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[3H]dihydroalprenolol binding to beta adrenergic receptors in multiple sclerosis brain
Neuroscience Letters 289 (2000) 75±77
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[ 3H]dihydroalprenolol binding to beta adrenergic receptors in multiple sclerosis brain Esther Zeinstra, Nadine Wilczak, Jacques De Keyser* Department of Neurology, Academisch Ziekenhuis Groningen, PO Box 30.001, 9700 RB Groningen, The Netherlands Received 3 May 2000; received in revised form 24 May 2000; accepted 8 June 2000
Abstract By using immunocytochemistry we previously reported the absence of b2 adrenergic receptors on astrocytes in multiple sclerosis (MS) white matter. Here, we measured b1 and b2 adrenergic receptor concentrations in postmortem brain sections of six MS patients and six controls by using quantitative autoradiography with [ 3H]dihydroalprenolol. White matter contained no b1 adrenergic receptors. In white matter of controls low levels of b2 adrenergic receptors were detected. In agreement with the immunohistochemical study, we were unable to detect b2 adrenergic receptors in both normal appearing white matter and astrogliotic plaques in MS. Concentrations of b1 and b2 adrenergic receptors in cerebral cortex were not different between controls and MS patients. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Multiple sclerosis; b2 Adrenergic receptors; Autoradiography; Plaques; Neurons; [ 3H]dihydroalprenolol
Astrocytes in optic nerve, and white matter of brain and spinal cord normally express b2 adrenergic receptors [7]. Expression usually increases after either acute or chronic injury [2,7,11]. By using immunohistochemistry we found that astrocytes in plaques and normal appearing white matter in brains of multiple sclerosis (MS) patients lack b2 adrenergic receptors [2]. An epiphenomenon is unlikely because the abnormality occurred on resting astrocytes in normal appearing MS white matter, and reactive astrocytes at the boundary of brain infarctions, which also display an in¯ammatory response, showed enhanced b2 adrenergic receptor expression [2]. The abnormality is likely to play an important role in the pathophysiology of MS because b2 adrenergic receptors are involved in suppressing the inducibility of major histocompatibility (MHC) class II molecules on astrocytes [3,5]. This suppressive mechanism explains why MHC class II expression is much more dif®cult to induce in vivo on astrocytes than on microglia [5]. The absence of astrocytic b2 adrenergic receptors may provide a good explanation why astrocytes in chronic active MS plaques express MHC class II [6,8,12,13]. The MHC class II molecules are required for presenting myelin antigen to anti-myelin T cells, which then become activated and are * Corresponding author. Tel.: 131-50-361-2430; fax: 131-50361-1707. E-mail address: [email protected] (J. De Keyser).
capable to initiate an autoimmune attack [1,5,10]. It has recently been shown that astrocytes have potential for processing and presentation of myelin antigen to proin¯ammatory T cells as well as to activate T cells [9]. The question arises whether neuronal b2 adrenergic receptors are also affected in MS. Immunostaining of MS brain sections revealed that b2 adrenergic receptors were present on neurons in cerebral cortex [2]. However, immunohistochemistry is not a suitable method to measure receptor concentrations. To quantify b1 and b2 adrenergic receptors in MS brain sections we performed radioligand binding experiments with [ 3H]dihydroalprenolol (DHA) [4]. Samples of cerebral cortex were obtained at autopsy from six patients with neuropathologically con®rmed MS (age 60 ^ 9 years; postmortem delay 8 ^ 2 h) with a disease duration of 30 ^ 8 years and six patients (age 63 ^ 13 years; postmortem delay 8 ^ 2 h) without evidence of neurologic disease (controls). Tissue samples were rapidly frozen in ethane-dry ice or liquid nitrogen and stored at 2808C until used. Cryostat sections of 20 mM thickness were thaw-mounted on gelatin-coated microscopic glass slides and dried overnight. Some sections were also stained with haematoxylin and eosin (H & E), luxol fast blue (myelin staining), and monoclonal antibodies to glial ®brillary acidic protein (GFAP; Innogenetics, Ghent, Belgium), which is a speci®c marker for astrocytes. In the MS brain sections twelve astrogliotic plaques were identi®ed.
0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 0) 01 25 4- 4
76
E. Zeinstra et al. / Neuroscience Letters 289 (2000) 75±77
Fig. 1. [ 3H]dihydroalprenolol competition binding with the selective b2 blocker ICI118551 in sections of frontal cortex. The curve displays two binding sites representing binding of the radioligand to b2 (high af®nity; arrow) and b1 (low af®nity) adrenergic receptors.
Binding experiments with [ 3H]DHA (speci®c activity 1.0 mCi/ml; Amersham, Buckinghamshire, UK), which labels both b1 and b2 adrenergic receptors [4], were done in duplicate. The slides were preincubated for 10 min at 208C with 75 mM Tris (pH 7.4) buffer containing 25 mM MgCl2, and subsequently incubated with 1.5 nM [ 3H]DHA for 60 min at 208C. Eight concentrations between 0.01 and 5 nM of [ 3H]DHA were used in saturation binding experiments, and 1.5 nM [ 3H]DHA was used for competition binding experiments. Incubations were terminated by the addition of ice-cold buffer. The tissues were wiped from the slides with Whatman GF/B glass ®ber ®lters and placed in scintillation vials with 5 ml scintillation ¯uid (Ready Safe,
Beckman, High Wycombe, UK), and radioactivity was measured in a liquid scintillation counter. Data from saturation and competition binding experiments were analyzed with a computerized non-linear ®tting program for multiple binding sites. The binding of [ 3H]DHA to cerebral cortex sections was saturable and Scatchard analysis revealed a KD value of 1.9 ^ 0.2 nM (not shown). [ 3H]DHA competition curves with the selective b2 blocker ICI118551 (TBI, USA) were biphasic, re¯ecting the presence of b2 receptors (Ki 1.2 ^ 0.2 nM) and b1 receptors (Ki 5.7 ^ 0.3 mM) (Fig. 1). At a concentration of 0.5 mM of ICI118551 all high af®nity sites, representing binding to b2 adrenergic receptors, were displaced. For the generation of autoradiographs, consecutive frozen brain slices were preincubated and subsequently incubated with 1.5 nM [ 3H]DHA as described above. After incubation, the sections were washed three times for 1 min each in the same buffer to remove unbound ligand, and then quickly dipped in distilled water. The sections were dried under a stream of cold air, placed in X-ray cassettes together with commercially available [ 125I] standards (Amersham), and exposed to [ 3H]Ultro®lm (Amersham, USA) for 6 weeks. The ®lms were developed with a D19 Kodak developer at 48C, and after drying they were scanned using an ARCUS plus scanner. The digitized images were analyzed and quanti®ed by computer-assisted densitometry using the program Image (National Institutes of Health Research Services Branch). A calibration curve was generated by ®tting of optical density and disintegrations per minute per milligram polymer values of the [ 125I] standards. The regions of interest were sampled and mean optical densities determined, and converted into fmol/mg protein, based on the experimentally determined relation between polymer and brain paste standards. Speci®c binding to b1 and b2 adrenergic receptors was obtained by subtracting non-speci®c binding, determined in the presence of 50 mM of the non-selective b
Fig. 2. b1 and b2 adrenergic receptor autoradiography. Consecutive sections of frontal lobe tissue from a patient with MS showing [ 3H]dihydroalprenolol total binding (a), binding in the presence of 0.5 mM of the selective b2 blocker ICI118551 (b), and non-speci®c binding in the presence of 50 mM propranolol (c). Note the absence of b1 and b2 adrenergic receptors in the astrogliotic plaque (pl).
E. Zeinstra et al. / Neuroscience Letters 289 (2000) 75±77 Table 1 b1 and b2 adrenergic receptor densities in cerebral cortex of MS patients and controls a
b1 receptors Outer cortical layers Inner cortical layers White matter Astrogliotic plaques b2 receptors Outer cortical layers Inner cortical layers White matter Astrogliotic plaques
Controls, n 6
MS patients, n 6
49.1 ^ 3.4 18.8 ^ 1.9 ND ±
45.9 ^ 4.3* 21.3 ^ 2.1* ND ND
33.8 ^ 1.7 18.6 ^ 1.3 2.3 ^ 0.8 ±
32.8 ^ 1.7* 20.8 ^ 2.3* ND ND
a Receptor densities are expressed in fmol/mg protein; values are means ^ SEM. ND, not detectable, * not signi®cantly different (Mann±Whitney test).
blocker propranolol (Sigma, St. Louis), from total binding. Binding to b2 adrenergic receptors was obtained by subtracting binding in the presence of 0.5 mM of ICI118551 from total binding. A representative autoradiograph is shown in Fig. 2. Quantitative analysis indicated that b1 and b2 adrenergic receptor densities in cerebral cortex of MS patients were not different from those of controls (Table 1). In white matter of controls we could detect very low levels of b2 adrenergic receptors. However, in accordance with our immunohistochemical ®ndings [2], we found that b2 adrenergic receptors were absent both in normal appearing white matter and astrogliotic plaques in MS. White matter of controls and MS patients contained no b1 adrenergic receptors. Immunohistochemically, b2 adrenergic receptors in cerebral cortex were found to be located on neuronal cell bodies [2]. Therefore, our results indicate that neuronal b2 adrenergic receptor concentrations in MS are not different from controls. Further research should concentrate on the mechanism by which these receptors disappear on astrocytes in MS white matter. Brain specimens were obtained from the National Neurological Research Specimen Bank (West Los Angeles, CA 90073; sponsored by NINDS/NIMH, National Multiple Sclerosis Society and Veterans Health Services and Research Administration of the Department of Veterans Affairs). We thank R. Leta for his help in performing the autoradiographs. This work was supported in part by a grant
77
of Van Lieshout Beheer and the `Beleidsruimte Onderzoek' of the Academic Hospital/Faculty of Medical Sciences of the University of Groningen (project 754501).
[1] Aschner, A., Astrocytes as mediators of immune and in¯ammatory responses in the CNS, Neurotoxicology, 19 (1998) 269±282. [2] De Keyser, J., Wilczak, N., Leta, R. and Stree¯and, C., Astrocytes in multiple sclerosis lack beta-2 adrenergic receptors, Neurology, 53 (1999) 1628±1633. [3] De Paermentier, F., Cheetham, S.C., Crompston, M.R. and Horton, R.W., Beta-adrenoreceptors in human brain labelled with [ 3H]dihydroalprenolol and [ 3H]CGP12177, Eur. J. Pharmacol, 167 (1989) 397±405. [4] Frohman, E.M., Vayuvegula, B., Gupta, S. and van den Noort, S., Norepinephrine inhibits gamma-interferoninduced major histocompatibility class II (Ia) antigen expression on cultured astrocytes via beta-2-adrenergic signal transduction mechanisms, Proc. Natl. Acad. Sci. USA, 85 (1988) 1292±1296. [5] Lee, S.H. and Brosnan, C.F., Molecular biology of glia: astrocytes, In W.C. Russell (Ed.), Molecular Biology of Multiple Sclerosis, Wiley, London, 1997, pp. 71±96. [6] Lee, L.C., Moore, G.R.W., Golenwsky, G. and Raine, C.S., Multiple sclerosis: a role for astroglia in active demyelination suggested by class II MHC expression and ultrastructural study, J. Neuropathol. Exp. Neurol., 49 (1990) 122±136. [7] Mantyh, P.W., Rogers, S.D., Allen, C.J., Catton, M.D., Ghilardi, J.R., Levin, L.A., Maggio, J.E. and Vigna, S.R., b2-adrenergic receptors are expressed by glia in vivo in the normal and injured central nervous system in the rat, rabbit, and human, J. Neurosci., 15 (1995) 152±164. [8] Ransohoff, R.M. and Estes, M.L., Astrocyte expression of major histocompatibility complex gene products in multiple sclerosis brain tissue obtained by stereotactic biopsy, Arch. Neurol., 48 (1991) 1244±1246. [9] Soos, J.M., Morrow, J., Ashley, T.A., Szente, B.E., Bikoff, E.K. and Zamvil, S.S., Astrocytes express elements of the class II endocytic pathway and process central nervous system autoantigen for presentation to encephalitogenic T cells, J. Immunol., 161 (1998) 5959±5966. [10] Storch, M. and Lassmann, H., Pathology and pathogenesis of demyelinating diseases, Curr. Opin. Neurol., 10 (1997) 186±192. [11] Sultin, J. and Shao, Y., Resting and reactive astrocytes express adrenergic receptors in the adult rat brain, Brain Res. Bull., 29 (1992) 277±284. [12] Traugott, U. and Lebon, P., Interferon-gamma and Ia antigen are present on astrocytes in active chronic multiple sclerosis lesions, J. Neurol. Sci., 84 (1988) 257±264. [13] Zeinstra, E., Wilczak, N., Stree¯and, C. and De Keyser, J., Astrocytes in multiple sclerosis express major histocompatibility class II molecules, NeuroReport, 11 (2000) 89±91.
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[ 3H]dihydroalprenolol binding to beta adrenergic receptors in multiple sclerosis brain Esther Zeinstra, Nadine Wilczak, Jacques De Keyser* Department of Neurology, Academisch Ziekenhuis Groningen, PO Box 30.001, 9700 RB Groningen, The Netherlands Received 3 May 2000; received in revised form 24 May 2000; accepted 8 June 2000
Abstract By using immunocytochemistry we previously reported the absence of b2 adrenergic receptors on astrocytes in multiple sclerosis (MS) white matter. Here, we measured b1 and b2 adrenergic receptor concentrations in postmortem brain sections of six MS patients and six controls by using quantitative autoradiography with [ 3H]dihydroalprenolol. White matter contained no b1 adrenergic receptors. In white matter of controls low levels of b2 adrenergic receptors were detected. In agreement with the immunohistochemical study, we were unable to detect b2 adrenergic receptors in both normal appearing white matter and astrogliotic plaques in MS. Concentrations of b1 and b2 adrenergic receptors in cerebral cortex were not different between controls and MS patients. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Multiple sclerosis; b2 Adrenergic receptors; Autoradiography; Plaques; Neurons; [ 3H]dihydroalprenolol
Astrocytes in optic nerve, and white matter of brain and spinal cord normally express b2 adrenergic receptors [7]. Expression usually increases after either acute or chronic injury [2,7,11]. By using immunohistochemistry we found that astrocytes in plaques and normal appearing white matter in brains of multiple sclerosis (MS) patients lack b2 adrenergic receptors [2]. An epiphenomenon is unlikely because the abnormality occurred on resting astrocytes in normal appearing MS white matter, and reactive astrocytes at the boundary of brain infarctions, which also display an in¯ammatory response, showed enhanced b2 adrenergic receptor expression [2]. The abnormality is likely to play an important role in the pathophysiology of MS because b2 adrenergic receptors are involved in suppressing the inducibility of major histocompatibility (MHC) class II molecules on astrocytes [3,5]. This suppressive mechanism explains why MHC class II expression is much more dif®cult to induce in vivo on astrocytes than on microglia [5]. The absence of astrocytic b2 adrenergic receptors may provide a good explanation why astrocytes in chronic active MS plaques express MHC class II [6,8,12,13]. The MHC class II molecules are required for presenting myelin antigen to anti-myelin T cells, which then become activated and are * Corresponding author. Tel.: 131-50-361-2430; fax: 131-50361-1707. E-mail address: [email protected] (J. De Keyser).
capable to initiate an autoimmune attack [1,5,10]. It has recently been shown that astrocytes have potential for processing and presentation of myelin antigen to proin¯ammatory T cells as well as to activate T cells [9]. The question arises whether neuronal b2 adrenergic receptors are also affected in MS. Immunostaining of MS brain sections revealed that b2 adrenergic receptors were present on neurons in cerebral cortex [2]. However, immunohistochemistry is not a suitable method to measure receptor concentrations. To quantify b1 and b2 adrenergic receptors in MS brain sections we performed radioligand binding experiments with [ 3H]dihydroalprenolol (DHA) [4]. Samples of cerebral cortex were obtained at autopsy from six patients with neuropathologically con®rmed MS (age 60 ^ 9 years; postmortem delay 8 ^ 2 h) with a disease duration of 30 ^ 8 years and six patients (age 63 ^ 13 years; postmortem delay 8 ^ 2 h) without evidence of neurologic disease (controls). Tissue samples were rapidly frozen in ethane-dry ice or liquid nitrogen and stored at 2808C until used. Cryostat sections of 20 mM thickness were thaw-mounted on gelatin-coated microscopic glass slides and dried overnight. Some sections were also stained with haematoxylin and eosin (H & E), luxol fast blue (myelin staining), and monoclonal antibodies to glial ®brillary acidic protein (GFAP; Innogenetics, Ghent, Belgium), which is a speci®c marker for astrocytes. In the MS brain sections twelve astrogliotic plaques were identi®ed.
0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 0) 01 25 4- 4
76
E. Zeinstra et al. / Neuroscience Letters 289 (2000) 75±77
Fig. 1. [ 3H]dihydroalprenolol competition binding with the selective b2 blocker ICI118551 in sections of frontal cortex. The curve displays two binding sites representing binding of the radioligand to b2 (high af®nity; arrow) and b1 (low af®nity) adrenergic receptors.
Binding experiments with [ 3H]DHA (speci®c activity 1.0 mCi/ml; Amersham, Buckinghamshire, UK), which labels both b1 and b2 adrenergic receptors [4], were done in duplicate. The slides were preincubated for 10 min at 208C with 75 mM Tris (pH 7.4) buffer containing 25 mM MgCl2, and subsequently incubated with 1.5 nM [ 3H]DHA for 60 min at 208C. Eight concentrations between 0.01 and 5 nM of [ 3H]DHA were used in saturation binding experiments, and 1.5 nM [ 3H]DHA was used for competition binding experiments. Incubations were terminated by the addition of ice-cold buffer. The tissues were wiped from the slides with Whatman GF/B glass ®ber ®lters and placed in scintillation vials with 5 ml scintillation ¯uid (Ready Safe,
Beckman, High Wycombe, UK), and radioactivity was measured in a liquid scintillation counter. Data from saturation and competition binding experiments were analyzed with a computerized non-linear ®tting program for multiple binding sites. The binding of [ 3H]DHA to cerebral cortex sections was saturable and Scatchard analysis revealed a KD value of 1.9 ^ 0.2 nM (not shown). [ 3H]DHA competition curves with the selective b2 blocker ICI118551 (TBI, USA) were biphasic, re¯ecting the presence of b2 receptors (Ki 1.2 ^ 0.2 nM) and b1 receptors (Ki 5.7 ^ 0.3 mM) (Fig. 1). At a concentration of 0.5 mM of ICI118551 all high af®nity sites, representing binding to b2 adrenergic receptors, were displaced. For the generation of autoradiographs, consecutive frozen brain slices were preincubated and subsequently incubated with 1.5 nM [ 3H]DHA as described above. After incubation, the sections were washed three times for 1 min each in the same buffer to remove unbound ligand, and then quickly dipped in distilled water. The sections were dried under a stream of cold air, placed in X-ray cassettes together with commercially available [ 125I] standards (Amersham), and exposed to [ 3H]Ultro®lm (Amersham, USA) for 6 weeks. The ®lms were developed with a D19 Kodak developer at 48C, and after drying they were scanned using an ARCUS plus scanner. The digitized images were analyzed and quanti®ed by computer-assisted densitometry using the program Image (National Institutes of Health Research Services Branch). A calibration curve was generated by ®tting of optical density and disintegrations per minute per milligram polymer values of the [ 125I] standards. The regions of interest were sampled and mean optical densities determined, and converted into fmol/mg protein, based on the experimentally determined relation between polymer and brain paste standards. Speci®c binding to b1 and b2 adrenergic receptors was obtained by subtracting non-speci®c binding, determined in the presence of 50 mM of the non-selective b
Fig. 2. b1 and b2 adrenergic receptor autoradiography. Consecutive sections of frontal lobe tissue from a patient with MS showing [ 3H]dihydroalprenolol total binding (a), binding in the presence of 0.5 mM of the selective b2 blocker ICI118551 (b), and non-speci®c binding in the presence of 50 mM propranolol (c). Note the absence of b1 and b2 adrenergic receptors in the astrogliotic plaque (pl).
E. Zeinstra et al. / Neuroscience Letters 289 (2000) 75±77 Table 1 b1 and b2 adrenergic receptor densities in cerebral cortex of MS patients and controls a
b1 receptors Outer cortical layers Inner cortical layers White matter Astrogliotic plaques b2 receptors Outer cortical layers Inner cortical layers White matter Astrogliotic plaques
Controls, n 6
MS patients, n 6
49.1 ^ 3.4 18.8 ^ 1.9 ND ±
45.9 ^ 4.3* 21.3 ^ 2.1* ND ND
33.8 ^ 1.7 18.6 ^ 1.3 2.3 ^ 0.8 ±
32.8 ^ 1.7* 20.8 ^ 2.3* ND ND
a Receptor densities are expressed in fmol/mg protein; values are means ^ SEM. ND, not detectable, * not signi®cantly different (Mann±Whitney test).
blocker propranolol (Sigma, St. Louis), from total binding. Binding to b2 adrenergic receptors was obtained by subtracting binding in the presence of 0.5 mM of ICI118551 from total binding. A representative autoradiograph is shown in Fig. 2. Quantitative analysis indicated that b1 and b2 adrenergic receptor densities in cerebral cortex of MS patients were not different from those of controls (Table 1). In white matter of controls we could detect very low levels of b2 adrenergic receptors. However, in accordance with our immunohistochemical ®ndings [2], we found that b2 adrenergic receptors were absent both in normal appearing white matter and astrogliotic plaques in MS. White matter of controls and MS patients contained no b1 adrenergic receptors. Immunohistochemically, b2 adrenergic receptors in cerebral cortex were found to be located on neuronal cell bodies [2]. Therefore, our results indicate that neuronal b2 adrenergic receptor concentrations in MS are not different from controls. Further research should concentrate on the mechanism by which these receptors disappear on astrocytes in MS white matter. Brain specimens were obtained from the National Neurological Research Specimen Bank (West Los Angeles, CA 90073; sponsored by NINDS/NIMH, National Multiple Sclerosis Society and Veterans Health Services and Research Administration of the Department of Veterans Affairs). We thank R. Leta for his help in performing the autoradiographs. This work was supported in part by a grant
77
of Van Lieshout Beheer and the `Beleidsruimte Onderzoek' of the Academic Hospital/Faculty of Medical Sciences of the University of Groningen (project 754501).
[1] Aschner, A., Astrocytes as mediators of immune and in¯ammatory responses in the CNS, Neurotoxicology, 19 (1998) 269±282. [2] De Keyser, J., Wilczak, N., Leta, R. and Stree¯and, C., Astrocytes in multiple sclerosis lack beta-2 adrenergic receptors, Neurology, 53 (1999) 1628±1633. [3] De Paermentier, F., Cheetham, S.C., Crompston, M.R. and Horton, R.W., Beta-adrenoreceptors in human brain labelled with [ 3H]dihydroalprenolol and [ 3H]CGP12177, Eur. J. Pharmacol, 167 (1989) 397±405. [4] Frohman, E.M., Vayuvegula, B., Gupta, S. and van den Noort, S., Norepinephrine inhibits gamma-interferoninduced major histocompatibility class II (Ia) antigen expression on cultured astrocytes via beta-2-adrenergic signal transduction mechanisms, Proc. Natl. Acad. Sci. USA, 85 (1988) 1292±1296. [5] Lee, S.H. and Brosnan, C.F., Molecular biology of glia: astrocytes, In W.C. Russell (Ed.), Molecular Biology of Multiple Sclerosis, Wiley, London, 1997, pp. 71±96. [6] Lee, L.C., Moore, G.R.W., Golenwsky, G. and Raine, C.S., Multiple sclerosis: a role for astroglia in active demyelination suggested by class II MHC expression and ultrastructural study, J. Neuropathol. Exp. Neurol., 49 (1990) 122±136. [7] Mantyh, P.W., Rogers, S.D., Allen, C.J., Catton, M.D., Ghilardi, J.R., Levin, L.A., Maggio, J.E. and Vigna, S.R., b2-adrenergic receptors are expressed by glia in vivo in the normal and injured central nervous system in the rat, rabbit, and human, J. Neurosci., 15 (1995) 152±164. [8] Ransohoff, R.M. and Estes, M.L., Astrocyte expression of major histocompatibility complex gene products in multiple sclerosis brain tissue obtained by stereotactic biopsy, Arch. Neurol., 48 (1991) 1244±1246. [9] Soos, J.M., Morrow, J., Ashley, T.A., Szente, B.E., Bikoff, E.K. and Zamvil, S.S., Astrocytes express elements of the class II endocytic pathway and process central nervous system autoantigen for presentation to encephalitogenic T cells, J. Immunol., 161 (1998) 5959±5966. [10] Storch, M. and Lassmann, H., Pathology and pathogenesis of demyelinating diseases, Curr. Opin. Neurol., 10 (1997) 186±192. [11] Sultin, J. and Shao, Y., Resting and reactive astrocytes express adrenergic receptors in the adult rat brain, Brain Res. Bull., 29 (1992) 277±284. [12] Traugott, U. and Lebon, P., Interferon-gamma and Ia antigen are present on astrocytes in active chronic multiple sclerosis lesions, J. Neurol. Sci., 84 (1988) 257±264. [13] Zeinstra, E., Wilczak, N., Stree¯and, C. and De Keyser, J., Astrocytes in multiple sclerosis express major histocompatibility class II molecules, NeuroReport, 11 (2000) 89±91.