro, 7. lmmunol., 145 (1990) 2474-2481. 16 Sekaly, R.P., Tonnelle, C., Strubin, M., Mach, B. and Long, E.O., Cell surface expression of class I1 histocompatibility antigens occurs in the absence of the invariant chain, J. EXp. Mad,, 164 (1986) 1490-1504. 17 Simmons, R,D,, Buzbee, T.M., Linthicum, D.C., Mandy, W,J,, Chen, O, and Wang, C., Simultaneous visualization of vascular permeability change and leukocyte egress in the central nervous system during autoimmune encephalomyelitis, Acta Neuro. pathol., 74 (1987) 191-193. 18 Sobel, R.A., Blanchette, B.W., Bhan, A.K, and Colvin, R,B,, The immunopathology of experimental allergic encephalomyelitis. If. Endothelial cell Ia increase prior to inflammatory cell infiltration, 7. lmmunol., 132 (1984) 2402-2407. 19 Sternberger, N.H., Stemberger, L.A., Kies, M.W. and Shear, C:R., Cell surface endothelial proteins altered in experimental allergic encephalomyelitis, 7. Neurolmmunol., 21 (1989) 241-248. 20 Tanaka, M. and McCarron, R.M., The inhibitory effect of tumor necrosis factor and interleukin-1 on la induction by interferon-y on endothelial cells from routine central nervous system microvessels, 7. Neuroimmunol., 27 (1990) 209-215. 21 Theofilpoulis, A.N., Dixon, F.J. and Bokisch, V.A., Binding of soluble immune complexes to human lymphoblastoid cells. I. Characterizatton of receptors for IgG 1:'6and complement and description of the binding mechanism, 7. Exp. Mad., 140 (1974) 877-894. 22 Traugott, U., Ralne, C.S. and McFarlin, D.E., Acute experimental allergic encephalomyelitis in the mouse: immunopathologyof the developing lesion, Ca//. lmmunol., 91 (1985) 240-.254. 23 Traugott, U., Scheinber8, L.C. and Raine, C.S., On the presence of la-positive endothelial cells and astrocytes in multiple sclerosis lesions and its relevance to antigen presentation, 7. Neuroimmunol., 8 (1985) 1-14. 24 Wilcox, C.E., Healey, D.G., Baker, D., Willoughby, D.A. and Turk, J.L., Presentation of myelinbasic protein by normal guineapig brain endothelial cells and its relevance to experimental after8ic encephalomyelitis, 7. lmmunol., 67 (1989) 435--440.
The authors thank Devera G. Schoenberg, M.S. for careful preparation of this manuscript.
325
Elsevier
BRES 24946
Class II MHC antigen expression by cultured human cerebral vascular endothelial cells* R i c h a r d M . M c C a r r o n 1, L a n W a n g 1, E l l i o t P. C o w a n 2 a n d M a r i a S p a t z 1 SStroke Branch and 2Neuroimmunology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892 (U.S.A.)
(Accepted 27 August 1991) Key words: Endothelium; Brain; Class II MHC; Blood-brain barrier; HLA-DR
Cerebral vascular endothelial cells (EC) isolated from human brain do not constitutivelyexpress class II MHC antigens. However, incubation in the presence of human interferon-gamma(IFNy) resulted in the expression of both HLA-DR and -DP antigens. FACS analysis revealed that approximately40% of the EC population expressed HLA-DR antigen. Ouantitation by ELISA demonstrated that maximum expression was observed with 100 Ulml IFN~ for 4 days. Treatment with IFN~ also increased class II mRNA levels in all EC cultures tested.
The cerebral capillary endothelial cell (EC) is strategically located to play a critical role(s) in regulating peripheral blood cell trafficking into the central nervous system (CNS). The cerebral microvascular endothelium differs from other endothelium in several respects, e.g., they form tight continuous junctions, have fewer vesicles, and also possess a variety of highly specific transport mechanisms. These characteristics are at least partially responsible for the lack or limited amount of lymphocytes trafficking into the CNS, and account for the term blood-brain barrier (BBB). Numerous studies have identified alterations in the BBB permeability in CNS diseases such as multiple sclerosis (MS) and the model disease experimental allergic encephalomyelitis (EAE) s'7,t2'17'19. The mechanism(s) by which these alterations in the BBB occur allowing the entry (extravasation) of inflammatory cells into the brain parenchyma are not known. One factor which plays a well.documented role in interactions with immune cells is the class II MHC (la) molecule. The expression of these molecules on EC has implicated these cells in a variety of phenomena including antigen presentation 9'11'24, lymphocyte adhesion 3's and cytolytic T cell interactions I°'13'15. It is shown here that cerebral capillary EC isolated from human brain do not constitutively express class II MHC (HLA-DR, -DP, -DO) antigens. However, these cells can be induced to express both HLA-DR and HLA-DP antigens by in vitro culture with recombinant human interferon-gamma
(IFNy). This is the first demonstration of class II MHC antigen expression by cultured human cerebral vascular EC, and indicates a possible mechanism by which these cells interact with immune cells at the site of the BBB. E C culture. EC were prepared from small samples of human brain tissue surgically removed for treatment of idiopathic epilepsy. Microvessels were isolated by mechanical dispersion (homogenization) and filtration techniques similar to the method by Gerhard et al. 4. Cells were passaged by trypsinization and were utilized between passages 4 and 12. EC purity was assessed with antibody to human factor VIII-related antigen, and contamination by astrocytes measured with antibody to glial fibrillary acidic protein (GFAP). EC were cultured on gelatin-coated coverslips, fixed (3.7% formalin at room temperature for I0 min), permeabilized (0.1% Triton X.100, room temperature for 5 rain), and stained with rabbit anti-human factor VIII-related antigen (I :50) (Accurate Chemical and Scientific Corporation, Burlingame, CA) or rabbit anti-GFAP (Accurate) (1:200) in a humidified chamber for 45-60 rain at room temperature. After washing, secondary antibody [rhodamine-conjugated goat F(ab')2anti-rabbit IgG (H + L) (1:200)] was added at room temperature for 30-45 min. Staining for HLA-DR, -DP and -DQ antigens was performed with mouse antibodies to HLA-DR antigen (clone L243; 1:50), HLA-DP antigen (clone B7/21, 1:50) and HLA-DQ antigen (clone SK10, 1:25) (Becton Dickinson; Mountain View, CA). Cells were fixed as described above, but were not per-
* Presented in part at the American Associationof Immunologists/AmericanSociety for Biochemistryand Molecular BiologyJoint Meeting, New Orleans, LA, June 7, 1990. Correspondence: R.M. McCarron, National Institutes of Health, 9000 RockvillePike, Building36, Room 4D04 Bethesda, MD 20892, U.S.A.
326 meabilized. Single cell suspensions of EC populations were stained as described above and analyzed by flow cytometry on a FACS IV analyzer (Becton-Dickinson FACS Systems). ELISA. Cell surface expression of HLA-DR molecules was measured by ELISA essentially as previously described 2°. EC (2 × 104 cells/0.1 ml/well in 0.3 cm2 well (pre-coated with type I collagen; Collaborative Research, Bedford, MA) were incubated for 24-96 h at 37 °C in a 5.0% CO2 atmosphere in the absence or presence of indicated concentrations of recombinant human IFN7 (Interferon Sciences, Rockviile, MD). Nonspecific binding sites of cells were blocked by the addition of ~00/~1 phosphate buffered saline (PBS) containing 2% bovine serum albumin and 50/~g aggregated rabbit IgO, prepared by heating monomeric IgO at 63 °C for 20 min21. After incubation with anti-HLA-DR or anti-HLA-DP antibody (1:1000), cells were treated with biotin-conjugated antimurine IgO (Tago, Burlingame, CA) (1:100; not crossreactive with rabbit IgO) followed by avidin horseradish peroxidase (5 mg/ml; Sigma Co., St. Louis, MO) diluted
1:3000. Following development by addition of phosphate citrate buffer containing 0.04% O-phenylenediamine and 0.012% H202, pH 5.0, the reaction was stopped with 4 N HCI, and the optical density (OD) was read at 490 nm (reference, 3 nm). Six wells of each experimental condition were routinely used. Northern blot analysis. Cultured cells were washed twice with PBS, and total cellular RNA was isolated by the acid guanidine:phenol:chloroform method I. RNA was separated on agarose-formaldehyde gels, transferred to nylon membranes (Nytran, Schleicher and Schuell, Keene, NH) and hybridized with probes for HLA-DRa and human p-actin sequentially as previously described 2. The DRa probe was a 600 bp 3" Pst I fragment of cDNA clone DRa-10 x6 (generously provided by Dr. E. Long, NIH). The actin probe was a 1.9 kb Eco RI-Hind III fragment of human/~-actin (a gift from Dr. Herbert Cooper, NIH). The blots were analyzed by either auto-radiography or signal quantitation on an Ambis Radioanalytic Imaging System (Ambis, San Diego, CA) using a 1.6 x 3.2 x 0.5 mm resolution plate. All DRa signals
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10 0 101 102 10 3 10 4 10 s Fluorescence Intensity Fig. I. Fluorescence microscopy of cultured human cerebral vascular endothelial cells(EC) incubated in the presence of I00 U/ml human interferon-gamma (IFN},) and stained with antibody to Factor Vlll-relatedantigen (A) or antibody to H L A - D R antigen (B); Bar = 2 ~m, C: fluorescenceflow cytometric analysisof the H L A - D R antibody binding to EC, The binding of H L A - D Q (top panel) and H L A - D R (bottom panel) is demonstrated as histograms plottingcell number (X-axis) vs fluorescence intensity(Y-axis), Identical cultures treated with
secondary antibody alone are indicated by dotted lines.
327 quantitated on the Imaging System were corrected for lane to lane differences in the amounts of R N A present by determining the relative intensities of the signal obLained with the actin probe and dividing this factor into the number of counts obtained using the D R a probe. Confluent cultures examined by light microscopy exhibited the 'cobblestone' appearance characteristic of EC cultures. Greater than 95% of the cells in culture were positively stained for yon WiUebrand Factor VIII antigen (Fig. 1A). Less than 2% of initially passaged cells expressed the astrocyte-specific marker, GFAP. In latepassaged cell cultures, no GFAP-positive cells were detected. These cells were obtained from the fifth passage of cultured cells obtained from one brain tissue specimen. In all passages tested and in identical cultures obtained from other brain biopsy tissue, no constitutive expression of class II MHC antigen was observed. Incubation of EC cultures for 4 days in the presence of human IFN), resulted in expression of class II MHC antigens in all human cerebral EC cultures (Fig. 1B). In addition to the positive staining observed with HLADR-specific antibody, all cultures also stained positively for HLA-DP antigen. Little to no staining was observed with antibody to H L A - D Q antigen, which positively stained B cells (results not shown). Analysis of IFNy-treated (100 U/ml, 4 days) cells with FACS revealed that as much as 40% of the EC population expressed H L A - D R antigen (Fig. 1C). In FACS analysis of 3 separate EC populations, an average of 40.7% .4. 4.5% of the EC expressed HLA-DR antigen. The mean fluorescence intensity of the H L A - D R antigen staining in these experiments was 139.7 ± 36.5.
To quantitate the degree of class II MHC antigen staining by EC, an ELISA was performed using HLADR, -DP and -DQ specific antibodies (Fig. 2). In all experiments, the magnitude of staining was greatest for H L A - D R > HLA-DP > > HLA-DQ. Very little HLA-DQ antigen was detected in any of the EC cultures, confirming the results obtained by examination with fluorescent microscopy (results not shown). The extent of antigen expression was proportional to the concentration of exogenous IFN), with maximum staining at 100 U/ml IFN),. No class II MHC antigen expression was detected on EC incubated in the presence of < 10 U/ml of IFN~, (data not shown). Although class II MHC antigen could be detected after culture with IFN), for 48 or 72 h, maximal expression was seen after 4 days in culture. IFNT-induced expression of HLA-DR antigen on the surfaces of cerebral vascular EC was also reflected at the RNA level. Total cellular R N A extracted from EC incubated in the absence or presence of IFN7 for two days was examined by Northern blot analysis using probes for H L A - D R a and actin (Fig. 3). In all experiments, signals were quantitated using a radioanalytic imaging system and normalized to the actin signal. The results indicate that IFNT-treatment effectively increased class II mRNA levels in all EC cultures tested. The results described here provide the first demonstration of class II MHC antigen on pure cultures of cerebral vascular EC isolated from human brain. The ability to express class II MHC antigens implicates these cells as potential participants in diseases characterized by immune-mediated reactions which ultimately alter the
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Fig. 2. Cell surface expression of HLA-DR and DP molecules as measured by ELISA. EC were cultured in the presence of indicated concentrations of recombinant human IFNy for 4 days at 37 °C. The data represent the HLA-DR or -DP specific OD value of each experimental condition minus the HLA-DR or -DP specific OD value obtained in the absence of IFNT. Background OD values (no primary antibody) were similar in all experiments (0.113 "" 0.24) and were subtracted from all data.
Fig. 3. Effect of IFNy on levels of HLA.DRa transcripts. A: total cellular RNA was isolated from microvessel EC that were either untreated (UnRx) or treated with IFNT,(100 U/ml) (IFN) for 2 days in two separate experiments, and analyzed by Northern blotting with a probe for HLA-DRa (top) and fl-actin (bottom). Results from two separate experiments are shown. B: results from A. quantitated by means of an Ambis Radioanalytic Imaging System, and normalized to actin counts as described in the text.
328 permeability of the BBB (i.e., autoimmune disorders such as MS). Class II MHC-positive EC could function as accessory cells and present antigen at the site of the BBB to antigen-specific T lymphocytes. This interaction could lead to the elaboration and release of both lymphokines and endothelium-derived factors (endokines) which may affect BBB permeability by either opening tight junctions or increasing endothelial transport mechanisms. The fact that encephalitogenic T cells induce permeability changes in la.positive but not la-negative EC provides additional evidence for the potential role of class II MHC-positive EC in pathogenic mechanisms t°. T cell-mediated alterations in permeability of cerebral vascular EC at the BBB could permit the localized infiltration of peripheral blood components into the CNS. Further evidence implicating class II MHC antigens in
pathogenic mechanisms includes observations in the E A E model system. Cerebral capillary EC from mice with E A E express la antigen, but EC from normal mice are Ia-negative 11'22. There is also evidence that la antigen expression in E A E precedes extravasation of lymphocytes into the CNS 14'm. Interestingly, H L A - D R antigen expression has been shown in situ on human EC and is believed to be associated with the development of MS lesions 23. Understanding the mechanism of class II MHC antigen expression on EC and its modulation may enlighten our knowledge on the influence of this antigen and these cells on BBB permeability and their role in neuroimmunological disorders.
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The authors thank Devera G. Schoenberg, M.S. for careful preparation of this manuscript.
