Virchows Archiv B Cell Pathol (1991) 60:329-336

Vir ows Arch&B CellPathology

IncludingMolecu(m"Pathology

9 Springer-Verlag 1991

Cerebral amyloid plaques in Alzheimer's disease but not in scrapie-affected mice are closely associated with a local inflammatory process* P. Eikelenboom 1, J.M. Rozemuller 2, G. Kraal 3, F.C. Stam 2, P.A. McBride 4, M.E. Bruce 4, and H. Fraser 4 Departments of 1Psychiatry, 2Neuropathology, 3Cell Biology, Medical Faculty, Free University, Amsterdam, The Netherlands 4AFRC & MRC, Neuropathogenesis Unit, West Mains Road, Edinburgh, UK Received November 2, 1990 / Accepted April 24, 1991

Summary. Complement proteins of the classical pathway can be immunohistochemically identified in cerebral amyloid plaques in Alzheimer's disease. Microglial cells in and around amyloid plaques express class II major histocompatibility (MHC) antigens and complement receptors CR3 and CR4. Negative immunostaining for immunoglobulins and for T-cell subsets in the brain parenchyma demonstrates a lack of evidence for the involvement of specific immune responses (such as an immune complex-mediated complement activation or a cell-mediated immune response) in cerebral amyloid deposits in Alzheimer's disease. Cerebral amyloid plaques in scrapie-affected mice (slow-virus induced encephalopathy) do not contain complement factors Clq and C3c and are not clustered with microglial cells expressing MHC class II molecules or complement receptor CR3. The data presented suggest the induction of a reactive inflammatory process by fl/A4 amyloid in the human brain, but not by scrapie-induced PrP amyloid in mice. Our findings do not support the hypothesis that the immune system is involved in the generation of amyloid plaques in Alzheimer's disease. Key words: Amyloid plaques - Complement factors Immune system - Alzheimer's disease and scrapie

Introduction Cerebral amyloid deposition is a prominent feature of the pathological process in Alzheimer's disease. Extracellular amyloid fibrils, composed of a 4 kd peptide termed fl/A4 protein, accumulate in cerebral and meningeal blood vessels (Glenner and Wong 1984) and in the cores of senile plaques (Masters et al. 1985; Selkoe * This study was partly supported by a grant from the Praeventiefonds, project 28-1945 Offprint requests to: P. Eikelenboom, Valeriuskliniek, Valeriusplein 9, NL-1075 BG Amsterdam, The Netherlands

et al. 1986). The fl/A4 protein appears to be derived from a much larger precursor protein (fl-amyloid precursor protein) by proteolytic cleavage (Kang et al. 1987). With antibodies against fl/A4 protein, two basic types of plaques can be detected in brain tissue from patients with Alzheimer's disease and Down's syndrome (Tagliavini et al. 1988; Yamaguchi et al. 1988; Ikeda et al. 1989. Joachim et al. 1989; Rozemuller et al. 1989 b). These are: a) amorphous or diffuse non-congophilic plaques with deposition of fl/A4 protein not associated with altered neurites and reactive glial cells, and b) classical plaques with a fl/A4-positive congophilic core and with a corona of dystrophic neurites with glial alteration. Much progress in the characterization of cerebral amyloid fibrils has been made during the past few years, but information about other factors involved in the process of amyloid deposition is relatively sparse. Using immunohistochemical techniques complement factors Clq, C4 and C3, but not C5 (Eikelenboom and Stare 1982, 1984; Ishii and Haga 1984; Pouplard and Emile 1985), P component (Coria et al. 1988), and ~tl-antichymotrypsin (Abraham et al. 1988) have been found in both amorphous and classical plaques (Rozemuller et al. 1989b). Studies with antibodies raised against subunits of individual complement components and inactivated complement products indicate that the presence of complement factors is due to an activation that has occurred in senile plaques (Eikelenboom et al. 1989). Reports of the presence of immunoglobulins and other serum proteins in senile plaques present contradictory results (Stam 1965; Ishii etal. 1975; Ishii and Haga 1976; Powers et al. 1981 ; Mann et al. 1982; Eikelenboom and Stam 1982, 1984; Stam and Eikelenboom 1985; Alafuzoffet al. 1987; Rozemuller et al. 1988). As well as serum proteins, immune system-related cellular markers have been demonstrated in association with amyloid plaques. Immunoreactivity for MHC class II glycoprotein HLADR (Pouplard-Barthelaix etal. 1986; McGeer etal. 1987; Rogers et al. 1988; Itagaki et al. 1989; Rozemuller et al. 1989b) and for leucocyte adhesion molecules of the LFA-1 family (leucocyte function-associated anti-

gens) (Rozemuller et al. 1989 a) has been demonstrated on small glial cells (referred to as microglial cells) in the corona of senile plaques. T-helper cell (CD4) and T-suppressor cell (CD8) antigens are reported on cells in the brain parenchyma of patients with Alzheimer's disease (Itagaki et al. 1988; Rogers et al. 1988). The expression of immune system-associated antigens in the cortex of patients with Alzheimer's disease and their relationship with amyloid plaques suggest that brain-immune interactions are involved in the pathogenetic mechanism of Alzheimer's disease and, in particular, that amyloid deposition may occur in an immunological context (Ishii etal. 1988; Pouplard-Barthelaix 1988; Rogers et al. 1988; McGeer et al. 1989 b). Cerebral amyloid plaques can also be found in unconventional slowvirus induced encephalopathies, including CreutzfeldtJakob disease, Gerstmann-Str~ussler disease and scrapie. The amyloid present is derived from the glycoprotein, PrP, which is produced in neurons (Kretschmar et al. 1986; Roberts etal. 1986). These cerebral amyloid plaques in Creutzfeldt-Jakob disease (Pouplard-Barthelaix 1988) and in scrapie-affected mice (Eikelenboom et al. 1987) lack immunoreactivity for complement factors. In the present study we have investigated, firstly, the nature of the neuroimmunological context in which amyloid plaques in Alzheimer's disease would occur and, secondly, whether or not the neuroimmunological findings are related to a disease-specific phenomenon. Particular attention has been paid to the occurrence of complement proteins, immunoglobulins and MHC class II antigens with T-cell antigens. The presence of these molecules should indicate the possible involvement of specific immune responses (such as an immune complex-mediated complement activation or a cell-mediated immune response) in cerebral amyloid deposition. Using a panel of antibodies against both humoral and cellular components of the immune system, we examined cerebral amyloid plaques for any association with immune systemrelated markers in both Alzheimer's disease and scrapie.

on the same slides as the brain sections to serve as positive controls for hemopoietic cell markers.

hnmunoperoxidase technique. For immunohistochemical staining on frozen tissue, 8 p.m thick cryostat sections were mounted on poly-l-lysine coated glass slides, air-dried and fixed in acetone for 10 min before use. The primary antibodies used in this study are listed in Tables 1 and 2, together with their specificity, sources, selected references and the immunocytochemical technique which was used in this study. The specificity of each antibody against lymphoid or non-lymphoid cells was evaluated in lymphoid tissue (thymus, spleen, tonsil). All antibodies were appropriately diluted in phosphate-buffered saline (PBS), pH 7.4, containing 1% bovine serum albumin. Secondary antisera and reagents were tested for lack of cross reactivity and nonspecific staining. Each incubation was performed at room temperature and followed by repeated washes in PBS. Peroxidase activity was visualised using 3,3 diamino-benzidine (DAB) (5 mg DAB in 10 mg PBS, pH 7.4, containing 0.02% H202 for 3-5 min), and the staining was intensified with copper sulphate (0.5% in 0,9% NaC1 for 5 min (Hsu and Soban 1982). All sections were counterstained with Congo red (Puchtler et al. 1962) to visualize the amyloid. After Congo red staining, sections were dehydrated and mounted in malinol. The indirect peroxidase conjugate technique was as follows: acetone-fixed cryostat sections were washed in PBS and incubated with the primary antibodies for 60 rain and in the second step with peroxidase-labeled rabbit anti-mouse antisera (DAKO) or rabit anti-rat-antisera (DAKO) for 30 rain. Peroxidase was revealed by the DAB method. In the peroxidase-anti-peroxidase (PAP) technique, acetone-fixed cryostat sections were pre-incubated with normal swine serum and incubated with the primary rabbit antisera for 60 min. After a second pre-incubation with normal swine serum, sections were incubated with swine anti-rabbit immunoglobulins (DAKO) for 30 min. In the third step sections were incubated with rabbit peroxidase-anti-peroxidase (PAP) complex, (DAKO) for 30 rain. Peroxidase activity was revealed by the DAB method. Double staining was performed by incubating overnight simultaneously with anti-A4 rabbit polyclonal antibodies and anti-C3d monoclonal antibodies, followed in the second step by an incubation with alkaline-phosphatase-labeled goat-anti-rabbit (Tago) and biotinylated horse anti-mouse antibodies (Vector). In the third step sections were incubated with peroxidase-labeled avidin-biotin-complex (Vector elite kit). Peroxidase activity was visualised using the DAB method. Alkaline phosphatase was revealed using naphtol AS-MX phosphate as substrate and Fast Blue BB as coupling agent. Secondary antisera and reagents were tested for lack of cross-reactivity and nonspecific staining.

Material and methods Brain tissue was obtained from five patients (age 48-81 years) with a clinical and pathological diagnosis of Alzheimer's dementia. Small pieces of parietal and temporal cortex were frozen 3-7 h after death in liquid nitrogen for immunohistochemical studies. From one patient frozen brain tissue was also available from a brain biopsy performed 2 years before death. The pathological diagnosis was based on histological staining (haematoxylin and eosin, Congo red, Bodian staining, KlueverBarrera) performed on prolonged formalin-fixed, paraffin-embedded tissue (10% formalin for 4-5 weeks). Four serapie-affected mice with cerebral amyloid plaques were studied. Inbred mice of the IM/Dk strain were infected with the 87V strain of scrapie (Bruce et al. 1976) as follows. An inoculum was prepared from the brain of an infected mouse by homogenisation at a dilution of 5 • 10-2 in physiological saline and centrifugation at 500 g for 10 rain. Each mouse was injected intracerebrally with 0.02 ml of the inoculum. Mice were killed by cervical fracture when showing signs of advanced scrapie, after an incubation period of about 300 days. Brains were removed immediately and frozen within seconds in liquid nitrogen. Thymus sections were mounted

Results

The immunohistological findings on amyloid plaques in Alzheimer's disease and scrapie-affected mice for serum proteins and hemopoietic cell markers are summarized in Table 3.

Alzheimer's disease Antisera against fl/A4 protein showed two basic types of plaques: amorphous non-congophilic plaques and classical plaques with a fl/A4 positive central core and a fl/A4 negative corona (see Rozemuller et al. 1989b) (Fig. 1). The amorphous plaques were present in all cortical layers and outnumbered the classical plaques. Complement factors Clq and C3 were present in both amorphous and classical plaques (Fig. 2). Immunoglobulins

Table 1. Specificity of used markers in Alzheimer's disease and selected references Antigen

Antibody

Source

Dilution

Technique

References

I Amyloid fl/A4 protein

Rabbit polyclonals

gift 1

1:400

PAP

Masters et al. (1985)

II Serum proteins Clq C3c C3d IgG (7 specific) IgM (/~ specific) IgA (ct specific) Kappa light chain Lambda light chain

Rabbit polyclonals Rabbit polyclonals Mouse monoclonals Rabbit polyclonals Rabbit polyclonals Rabbit polyclonals Rabbit polyclonals Rabbit polyclonals

DAKO DAKO gift 2 DAKO DAKO DAKO DAKO DAKO

1:400 1:200 1 : 1000 1 : 600 1 : 50 1:400 1 : 1600 1:1600

PAP PAP ABC PAP PAP PAP PAP PAP

III Cell markers CD4 T cell helper CD8 T cell suppressor CD1 la (LFA-lct) CDI l b (CR3c0 CDllc (p150, 95) CD18 (LFA-1B) CD21 (C3d rec) (CR2) CD35 (C3b rec) (CR1) ICAM-1 HLA-DR (classlI MHC)

Mouse monoclonals Leu 3a Leu 2a TB 133 Bear 1 Leu M5 TB-133 HB 5 To/5 RR-1/1 OK Ia

B&D B&D gift 2 gift 2 B&D gift 2 B&D B&D

1:25 1 : 100 1:1500 1:250 1:25 1:1000 1:40 1:25 1:100 1:80

IP IP IP IP IP IP IP IP IP IP

DAKO

Hack et al. (1988)

Evans et al. (1981) Evans et al. (1981) Keizer et al. (1985) Keizer et al. (1985) Schwarting et al. (1985) Miedema et al. (1984) Tedder et al. (1984) Gerdes et al. (1982) Dustin et al. (1986) Reinherz et al. (1979)

PAP: peroxidase-antiperoxidase method IP: indirect peroxidase method ABC: Avidin-Biotin Complex method gift 1: C.L. Masters, University of Melbourne B & D : Becton & Dickinson, Inc. gift 2 : Central Lab. Netherlands Red Cross Transfusion Service, Amsterdam

Table 2. Specificity of used markers in scrapie affected mice and selected references Directed against I Serum proteins Clq C3 IgG (heavy and light chains) IgG (7 specific) IgM (# specific) IgA (e specific) II Cell markers T helper cells T suppressor cell CR3 LFA-lfl Ia region H2 complex

Antibody

Source

Dilution

Technique

Rabbit polyclonals Rabbit polyclonals Goat polyclonals

girl 1 gift 1 Cappel

1:100 1:25 1:100

IP IP DP

Goat polyclonals Sheep polyclonals Goat polyclonals

Cappel Serotec Nordic

1:100 l:100 1:100

DP DP DP

1 :I 1:1 1: 1

IP IP IP IP IP

Rat monoclonals MT4 lyT2 M 1/70 (Mac-l) M/18 M 5/114

1:5 1 :1

Reference

Pierres et al. (1984) Ledbetter et al. (1979) Springer et al. (1979) Pierres et al. (1982) Bhattacharya et al. (1981)

IP: indirect peroxidase method DP : direct peroxidase method gift ~: van Dijk, Rijksuniversiteit Utrecht

were n o t detected in a m o r p h o u s and classical plaques except that the c o r o n a o f an occasional plaque showed weak peroxidase staining for IgG. D o u b l e i m m u n o s t a i n ing for fl/A4 protein and c o m p l e m e n t factor C3d shows that all fl/A4 depositions were also i m m u n o l a b e l e d for C3d (Fig. 3). Plaque-like depositions, stained only for

C3d and not for fl/A4 protein, were not seen in the neuropil. In classical plaques clusters o f small glial cells (referred to as microglial cells) were i m m u n o s t a i n e d for C D l l a , C D l l b , C D l l c , C D 1 8 and M H C class II antigens but n o t for CD21 and CD35. These cells were m o s t

Fig. 1, 2. Parietal cortex of a 55-year-old man with Alzheimer's disease. Frozen sections stained for /?/A4 (Fig. l) and complement C3c (Fig. 2) showing plaques. Bar is 20 p.m Table 3. Immunostaining of amyloid plaques for immune system

related markers in Alzheimer's disease and scrapie affected mice Alzheimer Serum proteins Clq C3 Ig

Scrapie

+ + B

Lymphocytes T helper cells T suppressor cells Microglial cells Class II MHC antigen CR1 CR2 CR3 CR4 LFA-lcr LFA-I~ Adhesion molecules ICAM Symbols: - no labelling + positive labelling ND not determined

frequently f o u n d at the b o r d e r between the congophilic core and the c o r o n a (Fig. 4, 5 and 6). These i m m u n o l a beled cells were n o t f o u n d in association with the a m o r p h o u s plaques. In addition, small b r a n c h e d glial cells dispersed in the subcortical white m a t t e r were also stained. Microglial cells positive for M H C class II and C D 11 c frequently o u t n u m b e r e d the i m m u n o l a b e l e d cells for C D l l b in the c o r o n a o f classical plaques. A m o r p h o u s plaques and the c o r o n a o f classical plaques showed a diffuse i m m u n o s t a i n i n g for I C A M - 1 . With a n t i - C D 4 and C D - 8 r o u n d positive cells were seen in the lumen o f b l o o d vessels but not outside the vessels

+ ND ND + + + + +

ND ND

Fig. 3. Parietal cortex of a 49-year-old man with Alzheimer's disease. Double immunostaining for fl/A4 (blue) and complement C3d (brown) show that the fl/A4 plaques are also immunolabeled for C3d. Bar is 20 gm

ND

Fig. 4-6. Parietal cortex of a 55-year-old man with Alzheimer's disease. Figure 4 shows a classical plaque immunostained for HLADr (brown) followed by congo red. Bar is 13 pm Fig. 5--6. Show two adjacent sections immunostained for CDllc

followed by congo red. Classical plaques with immunolabeled glial cells (brown) around the amyloid core (red). Arrows point to the same plaque in both figures. Bar is respectively 20 and 13 Ixm

in the neuropil. Immunolabeling for hemopoietic cell markers in human lymphoid tissues confirmed the specificity for each primary antibody (see Table 1).

Scrapie Amyloid plaques did not show immunostaining for complement factors Clq and C3. Only a few branched glial cells in the white matter stained faintly for Mac-1. Matsumoto et al. (1985) have reported the same immunostaining pattern for Mac-1 in normal adult mouse brain. Amyloid plaques sometimes showed a weak diffuse immunostaining for Mac-1 but no positively labeled cells were found associated with amyloid plaques. No immunolabeled cells for MHC class II molecules were observed around amyloid plaques. Immunolabeling in thymus sections confirmed the specificity of each antibody (Table 2).

Discussion

A possible involvement of specific immune responses in cerebral amyloid deposition in Alzheimer disease has been suggested from two types of observation (Ishii et al. 1988 ; Pouplard-Barthelaix 1988; McGeer et al. 1989 b). Firstly, the immunohistochemical demonstration of complement components of the classical pathway in association with amyloid deposits indicates the presence of immunoglobulins and the involvement of a humoral immune response. Secondly, an enhanced MHC class II antigen expression on microglial cells has been reported together with the occurrence of helper (T4) and suppressor (T8) lymphocytes (Itagaki et al. 1988; Rogers et al. 1988; McGeer et al. 1989 b) which indicates a cellular immune response. In the present study we did not find immunostaining for immunoglobulins in either amorphous or classical plaques. These findings are in line with recent reports on the absence of immunostaining for immunoglobulins in classical plaques (PouplardBarthelaix 1988; Rozemuller et al. 1988). The presence of Clq and absence of properdin in amyloid plaques indicate that the alternative complement pathway is not involved in the complement activation (Eikelenboom and Stare 1982; Ishii and Haga 1984; Pouplard and Emile 1985; McGeer etal. 1989a, b). However, nonimmunological factors such as trypsin-like enzymes can also initiate the classical complement reaction (Cooper 1985). Several authors have emphasized the possible role of microglial cells in the formation of senile plaques. In an ultrastructural, three-dimensional reconstruction of classical plaques it has been recently demonstrated that microglial cells form together with the amyloid star the central complex of the classical plaques (Wegiel and Wisniewski 1990). These cells showed a broad variety in size and shape. Colocalization of clusters of microglial cells expressing MHC class II surface glycoprotein HLADR and glycoproteins of the LFA-1 family are found with classical amyloid plaques but not with amorphous

plaques (Itagaki et al. 1989; Rozemuller et al. 1989a, b). The expression of HLA-DR is considered an essential prerequisite for T-cell participation in an immune response, although HLA-DR expression can accompany a non-immunological mediated reaction. Data about the presence of T-cell subsets in the brain parenchyma in Alzheimer disease are contradictatory (Itagaki et al. 1988; Pouplard-Bartelaix 1988; Rogers et al. 1988; this report). As well as positive staining for HLA-DR, small glial cells (referred to as microglial cells) in the corona of amyloid plaques also show positive staining for leucocyte adhesion molecules belonging to the LFA-1 family (Rozemuller et al. 1989 a). This family of adhesion molecules consists of the membrane glycoproteins LFA-1, iC3b receptor (Mac-l) and P 150/95. These molecules are non-covalent associated ~fl heterodimers with homologous ~ subunits and a common fl subunit. Immunostaining for the molecules of the LFA-1 family demonstrates the presence of complement receptors CR3 (CD 1lb/CD 18) and CR4 (CD 11c/CD 18). The ligand for both CR3 and CR4 is the complement factor iC3b. We did not find immunostaining for complement receptor CRI (CD35) and CR2 (CD21), which are respectively directed against the complement products C3b/C4b and C3d, g. Immunostaining for ICAM, a ligand for LFA-I and induced during inflammation (Kishimoto et al. 1989), was found in both amorphous and classical plaques. Hence, leucocyte cell adhesive molecules belonging to the LFA-1 family as well as their ligands can be demonstrated in classical plaques in Alzheimer's disease. In contrast, amyloid plaques in scrapie-affected mice do not contain complement factors Clq and C3c and are not associated with clusters of microglial cells expressing MHC class II molecules or molecules of the LFA-1 family. These findings indicate that amyloid plaques in Alzheimer's disease, but not in scrapie-affected mice, are associated with molecules indicating an inflammatory process. The absence of immunoglobulins and T-cell subsets suggests that an immune complexmediated complement activation and/or a cellular immune-mediated response are not involved in cerebral amyloid formation in Alzheimer's disease. Thus, the amyloid deposition in Alzheimer's disease is closely associated with a locally induced inflammatory process. This view is in line with some recently reported findings and opinions: (1) the presence of the acute phase proteins complement factors, P-component and ~l,-antichymotrypsin in both amorphous and classical plaques (Rozemuller et al. 1989b) (2) the increase of ~1, antichymotrypsin mRNA in Alzheimer's disease brain indicating a brain "acute phase response" (Abraham and Potter 1989), (3) brain interleukin-1 immunoreactivity is elevated in Alzheimer's disease (Griffin et al. 1989), (4) interleukin-1 regulates the synthesis of amyloid precursor protein mRNA (Goldgaber et al. 1989), (5) the promoter region of the amyloid precursor protein region contains acute phase and heat shock elements (Salbaum et al. 1989). In contrast to classical plaques in Alzheimer's disease, little or no neuritic change is seen around the amyloid

core in the u n c o n v e n t i o n a l virus encephalopathies such as scrapie (Bruce and Fraser 1975) and Creutzfeldt-Jacob disease (Boellaard and Schlote 1981). T h e tight association o f cerebral amyloid deposition with a local inf l a m m a t o r y process in Alzheimer's disease, but not in slow virus induced e n c e p h a l o p a t h y , suggests that this process m a y be neurotoxic and thus play a role in the f o r m a t i o n o f dystrophic neurites s u r r o u n d i n g the amyloid cores in Alzheimer's disease.

Acknowledgement. Brain specimens were obtained from the brain bank in the Netherlands institute for Brain Research Amsterdam. We are especially grateful to Drs W. Kamphorst and R. Ravid for providing brain tissue.

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