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Journal of Alzheimer's Disease 7 (2005) 221­232 IOS Press

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Ineffective phagocytosis of amyloid- by macrophages of Alzheimer's disease patients

Milan Fialaa, , Justin Lina , John Ringman b , Vali Kermani-Arabc, George Tsao a, Amish Patela , Albert S. Lossinskyd, Michael C. Gravesb , Andrew Gustavson b, James Sayre e , Emanuela Sofronia , Tatiana Suareza, Francesco Chiappelli f and George Bernard f

Department of Medicine, Greater LA VA Medical Center and UCLA School of Medicine, Los Angeles, CA 90095, USA b Department of Neurology, UCLA School of Medicine, Los Angeles, CA 90095, USA c Immuno-Biogenetic Institute, Inc, West Hills, CA 91307, USA d Neural Engineering Program, Huntington Medical Research Institutes, 743 Fairmount Avenue, Pasadena, CA 91105, USA e Department of Biostatistics, UCLA School of Public Health, Los Angeles, CA 90095, USA f Division of Oral Biology and Medicine, UCLA School of Dentistry, Los Angeles, CA 90095, USA

a

Abstract. The defective clearance of amyloid- (A) in the brain of Alzheimer's disease (AD) patients is unexplained. The immunohistochemical studies of the frontal lobe and hippocampus show perivascular and intraplaque infiltration by blood-borne macrophages containing intracellular A but only inefficient clearance of A deposits. Neurons and neuronal nuclei, respectively, express interleukin-1 and the chemokine RANTES, which could induce the inflammatory cell infiltration. To clarify the pathophysiology of A clearance, we examined A phagocytosis by monocytes and macrophages isolated from the blood of age-matched patients and controls. Control monocytes display excellent differentiation into macrophages and intracellular phagocytosis of A followed by A degradation or export. AD monocytes show poor differentiation and only surface uptake of A and suffer apoptosis. HLA DR and cyclooxygenase-2 are abnormally expressed on neutrophils and monocytes of AD patients. AD patients have higher levels of intracellular cytokines compared to controls. Thus A clearance is not restricted to brain microglia and involves systemic innate immune responses. In AD, however, macrophage phagocytosis is defective, which may elicit compensatory response by the adaptive immune system. Keywords: Macrophage/monocyte, amyloid-beta phagocytosis, macrophage apoptosis, innate immunity, inflammation and Alzheimer's disease

1. Introduction According to the modified amyloid hypothesis, oligomeric amyloid- (A) accumulation and chronic inflammation lead to neuronal demise [20]. Neurons are damaged by oxidative stress in combination with cell-cycle dysregulation according to the "two-hit" hypothesis of Alzheimer's disease (AD) [57]. The role

Corresponding author: Milan Fiala, M.D., Oral Biology and Medicine, UCLA 63-090, Los Angeles, CA 90095-1668, USA. Tel.: +1 310 206 6392; Fax: +1 310 825 2042; E-mail: [email protected]

of inflammation in AD is supported by AD risk reduction in patients using certain non-steroidal antiinflammatory drugs [51]; however, prospective trials with anti-inflammatory drugs have not shown clear therapeutic effects [1]. Inflammation has been postulated to be induced through activation of astrocytes [6] and microglia [8,46] by A deposits and has been ascribed to multiple immunologic mechanisms, including activated components of complement [10,28,41], acute phase reactant -1 antichymotrypsin [26], and alteration of cytokine levels, in particular of tumor necrosis factor- [9], interleukin (IL)-1 [36], IL-6 [45], trans-

ISSN 1387-2877/05/$17.00 © 2005 ­ IOS Press and the authors. All rights reserved

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forming growth factor- [49], chemokines [54], and altered enzyme activities, including cyclooxygenase 2 (COX-2) [14,38,40,55,56], and inducible nitric oxide synthase (iNOS) [19,25]. While the inflammation in AD brain has been assumed to be locally-mediated inflammatory response by microglia to A deposits [2], systemic immune responses may be present, both adaptive [35] and innate, as shown in this study. We previously showed by immunohistochemistry (IHC) that the effector cells of the innate immune system in AD brain, monocyte/macrophages (MM's), are distinct from microglia by their size and shape, and the ability to disrupt the tight junction protein ZO-1 in microvessels penetrate the blood-brain barrier (BBB), infiltrate perivascular spaces and neuritic plaques and phagocytize A [14]. In addition, CD8 T cells, which are the effector cells of adaptive immunity, also infiltrate perivascular spaces in AD brain [14]. We speculate that the accumulation of the metabolic end-product A in AD brain might be due to the failure of the innate immune system [17]; and the activation of the adaptive immune system might be a response to this failure. In order to examine this hypothesis, we have examined MM's in brain tissues and peripheral blood of AD patients and compared ADMM's to control MM's. We have confirmed previously observed robust perivascular and intraplaque infiltration by MM's of AD brain in frontal and temporal lobes and hippocampus. To evaluate the role of ADMM's in clearance of A, we have investigated the relation between MM infiltration and residual A in each plaque; and to clarify the causes of inflammatory cell infiltration, we have examined the expression of the chemokine RANTES and interleukin1 (IL-1). In parallel with IHC studies of AD brains, we have investigated in vitro phagocytosis of A by MM's of AD patients and control subjects. In addition, we examined cytokine expression by circulating mononuclear cells.

teria, CA). With primary goat antibodies, we used the Biotin blocking system (after Dual Endogenous Enzyme block) and the LSAB plus kit (DAKO). We used the following antibodies: anti-RANTES, either goat (R&D Systems, Minneapolis, MN) or rabbit (Torrey Pines BioLabs, Houston, TX); goat anti-IL-1 (Santa Cruz Biotechnology, Santa Cruz, CA); mouse antiNeuN and rabbit anti-A 1­42 (Chemicon, Temecula, CA); mouse anti-A 1­42 (4G8, Signet Labs, Dedham, Mass). All positive staining results were validated by negative IgG isotype results. 2.2. Patients and control subjects ­ Diagnostic criteria, blood specimens, and immune studies Twenty-four patients with diagnosis of possible or probable AD and 20 control subjects (the participants in a study of cognitively normal older subjects or the staff) were recruited through the UCLA Alzheimer's Disease Research Center from November 2000 until May 2004. All AD patients and controls in this study were ambulatory at the time of blood collection. The patients who were classified as AD met the National Institute of Neurological and Communicative Disorders and the Alzheimer's Disease and Related Disorders Association (NINCDS/ADRDA) criteria for probable Alzheimer's disease [31]. All have undergone diagnostic evaluation including complete medical history and physical examination, routine blood tests (thyroid stimulating hormone level, complete blood count, electrolytes, renal function, liver function, vitamin B12 level and serological test for syphilis), and neurologic, neuroradiological and neuropsychological examinations. The latter included Mini Mental Status Exam [16], Consortium to Establish a Registry for AD [47] (CERAD) ten word memory test and visual spatial coping task, modified 15 item Boston naming test, a timed word generation verbal fluency task. The diagnosis was established though consensus of a panel comprised of behavioral neurologists, geriatric psychiatrists and neuropsychologists based on NINCDS/ADRDA criteria. All experimental procedures were reviewed and approved by the UCLA IRB Human Subjects Committee and all subjects gave informed consent prior to participation. The study was performed since June 2001 until May 2004. Approximately 20 cc of venous blood was collected from each subject. For cytospin studies of neutrophils and monocytes, peripheral blood mononuclear cells (PBMC's) with neutrophils were separated from EDTA-anticoagulated blood by centrifugation on

2. Methods 2.1. Immunohistochemistry (IHC) of brain tissues Paraffin embedded brain tissues of eight AD patients (mean age 77.6 years) and 5 control subjects (mean age 74.6 years) were obtained from the UCLA ADRC Brain Bank. Antigen retrieval, Dual endogenous enzyme block, Protein block serum free, and immunostaining were performed according to manufacturer's instructions by the EnVision technique (DAKO, Carpin-

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Fig. 1. Macrophages infiltrate AD frontal lobe and hippocampus, the areas with neurons expressing RANTES and IL-1 but do not efficiently phagocytize A. A. Macrophage infiltration of frontal lobe perivascular areas (a,b,c anti-CD68; d IgG isotype; a × 40, b,c,d × 20) and the hippocampus (e,f,g anti-CD68; h IgG isotype; e,h × 20, f × 10, g × 40); B. Neuronal expression of RANTES by frontal lobe neurons (a anti-RANTES (blue) and anti-NeuN (red) double staining; b, c anti-RANTES (brown), d IgG isotype; a × 40, b,c,d × 20, inset × 100) and hippocampal pyramidal neurons (e,f,g anti-RANTES (brown), h IgG isotype; e-h × 20; inset × 100); C. Neuronal expression of IL-1 by frontal lobe (a and b anti-IL-1 brown)) and hippocampal pyramidal neurons (c anti-IL-1; d IgG isotype; a-c ×20, d ×40, inset ×100) D. Residual A in each plaque does not correlate with macrophage density or macrophage area (anti-A 1­42 (red), anti-CD68 (brown), a-d ×40; areas and densities of 30 plaques were scanned by Image-Pro and analyzed as described in the text).

Ficoll-Hypaque gradient in IEC 4B centrifuge (rotor radius of 8.5 cm) at 2200 RPM for 20 min. For other studies, pure PBMC's were separated using centrifugation at 3000 RPM. We performed three immunological studies (A,B,C): Study A: A phagocytosis by monocytes: Blood

samples of 17 AD patients (mean age 66.3 years and mean Mini Mental Status exam (MMSE) score 23/30) and 16 cognitively normal control subjects (mean age 61.8 years) were analyzed. Study B: A phagocytosis by differentiated macrophages: Blood samples of 7 AD patients (mean age

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81 years and mean MMSE score 20.8/30) and 5 cognitively normal control subjects (mean age 69 years) were analyzed. Study C: Human leukocyte antigen DR (HLA DR) expression on neutrophils: Blood samples from six AD patients (mean age 71.8 years and mean MMSE score 21.8/30) and seven controls (mean age 61 years) were analyzed by flow cytometry and immunofluorescence. 2.3. Amyloid- phagocytosis and apoptosis (a) To test phagocytosis of A by freshly isolated monocytes, 100,000 PBMC's were incubated overnight in RPMI medium (GIBCO) with 10% fetal calf serum and A 1­42 (California Peptide Research, Inc, Napa, CA) (dissolved in dimethylsulfoxide and diluted to the final concentration 5 µg/ml in the medium [15]), cytospun, fixed with 4% paraformaldehyde, permeabilized using 0.1% Triton X-100, blocked with 1% bovine serum albumin, and stained using rabbit antibody to A 1­42 (Chemicon International, Inc) (1:200) specific for 6 amino acid sequence from the C-terminal of human A (1­42) and monoclonal anti-CD68 (1:500) (DAKO), followed by goat anti-mouse Alexa 488 IgG (1:100) and anti-rabbit Texas Red (TR) IgG (1:100). The nuclei were stained with dilute stock DAPI solution (1:300). In some experiments we used monoclonal anti-A (1­42) (Signet). (b) To test A phagocytosis and apoptosis by macrophages, monocytes were first differentiated into macrophages by 7- to 14-day cultivation of 20,000­ 50,000 PBMC's suspended in RPMI medium with 10% autologous human serum in 8 chamber polystyrene vessel TC treated glass slides (BD Falcon) (or in 24-well plates with glass coverslips). Differentiated macrophages were then exposed to A 1­42 conjugated to TR or fluorescein isothiocyanate (FITC) (5 µg/ml), incubated for 24 or 48 h and examined by fluorescence or confocal microscopy for A uptake and apoptosis. Apoptosis was determined using the FLICA DEVD-FMK caspase 3/7-assay kit (Immunohistochemistry Technologies, Bloomington, MN), which demonstrates a positive result in the cells comprising activated caspase-3 by the reaction with a fluorescent DEVD substrate. 2.4. HLA DR and COX-2 immunofluorescence Cytospun leukocytes were fixed in 4% paraformaldehyde and stored in 0.5% paraformaldehyde. They were washed with PBS, permeabilized with 0.1% Tri-

ton in PBS for 10 minutes, rewashed, blocked with 1% bovine serum albumin, and stained with the primary antibody monoclonal anti-COX-2 IgG1 (Cayman Chemical) (1:100), monoclonal anti-HLA DR (#297) (1:20) or normal mouse IgG isotype (the latter two from S. Ferrone, Roswell Park Cancer Institute), and the secondary Alexa 488-conjugated (1:100) goat anti-mouse IgG (Molecular Probes). Normal IgG1 isotype mouse serum produced negative results with AD neutrophils. The nuclei were stained with dilute stock DAPI solution (1:300). The preparations were examined using Olympus Bmax fluorescence microscope and Image Pro software for automatic scanning and measuring the areas of immunostaining.

2.5. Transmission electron microscopy (TEM) Macrophages, which differentiated on plastic coverslips (Ted Pella, Inc, Redding, CA) in RPMI medium with autologous serum, were fixed with 3% glutaraldehyde in 0.1 M sodium cacodylate buffer, dehydrated in an increasing ethanol gradient and embedded in conventional epon plastic. Thin sections were stained with lead and uranyl salts and examined with the FEI Morgagni TEM, as described [22].

2.6. Intracellular cytokine flow cytometry Multicolor fluorescent staining was performed using Intracellular Cytokine Staining Kit (BD Pharmingen, San Diego, CA), as recommended by the manufacturer. PBMC's were isolated by the Ficoll-Hypaque gradient technique. One million cells were cultured for 4 h at 37 C in 5% CO2 in RPMI medium with 10% fetal calf serum in the presence of 2 µL of activation cocktail (brefeldin (500 µg/ml), phorbol esters (2.5 µg/ml) plus ionomycin (25 µg/ml)). Neutrophils were isolated from the buffy coat of anticoagulated blood. After a wash, the cells were fixed using Cytofix/Cytoperm solution for 20 min, permeabilized in Perm/Wash solution and stained for 30 min using FITC-conjugated antibodies to cell surface markers, CD14 or CD15, and phycoerythrin (PE)-conjugated HLA-DR antibody (BD), or PE-conjugated anti-COX-2 (Cayman Co., Ann Arbor, Mich). After washing with Perm/Fix solution, the cells were resuspended in Staining Buffer and processed by flow cytometric analysis.

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Fig. 2. Monocytes of control subjects, but not those of AD patients, avidly phagocytize amyloid- in representative pictures of three control subjects' PBMC cytospins (a,b,c) and three AD patients' PBMC cytospins (d,e,f). One million of PBMC's of 17 AD patients and 16 control subjects were each incubated overnight in one ml of RPMI medium with 10% autologous serum and A (5 µg/ml); the cells were then cytospun onto glass slides, immunostained with anti-A and anti-CD68, examined by immunofluorescence microscopy, photographed at 40×, scanned by Image-Pro, and the areas and densities analyzed by t-test and U test, as described in the text. (Difference in the intensity of A fluorescence between patients and controls, P < 0.0001).

2.7. Statistical analysis The significance of monocyte data (Fig. 2) was determined by t-test analysis for equality of means with equal variances not assumed when Levene's test for equality of variances was found significant, or with equal variances assumed when Levene's test was not significant. The significance results were corroborated by non-parametric analysis using the Mann-Whitney U test (U test). The significance of cytokine data (Table 1) was performed using the Mann-Whitney U test (U test). Statistical testing was performed with the statistical software SPSS, Version 10.0 (SPSS, Chicago).

3. Results 3.1. Monocytes/macrophages infiltrate AD frontal lobe and hippocampus, the brain areas with neurons expressing RANTES and IL-1 CD68-positive macrophages infiltrated AD frontal lobe (Fig. 1A a,b,c) and hippocampus (Fig. 1A e,f,g) in conformity with previous findings [14]. To explain MM migration across the blood-brain barrier, we searched for the chemokine RANTES. The frontal lobe and hippocampal neurons expressed RANTES as shown with two different RANTES antibodies. RANTES was expressed in neuronal nuclei (Fig. 1B a,c) or cytoplasm

(Fig. 1B b). Some CD68-positive macrophages also expressed RANTES (not shown). Frontal lobe and hippocampal neurons also expressed the key innate immunity cytokine IL-1 but, unlike RANTES, the expression was cytoplasmic (Fig. 1C). To clarify the role of macrophages in clearance of A plaques, we examined the relation between the macrophage area and the residual A area in each plaque and found that the area or density of macrophage infiltration was not related to the residual A area and/or the density in each plaque (Pearson correlation, Spearman' rho, Kendall's tau of A area, density or area x density vs. macrophage area, density or area x density were insignificant), suggesting that ADMM's attempt A phagocytosis but are inefficient in performing it. This lack of A clearance despite attempted A phagocytosis by macrophages in AD brain (cf. Fig. 6 in [14]), prompted us to study the phagocytic function of MM's in living AD patients and control subjects. 3.2. Monocytes of AD patients are defective in phagocytosis of A In the first part of the study (years 2001­2002), we tested phagocytosis of A by the monocytes of 17 AD patients and 16 control subjects. Cytospun monocytes and neutrophils freshly isolated from peripheral blood of AD patients and controls carried A as shown by

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M. Fiala et al. / Ineffective phagocytosis of amyloid- by macrophages Table 1 Intracellular cytokines IL-10, IFN- and IL-12 are increased in AD patients compared to control subjects Subject Age group Years IL-10/CD4 Unstimulated Stimulated P = 0.009 P = 0.003 0 0 0 0 0 0 5 10 5 10 4 16 2 3 5 6 4 9 2 6 1 2 25 11 12 18 % cells positive for intracellular cytokines INF-IFN-/CD4 IL-12/CD14 Unstimulated Stimulated Unstimulated Stimulated P = 0.009 P = 0.027 P = 0.065 P = 0.093 0 1 0 0 0 0 0 0 0 0 2 3 8 12 3 5 2 4 6 12 4 13 3 5 1 3 2 2 2 2 10 10 4 12 7 12 2 8 0 2 0 5 2 5 16 13 18 12 6 8 7 8

Control #1 #2 #3 AD Case #1 #2 #3 Control #4 #5 #6 #7 #8 AD Case #4 #5

We

60­70

60­70 >70

>70

tested the intracellular cytokines by flow cytometry as described in the text. We analyzed the data by Mann-Whitney U test, which showed significantly higher percentage of CD4 T cells or CD14 monocytes positive for intracellular cytokines in AD patients (the probabilities are indicated).

immunofluorescence (data not shown). To examine the ability of monocytes to phagocytize A, we cultured PBMC's for 24­48 hours in RPMI medium with 10% fetal bovine serum and A (5 µg/ml) and immunostained A and CD68 in cytospun monocytes. Many monocytes of control subjects appeared enlarged with bright staining for A, whereas monocytes of AD patients showed uniformly small size with dim staining for A (Fig. 2). To analyze the differences in uptake, we measured area x density of intracellular A in 30 AD and control monocytes and analyzed the differences by Mann-Wilcoxon U test or t-test for equality of means. Control monocytes ingested significantly more A compared to AD monocytes [(U test, P < 0.0001; t test, P < 0.0001)]. In the second part of the study (years 2003­2004),we examined the fate of intracellular A after phagocytosis by macrophages. 3.3. Macrophages of AD patients are defective in phagocytosis of A and suffer apoptotic cell death; control macrophages are efficient phagocytes of A After 1-3 weeks in culture in RPMI with 10% autologous serum, the control MM's showed excellent differentiation appearing as large adherent "fried-egg" macrophages with processes (see Figs 3A and B a,b,c,d) and vigorous phagocytosis of A into perinuclear vacuoles (resembling lysosomes) as seen by fluorescence and confocal microscopy (right panels in Figs 3A and 3B). In comparison, ADMM's showed poor differen-

tiation into fewer smaller macrophages and weak surface uptake of A(left panels in Figs 3A and 3B). TEM showed A phagocytosis by control MM's into cytoplasmic vacuoles (Fig. 3C). Upon 48-h exposure to A control macrophages showed a lack of apoptosis, complete clearance of A and aggregation into giant cells without any sign of apoptosis (Figs 3D d and e), whereas macrophages of AD subjects showed cell rounding and a strong apoptotic FLICA signal (Figs 3D a and b). To elucidate systemic immune activation, we examined expression of HLA DR and COX-2 on circulating monocytes and neutrophils and performed a pilot study of intracellular cytokines in PBMC's. 3.4. Neutrophils of AD patients over express HLA DR and COX-2 Freshly isolated cytospun neutrophils of six AD patients over expressed HLA DR in comparison to seven control subjects' neutrophils as demonstrated by both immunofluorescence of cytospun PBMC's (Fig. 4A) and flow cytometry (Fig. 4B). Neutrophils of AD patients also over expressed COX-2 compared to control neutrophils (Fig. 4B). 3.5. AD CD4 T cells and AD monocytes over express intracellular TH1 and TH2 cytokines We have tested intracellular cytokines, IL-10, IFN and IL-12, in a small sample of patients (n = 5) and control (n = 8) subjects and analyzed the data in

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Fig. 3. Monocytes of control subjects, but not those of AD patients, differentiate into macrophages, which efficiently phagocytize A A. Immunofluorescence microscopy and B. Confocal microscopy: 100,000 monocytes of 4 AD patients (left panel ­ each row represents a different AD patient) and 4 control subjects (right panel ­ each row represents a different subject.) were each differentiated in RPMI 1640 medium with 10% autologous serum for 7­14 days and then exposed to Texas Red-A (5 µg/ml) overnight and examined by phase contrast microscopy (a, b, c) (20 × objective), fluorescence microscopy (e,f,g) (20 × objective), or confocal microscopy (d is overlay of 20 sections, h is a middle z-section) (100 × objective) C. Transmission electron microscopy: 100,000 PBMC's were differentiated as described above on plastic coverslips in a 24-well TC plate and then were challenged with Texas Red-A (5 µg/ml) overnight and examined by transmission electron microscopy. D. Apoptosis of AD macrophages exposed to A; Differentiated macrophages of an AD patient (a,b) and a control subject (d,e) were exposed for 48 h to A (5 µg/ml) and examined by phase contrast microscopy (a,d) and the FLICA assay (b,e) (green fluorescence indicates caspase-3 reaction with the fluorescent substrate).

relation to age (Table 1). The results suggest that (a) intracellular cytokines increase with age, (b) in each age group, IL-10, IFN- and IL-12 are increased in AD patients compared to control subjects.

4. Discussion This paper outlines the roles of the innate and adaptive immune systems in AD pathogenesis. Here we

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Fig. 4. Peripheral blood neutrophils of AD patients over express HLA DR and COX-2: A. Representative cytospin preparations of three AD patients' leukocytes (left panel, each row is a different patient) and three control subjects (right panel, each row is a different subject) stained with DAPI (left columns in each panel) and HLA DR (right columns in each panel) B. Flow cytometry of AD (light color bar) and control (dark color bar) CD14 monocytes for COX-2 and HLA DR PBMC's of six AD patients and 7 control subjects, which were freshly isolated from peripheral blood, were either (A.) cytospun on glass slides and stained with anti HLADR and fluorescent anti-mouse IgG and photographed at 100×, or (B.) stained with FITC-anti-CD15 and PEanti-HLA-DR or PE- anti-COX-2, and examined by flow cytometry.

show in the AD brain inefficient A phagocytosis by macrophages and neuronal cytokine signaling, and in the blood defective innate immunity and dysregulated adaptive immunity. Our previous [13] and current IHC studies show that macrophages and, to a lesser extent, CD8 T cells and neutrophils infiltrate AD brain. MM's might be attracted into the neuropil by RANTES and IL-1 signals from neurons. Macrophages, in addition to previously considered microglia, infiltrate A plaques, but the clearance of A by these phago-

cytes is inefficient. Brain macrophages and microglia have been placed at the center of AD pathogenesis by two schools. One school considers microglia as perpetrators of the pathology through induction of cytokines and superoxide ions [23,24,32,48]. The other school contends that microglia might be beneficial because they could be induced to clear the brain of A burden [42]. Anti-A antibodies [53], low density lipoprotein (LDL)-receptor-related protein-1, and 2 macroglobulin- and apolipoprotein E [43] participate

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in the clearance of soluble A, but these mechanisms are clearly exceeded in AD brain. However, phagocytic cells may be capable of clearance of A deposits, possibly completely silently in normal subjects before the A build-up. Microglia show good phagocytosis through the scavenger receptors A and B [11, 39] but only poor degradation and a variable release of A [7]. Phagocytosis of A by microglia is increased by macrophage colony stimulating factor (MCSF) [34] and overexpression of the M-CSF receptor [33]. As shown herein, normal human monocytes and macrophages appear to have a remarkable ability to phagocytize and degrade or expel A. Inflammation in AD brain had been attributed mainly to local mechanisms involving in particular brain microglia, the purveyors of innate immunity in the brain [2,30]. Our current flow cytometric and immunofluorescence data in peripheral blood cells together with previous immunocytochemical data showing infiltration by macrophages and T cells in AD brain [14] suggest that the immune activation in AD involves both the brain-specific and systemic innate immunity as well as the adaptive immune system. An immunocytochemical study [50] revealed increased HLA DR immunoreactivity in AD brain involving "microglial" cells both in perivascular location and within senile plaques. Cytokine dysregulation in AD could explain HLA DR over expression, since major histocompatibility complex (MHC) class II expression can be induced on neutrophils by combined stimulation with granulocyte/macrophage colony stimulating factor, interferon- and IL-3 [18,44]. The intracellular cytokine assay results suggest that the cytokines IL-10 and IFN- are over expressed in T cells and the cytokines IL-12 are over expressed in monocytes of AD patients. IL-10 is a cytokine that strongly inhibits the function of monocytes/macrophages [5] and could play a role in the abnormal phagocytosis of A in AD. We propose the following hypothesis of AD pathogenesis: The neurons are stimulated by A deposits, oxidative stress, hyperphosphorylated or pathogenic microorganisms to signal for help and to attract MM's across BBB through chemokines and cytokines [15]. In AD, however, these MM's are defective and, instead of providing help, disrupt BBB, produce neuronotoxic cytokines, invade but only ineffectively phagocytize A deposits and suffer apoptotic cell death with release of A. Release of A at the vessel wall could be one of the factors in congophilic angiopathy. In normal subjects, MM's migrate and phagocytize A at a physiologic pace and thus forestall accumulation of A. Thus

physiological innate and adaptive immune responses to A are beneficial in clearance of A but, in pathological states either due to overproduction of A (familial AD) or deficiency of A clearance (sporadic AD), the detrimental effects and lack of beneficial effects tilt the balance to neuronal pathology. Our preliminary results suggest that a crucial defect of AD macrophages is not in binding A but in intracellular signaling through NFB. The proposed pathogenetic mechanism provides an approach to diagnosing and improving the immune system in AD by manipulation of blood cells, unlike the alternative longstanding hypothesis of localized intracerebral inflammation and local brain clearance mechanisms. Vaccination with A has been vigorously investigated in animal models [4] and AD patients (reviewed in [52]). Vaccination with A induced encephalitis in some patients, which could be related to complement activation [29] and other mechanisms. The phagocytic deficiency of the macrophage/microglial system in AD implies that the reliance on the adaptive immune system, B cells and antibodies, to increase A clearance will not work. The deficiencies observed in vitro may mean that in vivo the phagocytes of AD patients are unable to efficiently phagocytize A and exposure to A may actually damage the innate immune system. Our hypothesis highlights the role of the innate immune system in AD pathogenesis. In the rat brain, peripheral stimulation with bacterial lipopolysaccharide initiates a progressive innate immune response in the brain [21]. The innate immune response may exert both neuroprotection and neurodegeneration [37]. Neuroprotection is provided by microglia, which produce IL1 and potentiate the production of neurotrophic insulinlike growth factor I (IGF-I) by astrocytes [27] and other mechanisms. Neurodegeneration is caused by tumor necrosis factor- through its type 1 receptor [3]. Although the extrapolation from the rat pathology to human disease is difficult, the dual beneficial and detrimental effects of the innate immunity likely operate in the human brain. Our data show that the macrophage response is deficient whereas the T cell and monocyte production of cytokines is dysregulated. The implications of these perturbations could lead to novel approaches to the immunodiagnosis and therapy of AD emphasizing amelioration of macrophage function by hormonal and growth factors abundant in the serum in young age [12].

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Acknowledgement This study was supported by the grant "Blood-Brain Barrier in Alzheimer's Disease" from Alzheimer's Disease Association to M.F. We thank W. Yang for assistance with preparation of the manuscript. This work is indebted for advice to M. Witte, University of Arizona and H. Vinters, UCLA. The UCLA Brain Bank, Department of Pathology and Laboratory Medicine (Neuropathology) provided the brain tissues of patients with Alzheimer's disease and control patients. We thank S. Ferrone, Roswell Park Cancer Institute for HLA DR antibodies and C. Glabe, UCI, for amyloid- conjugates. Michael Graves receives support for research related to the topic of this article from several pharmaceutical companies.

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