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β-Amyloid-associated expression of intercellular adhesion molecule-1 in brain cortical tissue of transgenic Tg2576 mice
Neuroscience Letters 329 (2002) 111–115 www.elsevier.com/locate/neulet
b-Amyloid-associated expression of intercellular adhesion molecule-1 in brain cortical tissue of transgenic Tg2576 mice Jenny Apelt, Jacqueline Leßig, Reinhard Schliebs* Paul Flechsig Institute for Brain Research, Department of Neurochemistry, University of Leipzig, Jahnallee 59, 04109 Leipzig, Germany Received 12 April 2002; received in revised form 21 May 2002; accepted 29 May 2002
Abstract To study the relationship of b-amyloid-mediated micro- and astrogliosis and inflammation-induced proteins including intercellular adhesion molecule (ICAM-1), brain tissue from transgenic Tg2576 mice expressing the Swedish mutation of the human amyloid precursor protein were examined for ICAM-1 expression. Immunocytochemistry demonstrated a diffuse immunostaining of ICAM-1 in the corona around fibrillary b-amyloid plaques and an upregulation of ICAM-1 in activated microglial cells located in close proximity to the plaques, an ICAM-1 distribution pattern that partly mimics the situation in the brain of Alzheimer patients. The developmental time course revealed that the rate of cortical ICAM-1 induction was somewhat behind that of the progression of b-amyloid plaque deposition. The microglial expression of ICAM-1 is a further indicator of the presence of inflammatory reactions in aged transgenic Tg2576 mouse brain, and may be a result of plaque-mediated astrocytic interleukin-1b upregulation. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Alzheimer’s disease; Beta-amyloid; Microglia; Adhesion molecule; Immunocytochemistry; Inflammation; Transgenic mouse brain
From observations that senile plaques in Alzheimer brains are associated with reactive astrocytes and activated microglial cells, a role of inflammation in the pathogenesis of Alzheimer’s disease has been postulated [2]. Phagocytosis of neurons and neuronal regeneration require the interaction of microglial cells with degenerating axons and neuronal somata which is known to be mediated by the expression of cell adhesion molecules [4]. Adhesion molecules functionally represent cell surface receptors that enable cell–cell and cell–extracellular matrix interactions. Intercellular adhesion molecule-1 (ICAM-1) is a member of the immunoglobulin supergene family, and its expression is markedly upregulated by inflammatory mediators [9]. Pathological expression of cell adhesion molecules and functional impairments in related signal transduction mechanisms have been suggested to exhibit a key factor for the development of several neurodegenerative disorders [5]. Particularly in Alzheimer’s disease, the expression of ICAM-1 in b-amyloid-containing brain tissue, has been hypothesized to contribute to plaque formation, tissue re-
* Corresponding author. Tel.: 149-341-97-25734; fax: 149-341211-4492. E-mail address: [email protected] (R. Schliebs).
modelling and neurodegeneration [6,7,15]. The availability of a transgenic mouse (Tg2576) that produces human bamyloid peptides from birth and develops b-amyloid plaques in the aged brain [8] should represent a unique experimental approach to study in vivo the relationship of b-amyloid-mediated micro- and astrogliosis and inflammation-induced proteins including cell adhesion molecules. Therefore, brain tissue from transgenic Tg2576 mice at various ages was examined for ICAM-1 expression by immunocytochemistry and correlated with b-amyloid plaque load. The transgenic Tg2576 mice used in this study contained as a transgene the Swedish double mutation of the human amyloid precursor protein (APP695), as developed and described previously by Hsiao et al. [8]. The transgene is expressed in C57B6/SJL F1 mice (kindly provided by Dr Karen Hsiao, University of Minnesota), and was backcrossed to C57B6 breeders. N2 generation mice were studied at ages of 6, 7, 11, 13, 16, 21 and 24 months with three animals in each group. Age-matched non-transgenic littermates served as controls. Mice were deeply anaesthetized and transcardially perfused with saline followed by fixative (4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4). Brains
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were removed from the skull and immersion-fixed for an additional 4 h. Following equilibration in 30% sucrose in PB, brains were snap-frozen in n-hexane at 270 8C and stored at 220 8C. Thirty-micrometer sections were cut in the coronal plane and immersed in 0.1 M PB (pH 7.4). Immunocytochemical staining of ICAM-1 was performed using a monoclonal rat antibody against mouse ICAM-1 (1:500; Serotec, Oxford, UK). Following incubation with the antibody, immunoreactivity for ICAM-1 was visualized with biotinylated goat-anti-rat IgG (20 mg/ml; Dianova, Hamburg, Germany) followed by the avidin–biotin peroxidase complex (ABC)-technique using diaminobenzidine as substrate resulting in a brown reaction product. Sections treated in the same way but omitting the primary antibody were considered as controls. For double immunofluorescent staining of both ICAM-1 and b-amyloid, immunoreactivity for human b-amyloid peptide was detected using biotinylated rabbit AS720 raised against b-amyloid1–42 (kindly provided by Dr Ursula Mo¨ nning, Schering AG; dilution 1:500) as described recently [3], and was visualized with the red fluorescent Cy-3-conjugated donkey-anti-rabbit antibodies (20 mg/ml; Dianova, Hamburg, Germany). Subsequently, immunoreactivity for ICAM-1 was detected using the green fluorescent Cy2-conjugated donkey-antigoat rabbit antibody (20 mg/ml; Dianova, Hamburg, Germany). For double immunofluorescent labelling of ICAM-1 and glial fibrillary acidic protein (GFAP), sections were incubated subsequently with antibodies against ICAM-1 and GFAP (polyclonal rabbit-anti-GFAP; Sigma, Deisenhofen, Germany; 1:300), followed by visualization of ICAM-1 with the green fluorescent Cy-2-conjugated donkey-anti-rat antibodies and of GFAP with the red fluorescent Cy-3-conjugated donkey-anti-goat rabbit antibody (20 mg/ml each). Brain sections stained were analyzed using a Zeiss Axioplan 2 light microscope including a Sony DXC-930P colour video camera system. Doublelabelled immunofluorescent sections were scanned using a Zeiss LSM 510 confocal laser scanning microscope. ICAM1 expression and b-amyloid plaque load were semiquantitated by estimating the area covered by ICAM-1- and bamyloid-immunoreactivity (as corresponding reaction products), respectively, and referred to the total area of the corresponding cerebral cortical region, using a video camera-based, computer assisted imaging device and the software package of Imaging Research, Inc., MCID 4.0. Thresholds for object segmentation (of reaction product) were established in standard slides and remained constant throughout the analysis session. Regions analyzed comprised frontal and entorhinal cortex as well as hippocampal formation. Corresponding data obtained from three animals at each age were averaged and given as means ^ SEM. To determine whether b-amyloid deposits may induce the expression of adhesion molecules, immunocytochemistry for ICAM-1 was performed in brains of transgenic Tg2576 mice at various ages. Estimation of ICAM-1-immu-
nopositive areas revealed a late induction of ICAM-1 expression in transgenic mouse brain beginning between 11 and 13 months of age and increasing progressively up to the age of 24 months (Fig. 1A). The developmental course of ICAM-1 expression demonstrated that ICAM-1 induction was somewhat behind that of b-amyloid plaque deposition (Fig. 1B). Brain sections from non-transgenic littermates did not demonstrate any immunostaining for ICAM-1, except occasional labelling of some cortical capillary endothelial cells. Microscopic inspections revealed patchy and cluster-like accumulations of ICAM-1-immunoreactivity in cortical and hippocampal regions from transgenic mice with high plaque loads (Fig. 2B,C), while in brain sections of 7-month-old transgenic mice that did not demonstrate any significant b-amyloid deposition (Fig. 1B), no ICAM-1-immunoreactivity was observed (Fig. 2A). Counterstaining of brain sections with thioflavine-S, which labels fibrillary b-amyloid plaques, then disclosed a strong co-localization of senile plaques and ICAM-1-immunoreactivity (not shown). At higher magnification, a diffuse immunostaining for ICAM-1 around the b-amyloid plaques is detectable (Fig. 2D). Laser scanning microscopy of brain
Fig. 1. Age-related increases of cerebral cortical immunostaining for ICAM-1 (A) and b-amyloid (B) in transgenic Tg2576 mice. The reaction product area is given as a percentage of the total area (mean ^ SEM; N ¼ 3) of the brain region indicated (FC, frontal cortex; Ent, entorhinal cortex; HC, hippocampus). The rate of ICAM-1 induction is somewhat behind that of the progression of plaque deposition.
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Fig. 2. (A–C) Representative examples of ICAM-1 immunocytochemistry in brain sections obtained from 7- (A), 16- (B), and 24-month-old (C) Tg2576 mouse cortex (scale bar, 100 mm) demonstrating cluster-like accumulations of ICAM-1-immunoreactivity (brown reaction product visualized by the ABC-technique) that resemble senile plaque labelling. (D,E) High power magnification of cortical brain sections (scale bar 25 mm) from a 24-month-old transgenic mouse immunostained for ICAM-1 demonstrating a diffuse ICAM-1 staining around the senile plaque and the expression of ICAM-1 by activated microglia and brain capillaries in close proximity to the plaque. (F,G) Laser scanning micrograph of double immunofluorescent staining for both b-amyloid (red fluorescence) and ICAM-1 (green fluorescence) in parietal cortex of a 16- (F; scale bar, 50 mm) and 24-month-old transgenic Tg2576 mouse (G; scale bar, 25 mm), demonstrating strong labelling of the corona of b-amyloid plaques, while the core region is free of ICAM-1-immunoreactivity. At early stages of plaque development, only select b-amyloid deposits are associated with ICAM-1 (F), while in aged mice, almost each plaque demonstrates ICAM-1-attachments (G). (H) Laser scanning micrograph of double immunofluorescent staining for both ICAM-1 (green fluorescence) and GFAP (red fluorescence) in parietal cortex of a 24-month-old Tg2576 mouse (scale bar, 25 mm). Note the absence of ICAM-1immunoreactivity in reactive astrocytes, and the corona-like arrangement of ICAM-1-immunoreactivity around senile plaques.
sections subjected to double immunofluorescent labelling of both ICAM-1 and b-amyloid revealed that the monoclonal antibody against mouse ICAM-1 demonstrated a strong staining of the corona of fibrillary b-amyloid plaques, whereas the core region was nearly free of ICAM-1-immunoreactivity (Fig. 2G). While in aged transgenic mice,
nearly each plaque demonstrated ICAM-1-attachments (Fig. 2G), at early stages of plaque development (16 months), only select b-amyloid deposits are associated with ICAM-1 (Fig. 2F). The b-amyloid deposits not being attached by ICAM-1-immunoreactivity represented diffuse plaques that were not stained by thioflavine-S.
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Activated microglial cells and endothelial cells of brain capillaries in close proximity to the fibrillary b-amyloid plaques also demonstrated strong ICAM-1-immunoreactivity (Fig. 2D,E), while in regions more distal to the plaques, ICAM-1-immunoreactivity was only observed in endothelial cells of brain capillaries (Fig. 2E). However, reactive astrocytes surrounding b-amyloid plaques did not display any ICAM-1-immunoreactivity as revealed by double immunofluorescent labelling of ICAM-1 and GFAP, a marker protein for astrocytes (Fig. 2H). The distribution of ICAM-1 upon and around fibrillary, thioflavine-S-positive b-amyloid plaques, on cerebrovascular endothelium, and in activated microglial cells in close proximity to the plaques, detected in transgenic animals with significant b-amyloid plaque loads, partly mimics the situation observed in Alzheimer’s disease. Only a few studies report on the expression of ICAM-1 in brains from Alzheimer patients. In cerebral cortical regions and hippocampus, ICAM-1 was detected in all subsets of senile plaques including classic, diffuse and burned-out plaques, but was also found in tissue surrounding b-amyloid deposition [14], while in the cerebellum, ICAM-1 was only expressed in classic senile plaques in the granular and Purkinje cell layer and not in diffuse plaques of the molecular layer [15,16]. A strong immunolocalization of ICAM1 was observed on neuritic plaques and cerebrovascular endothelium as well as in the margin around the plaques within the extracellular matrix [6,7], but no ICAM-1-immunoreactivity could be detected on microglial cells or astrocytes [7]. However, another study described the presence ICAM-1-immunoreactivity on select GFAP-positive astrocytes in close proximity to senile plaques but not in every one [1]. The upregulation of ICAM-1 in aged transgenic mouse brain appears to be a plaque-induced phenomenon, since in 7-month-old transgenic mouse brain with no significant plaque depositions, no immunoreactivity for ICAM-1 is detectable. The presence of ICAM-1 within neuritic plaques and within the extracellular matrix around the plaques may suggest the occurrence of cell–cell and cell–matrix interactions as a result of plaque-induced neuritic degeneration, sprouting, and tissue-remodelling around the plaques [6,12]. However, the developmental time course of ICAM-1 expression indicates that the rate of ICAM-1 induction is somewhat behind that of the progression of b-amyloid plaque deposition. At early stages of plaque development, only select, thioflavine-S-positive b-amyloid deposits are associated with ICAM-1, while b-amyloidimmunoreactive, but thioflavine-S-negative plaques did not upregulate ICAM-1, indicating a major role of fibrillary plaques in inducing cell–cell and cell–matrix interactions. Already, in a previous report, we could demonstrate that b-amyloid plaque deposits mediate local inflammatory events including the induction of interleukin-1b (IL-1b) in reactive, plaque-surrounding, astrocytes [3]. As ICAM-1 is known to be upregulated in response to inflammation [5]
and by proinflammatory cytokines [10], the microglial expression of ICAM-1 is a further indicator of the presence of inflammatory reactions in aged transgenic Tg2576 mouse brain, and may represent a direct consequence of plaquemediated astrocytic IL-1b upregulation. Indeed, IL-1b may induce ICAM-1-gene transcription through activating nuclear factor kB, which is one of the transcription factors that has a binding site on the ICAM-1 promoter [11]. A similar relationship between b-amyloid, cell adhesion, and cytokines has been disclosed from stimulation experiments in cultured human vascular cells demonstrating that bamyloid may function as an inflammatory stimulator to activate vascular cells and to induce an inflammatory cascade, through interactions among adhesion molecules, CD40CD40L, and cytokines [13]. This study was supported by the Deutsche Forschungsgemeinschaft, grant number Schl 363/3-3, and the Interdisziplina¨ res Zentrum fu¨ r Klinische Forschung (IZKF) Leipzig, proj-no. TP C18, to R.S. The authors would like to express their gratitude to Dr Karen Hsiao Ashe, Department of Neurology, University of Minnesota, MN, USA, for kindly providing three Tg2576 F1 mice.
[1] Akiyama, H., Kawamata, T., Yamada, T., Tooyama, I., Ishii, T. and McGeer, P.L., Expression of intercellular adhesion molecule (ICAM)-1 by a subset of astrocytes in Alzheimer disease and some other neurological disorders, Acta Neuropathol. (Berl.), 85 (1993) 628–634. [2] Akiyama, H., Barger, S., Barnum, S., Bradt, B., Bauer, J., Cole, G.M., Cooper, N.R., Eikelenboom, P., Emmerling, M., Fiebich, B.L., Finch, C.E., Frautschy, S., Griffin, W.S., Hampel, H., Hull, M., Landreth, G., Lue, L., Mrak, R., Mackenzie, I.R., McGeer, P.L., O’Banion, M.K., Pachter, J., Pasinetti, G., Plata-Salaman, C., Rogers, J., Rydel, R., Shen, Y., Streit, W., Strohmeyer, R., Tooyoma, I., Van Muiswinkel, F.L., Veerhuis, R., Walker, D., Webster, S., Wegrzyniak, B., Wenk, G. and Wyss-Coray, T., Inflammation and Alzheimer’s disease, Neurobiol. Aging, 21 (2000) 383–421. [3] Apelt, J. and Schliebs, R., b-Amyloid-induced glial expression of both pro- and anti-inflammatory cytokines in cerebral cortex of aged transgenic Tg2576 mice with Alzheimer plaque pathology, Brain Res., 894 (2001) 21–30. [4] Brown, H.C. and Perry, V.H., Differential adhesion of macrophages to white and grey matter in an in vitro assay, Glia, 23 (1998) 361–373. [5] Cotman, C.W., Hailer, N.P., Pfister, K.K., Soltesz, I. and Schachner, M., Cell adhesion molecules in neural plasticity and pathology: similar mechanisms, distinct organizations? Prog. Neurobiol., 55 (1998) 659–669. [6] Eikelenboom, P., Zhan, S.S., Kamphorst, W., van der Valk, P. and Rozemuller, J.M., Cellular and substrate adhesion molecules (integrins) and their ligands in cerebral amyloid plaques in Alzheimer’s disease, Virchows Arch., 424 (1994) 421–427. [7] Frohman, E.M., Frohman, T.C., Gupta, S., de Fougerolles, A. and van den Noort, S., Expression of intercellular adhesion molecule 1 (ICAM-1) in Alzheimer’s disease, J. Neurol. Sci., 106 (1991) 105–111. [8] Hsiao, K., Chapman, P., Nilsen, S., Eckman, C., Harigaya, Y., Younkin, S., Yang, F. and Cole, G., Correlative memory defi-
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[13] Suo, Z., Tan, J., Placzek, A., Crawford, F., Fang, C. and Mullan, M., Alzheimer’s b-amyloid peptides induce inflammatory cascade in human vascular cells: the roles of cytokines and CD40, Brain Res., 807 (1998) 110–117. [14] Verbeek, M.M., Otte-Ho¨ ller, I., Westphal, J.R., Wesseling, P., Ruiter, D.J. and de Waal, R.M., Accumulation of intercellular adhesion molecule-1 in senile plaques in brain tissue of patients with Alzheimer’s disease, Am. J. Pathol., 144 (1994) 104–116. [15] Verbeek, M.M., Otte-Ho¨ ller, I., Wesseling, P., Ruiter, D.J. and de Waal, R.M., Differential expression of intercellular adhesion molecule-1 (ICAM-1) in the Ab-containing lesions in brains of patients with dementia of the Alzheimer type, Acta Neuropathol. (Berl.), 91 (1996) 608–615. [16] Zhan, S.S., Veerhuis, R., Kamphorst, W. and Eikelenboom, P., Distribution of b-amyloid associated proteins in plaques in Alzheimer’s disease and in the non-demented elderly, Neurodegeneration, 4 (1995) 291–297.
b-Amyloid-associated expression of intercellular adhesion molecule-1 in brain cortical tissue of transgenic Tg2576 mice Jenny Apelt, Jacqueline Leßig, Reinhard Schliebs* Paul Flechsig Institute for Brain Research, Department of Neurochemistry, University of Leipzig, Jahnallee 59, 04109 Leipzig, Germany Received 12 April 2002; received in revised form 21 May 2002; accepted 29 May 2002
Abstract To study the relationship of b-amyloid-mediated micro- and astrogliosis and inflammation-induced proteins including intercellular adhesion molecule (ICAM-1), brain tissue from transgenic Tg2576 mice expressing the Swedish mutation of the human amyloid precursor protein were examined for ICAM-1 expression. Immunocytochemistry demonstrated a diffuse immunostaining of ICAM-1 in the corona around fibrillary b-amyloid plaques and an upregulation of ICAM-1 in activated microglial cells located in close proximity to the plaques, an ICAM-1 distribution pattern that partly mimics the situation in the brain of Alzheimer patients. The developmental time course revealed that the rate of cortical ICAM-1 induction was somewhat behind that of the progression of b-amyloid plaque deposition. The microglial expression of ICAM-1 is a further indicator of the presence of inflammatory reactions in aged transgenic Tg2576 mouse brain, and may be a result of plaque-mediated astrocytic interleukin-1b upregulation. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Alzheimer’s disease; Beta-amyloid; Microglia; Adhesion molecule; Immunocytochemistry; Inflammation; Transgenic mouse brain
From observations that senile plaques in Alzheimer brains are associated with reactive astrocytes and activated microglial cells, a role of inflammation in the pathogenesis of Alzheimer’s disease has been postulated [2]. Phagocytosis of neurons and neuronal regeneration require the interaction of microglial cells with degenerating axons and neuronal somata which is known to be mediated by the expression of cell adhesion molecules [4]. Adhesion molecules functionally represent cell surface receptors that enable cell–cell and cell–extracellular matrix interactions. Intercellular adhesion molecule-1 (ICAM-1) is a member of the immunoglobulin supergene family, and its expression is markedly upregulated by inflammatory mediators [9]. Pathological expression of cell adhesion molecules and functional impairments in related signal transduction mechanisms have been suggested to exhibit a key factor for the development of several neurodegenerative disorders [5]. Particularly in Alzheimer’s disease, the expression of ICAM-1 in b-amyloid-containing brain tissue, has been hypothesized to contribute to plaque formation, tissue re-
* Corresponding author. Tel.: 149-341-97-25734; fax: 149-341211-4492. E-mail address: [email protected] (R. Schliebs).
modelling and neurodegeneration [6,7,15]. The availability of a transgenic mouse (Tg2576) that produces human bamyloid peptides from birth and develops b-amyloid plaques in the aged brain [8] should represent a unique experimental approach to study in vivo the relationship of b-amyloid-mediated micro- and astrogliosis and inflammation-induced proteins including cell adhesion molecules. Therefore, brain tissue from transgenic Tg2576 mice at various ages was examined for ICAM-1 expression by immunocytochemistry and correlated with b-amyloid plaque load. The transgenic Tg2576 mice used in this study contained as a transgene the Swedish double mutation of the human amyloid precursor protein (APP695), as developed and described previously by Hsiao et al. [8]. The transgene is expressed in C57B6/SJL F1 mice (kindly provided by Dr Karen Hsiao, University of Minnesota), and was backcrossed to C57B6 breeders. N2 generation mice were studied at ages of 6, 7, 11, 13, 16, 21 and 24 months with three animals in each group. Age-matched non-transgenic littermates served as controls. Mice were deeply anaesthetized and transcardially perfused with saline followed by fixative (4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4). Brains
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were removed from the skull and immersion-fixed for an additional 4 h. Following equilibration in 30% sucrose in PB, brains were snap-frozen in n-hexane at 270 8C and stored at 220 8C. Thirty-micrometer sections were cut in the coronal plane and immersed in 0.1 M PB (pH 7.4). Immunocytochemical staining of ICAM-1 was performed using a monoclonal rat antibody against mouse ICAM-1 (1:500; Serotec, Oxford, UK). Following incubation with the antibody, immunoreactivity for ICAM-1 was visualized with biotinylated goat-anti-rat IgG (20 mg/ml; Dianova, Hamburg, Germany) followed by the avidin–biotin peroxidase complex (ABC)-technique using diaminobenzidine as substrate resulting in a brown reaction product. Sections treated in the same way but omitting the primary antibody were considered as controls. For double immunofluorescent staining of both ICAM-1 and b-amyloid, immunoreactivity for human b-amyloid peptide was detected using biotinylated rabbit AS720 raised against b-amyloid1–42 (kindly provided by Dr Ursula Mo¨ nning, Schering AG; dilution 1:500) as described recently [3], and was visualized with the red fluorescent Cy-3-conjugated donkey-anti-rabbit antibodies (20 mg/ml; Dianova, Hamburg, Germany). Subsequently, immunoreactivity for ICAM-1 was detected using the green fluorescent Cy2-conjugated donkey-antigoat rabbit antibody (20 mg/ml; Dianova, Hamburg, Germany). For double immunofluorescent labelling of ICAM-1 and glial fibrillary acidic protein (GFAP), sections were incubated subsequently with antibodies against ICAM-1 and GFAP (polyclonal rabbit-anti-GFAP; Sigma, Deisenhofen, Germany; 1:300), followed by visualization of ICAM-1 with the green fluorescent Cy-2-conjugated donkey-anti-rat antibodies and of GFAP with the red fluorescent Cy-3-conjugated donkey-anti-goat rabbit antibody (20 mg/ml each). Brain sections stained were analyzed using a Zeiss Axioplan 2 light microscope including a Sony DXC-930P colour video camera system. Doublelabelled immunofluorescent sections were scanned using a Zeiss LSM 510 confocal laser scanning microscope. ICAM1 expression and b-amyloid plaque load were semiquantitated by estimating the area covered by ICAM-1- and bamyloid-immunoreactivity (as corresponding reaction products), respectively, and referred to the total area of the corresponding cerebral cortical region, using a video camera-based, computer assisted imaging device and the software package of Imaging Research, Inc., MCID 4.0. Thresholds for object segmentation (of reaction product) were established in standard slides and remained constant throughout the analysis session. Regions analyzed comprised frontal and entorhinal cortex as well as hippocampal formation. Corresponding data obtained from three animals at each age were averaged and given as means ^ SEM. To determine whether b-amyloid deposits may induce the expression of adhesion molecules, immunocytochemistry for ICAM-1 was performed in brains of transgenic Tg2576 mice at various ages. Estimation of ICAM-1-immu-
nopositive areas revealed a late induction of ICAM-1 expression in transgenic mouse brain beginning between 11 and 13 months of age and increasing progressively up to the age of 24 months (Fig. 1A). The developmental course of ICAM-1 expression demonstrated that ICAM-1 induction was somewhat behind that of b-amyloid plaque deposition (Fig. 1B). Brain sections from non-transgenic littermates did not demonstrate any immunostaining for ICAM-1, except occasional labelling of some cortical capillary endothelial cells. Microscopic inspections revealed patchy and cluster-like accumulations of ICAM-1-immunoreactivity in cortical and hippocampal regions from transgenic mice with high plaque loads (Fig. 2B,C), while in brain sections of 7-month-old transgenic mice that did not demonstrate any significant b-amyloid deposition (Fig. 1B), no ICAM-1-immunoreactivity was observed (Fig. 2A). Counterstaining of brain sections with thioflavine-S, which labels fibrillary b-amyloid plaques, then disclosed a strong co-localization of senile plaques and ICAM-1-immunoreactivity (not shown). At higher magnification, a diffuse immunostaining for ICAM-1 around the b-amyloid plaques is detectable (Fig. 2D). Laser scanning microscopy of brain
Fig. 1. Age-related increases of cerebral cortical immunostaining for ICAM-1 (A) and b-amyloid (B) in transgenic Tg2576 mice. The reaction product area is given as a percentage of the total area (mean ^ SEM; N ¼ 3) of the brain region indicated (FC, frontal cortex; Ent, entorhinal cortex; HC, hippocampus). The rate of ICAM-1 induction is somewhat behind that of the progression of plaque deposition.
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Fig. 2. (A–C) Representative examples of ICAM-1 immunocytochemistry in brain sections obtained from 7- (A), 16- (B), and 24-month-old (C) Tg2576 mouse cortex (scale bar, 100 mm) demonstrating cluster-like accumulations of ICAM-1-immunoreactivity (brown reaction product visualized by the ABC-technique) that resemble senile plaque labelling. (D,E) High power magnification of cortical brain sections (scale bar 25 mm) from a 24-month-old transgenic mouse immunostained for ICAM-1 demonstrating a diffuse ICAM-1 staining around the senile plaque and the expression of ICAM-1 by activated microglia and brain capillaries in close proximity to the plaque. (F,G) Laser scanning micrograph of double immunofluorescent staining for both b-amyloid (red fluorescence) and ICAM-1 (green fluorescence) in parietal cortex of a 16- (F; scale bar, 50 mm) and 24-month-old transgenic Tg2576 mouse (G; scale bar, 25 mm), demonstrating strong labelling of the corona of b-amyloid plaques, while the core region is free of ICAM-1-immunoreactivity. At early stages of plaque development, only select b-amyloid deposits are associated with ICAM-1 (F), while in aged mice, almost each plaque demonstrates ICAM-1-attachments (G). (H) Laser scanning micrograph of double immunofluorescent staining for both ICAM-1 (green fluorescence) and GFAP (red fluorescence) in parietal cortex of a 24-month-old Tg2576 mouse (scale bar, 25 mm). Note the absence of ICAM-1immunoreactivity in reactive astrocytes, and the corona-like arrangement of ICAM-1-immunoreactivity around senile plaques.
sections subjected to double immunofluorescent labelling of both ICAM-1 and b-amyloid revealed that the monoclonal antibody against mouse ICAM-1 demonstrated a strong staining of the corona of fibrillary b-amyloid plaques, whereas the core region was nearly free of ICAM-1-immunoreactivity (Fig. 2G). While in aged transgenic mice,
nearly each plaque demonstrated ICAM-1-attachments (Fig. 2G), at early stages of plaque development (16 months), only select b-amyloid deposits are associated with ICAM-1 (Fig. 2F). The b-amyloid deposits not being attached by ICAM-1-immunoreactivity represented diffuse plaques that were not stained by thioflavine-S.
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Activated microglial cells and endothelial cells of brain capillaries in close proximity to the fibrillary b-amyloid plaques also demonstrated strong ICAM-1-immunoreactivity (Fig. 2D,E), while in regions more distal to the plaques, ICAM-1-immunoreactivity was only observed in endothelial cells of brain capillaries (Fig. 2E). However, reactive astrocytes surrounding b-amyloid plaques did not display any ICAM-1-immunoreactivity as revealed by double immunofluorescent labelling of ICAM-1 and GFAP, a marker protein for astrocytes (Fig. 2H). The distribution of ICAM-1 upon and around fibrillary, thioflavine-S-positive b-amyloid plaques, on cerebrovascular endothelium, and in activated microglial cells in close proximity to the plaques, detected in transgenic animals with significant b-amyloid plaque loads, partly mimics the situation observed in Alzheimer’s disease. Only a few studies report on the expression of ICAM-1 in brains from Alzheimer patients. In cerebral cortical regions and hippocampus, ICAM-1 was detected in all subsets of senile plaques including classic, diffuse and burned-out plaques, but was also found in tissue surrounding b-amyloid deposition [14], while in the cerebellum, ICAM-1 was only expressed in classic senile plaques in the granular and Purkinje cell layer and not in diffuse plaques of the molecular layer [15,16]. A strong immunolocalization of ICAM1 was observed on neuritic plaques and cerebrovascular endothelium as well as in the margin around the plaques within the extracellular matrix [6,7], but no ICAM-1-immunoreactivity could be detected on microglial cells or astrocytes [7]. However, another study described the presence ICAM-1-immunoreactivity on select GFAP-positive astrocytes in close proximity to senile plaques but not in every one [1]. The upregulation of ICAM-1 in aged transgenic mouse brain appears to be a plaque-induced phenomenon, since in 7-month-old transgenic mouse brain with no significant plaque depositions, no immunoreactivity for ICAM-1 is detectable. The presence of ICAM-1 within neuritic plaques and within the extracellular matrix around the plaques may suggest the occurrence of cell–cell and cell–matrix interactions as a result of plaque-induced neuritic degeneration, sprouting, and tissue-remodelling around the plaques [6,12]. However, the developmental time course of ICAM-1 expression indicates that the rate of ICAM-1 induction is somewhat behind that of the progression of b-amyloid plaque deposition. At early stages of plaque development, only select, thioflavine-S-positive b-amyloid deposits are associated with ICAM-1, while b-amyloidimmunoreactive, but thioflavine-S-negative plaques did not upregulate ICAM-1, indicating a major role of fibrillary plaques in inducing cell–cell and cell–matrix interactions. Already, in a previous report, we could demonstrate that b-amyloid plaque deposits mediate local inflammatory events including the induction of interleukin-1b (IL-1b) in reactive, plaque-surrounding, astrocytes [3]. As ICAM-1 is known to be upregulated in response to inflammation [5]
and by proinflammatory cytokines [10], the microglial expression of ICAM-1 is a further indicator of the presence of inflammatory reactions in aged transgenic Tg2576 mouse brain, and may represent a direct consequence of plaquemediated astrocytic IL-1b upregulation. Indeed, IL-1b may induce ICAM-1-gene transcription through activating nuclear factor kB, which is one of the transcription factors that has a binding site on the ICAM-1 promoter [11]. A similar relationship between b-amyloid, cell adhesion, and cytokines has been disclosed from stimulation experiments in cultured human vascular cells demonstrating that bamyloid may function as an inflammatory stimulator to activate vascular cells and to induce an inflammatory cascade, through interactions among adhesion molecules, CD40CD40L, and cytokines [13]. This study was supported by the Deutsche Forschungsgemeinschaft, grant number Schl 363/3-3, and the Interdisziplina¨ res Zentrum fu¨ r Klinische Forschung (IZKF) Leipzig, proj-no. TP C18, to R.S. The authors would like to express their gratitude to Dr Karen Hsiao Ashe, Department of Neurology, University of Minnesota, MN, USA, for kindly providing three Tg2576 F1 mice.
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