Alzheimer disease macrophages shuttle amyloid-beta from neurons to vessels, contributing to amyloid angiopathy
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- Zaghi, J., Goldenson, B., Inayathullah, M. et al. Acta Neuropathol (2009) 117: 111. doi:10.1007/s00401-008-0481-0
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Neuronal accumulation of oligomeric amyloid-β (Αβ) is considered the proximal cause of neuronal demise in Alzheimer disease (AD) patients. Blood-borne macrophages might reduce Aβ stress to neurons by immigration into the brain and phagocytosis of Αβ. We tested migration and export across a blood-brain barrier model, and phagocytosis and clearance of Αβ by AD and normal subjects’ macrophages. Both AD and normal macrophages were inhibited in Αβ export across the blood-brain barrier due to adherence of Aβ-engorged macrophages to the endothelial layer. In comparison to normal subjects’ macrophages, AD macrophages ingested and cleared less Αβ, and underwent apoptosis upon exposure to soluble, protofibrillar, or fibrillar Αβ. Confocal microscopy of stained AD brain sections revealed oligomeric Aβ in neurons and apoptotic macrophages, which surrounded and infiltrated congophilic microvessels, and fibrillar Aβ in plaques and microvessel walls. After incubation with AD brain sections, normal subjects’ monocytes intruded into neurons and uploaded oligomeric Aβ. In conclusion, in patients with AD, macrophages appear to shuttle Aβ from neurons to vessels where their apoptosis may release fibrillar Aβ, contributing to cerebral amyloid angiopathy.
The amyloid hypothesis proposes that amyloid-β (Aβ) accumulation in the brain is the cause of Alzheimer disease . Aβ is potentially generated from Aβ precursor protein (APP) wherever APP and the β- and γ-secretases are located, such as the endoplasmic reticulum and Golgi apparatus, but most is produced at the plasma surface or in the secretory pathway . Aβ generation may also occur during autophagic turnover of APP-rich organelles . Increasing molecular weight assemblies of Aβ accumulate both extra- and intra-neuronally in Alzheimer disease (AD) brain ; of these assemblies, at least the intraneuronal oligomeric Aβ has pathological consequences . Aβ is cleared physiologically across the blood-brain barrier by low-density lipoprotein receptor-related protein-1 . Despite this physiological clearance, Aβ is found to accumulate in neurons and extracellular deposits since the first year of life. The pathological consequences in AD patients are intraneuronal accumulation of oligomeric Aβ in multivesicular bodies  and neuronal death. Surprisingly, the total neuronal load of Aβ is not predictive of neurofibrillary degeneration .
Brain amyloidosis of AD patients is considered to be related to insufficient clearance rather than over-expression of Aβ . Promising strategies for immune clearance of Aβ include Aβ vaccination , intravenous gamma-globulin , humanized anti-Aβ antibody (Bapineuzumab®), and transcriptional modulation of macrophages, such as by use of curcuminoids . Studies of AD brain tissues  and, recently, of APP transgenic mice brain tissues [28, 30, 37] suggest that blood-derived macrophages and microglia, rather than resident brain microglia, have a key role in the clearance of Aβ.
Previous studies have identified basic immune mechanisms necessary for clearance of Aβ by macrophages. Aβ-induced adhesion molecules and chemokines CCL3 (MIP-1 α), CCL4 (MIP-1β), and CCL2 [9, 17, 38] act on macrophages, microglia, and astrocytes, promoting monocyte migration, differentiation , and survival . In mouse macrophages and microglia, the class B scavenger receptor type 2 (CD36) is crucial for induction by Aβ of chemokines, cytokines, and reactive oxygen species [6, 34], and exposure to Aβ initiates the signaling cascade that links CD36 scavenger receptor to the actin cytoskeleton and diverse processes such as cellular migration, adhesion, and phagocytosis . Fucoidan inhibits both class A and B scavenger receptor interactions , but, in our experience, does not inhibit Aβ phagocytosis by human macrophages (Fiala et al. unpublished data). Recent studies suggest a role for Toll-like receptors (TLR’s) in Aβ clearance: TLR’s are transcriptionally downregulated in AD patients’ monocytes , and TLR-2 signaling enhances Aβ uptake by microglia . Importantly, mononuclear cells of most patients differ from those of normal subjects by transcriptional downregulation of β-1,4-mannosyl-glycoprotein 4-β-N-acetylglucosaminyltransferase (MGAT-3), an enzyme important in phagocytosis of Aβ .
Immunohistochemical studies brought new insights into the immune clearance of Aβ by macrophages in human brain. AD brain contains 11 times as many cyclooxygenase (COX-2)-positive macrophages as age-matched control brain , and a subset of these macrophages is inducible nitric oxide (NO) synthase (iNOS)-positive . Monocyte immigration might be orchestrated by neuronal and microglial chemokine RANTES and cytokine interleukin-1β (IL-1β) [13, 31]. In CCR2-deficient APP transgenic mice, clearance of Aβ is depressed . Despite the permeation of AD brain by macrophages, clearance of neuritic plaques is randomly incomplete, suggesting functional heterogeneity of AD macrophages .
AD is a human disease with specific immune and biochemical defects , which have not been engineered in APP transgenic mice. To clarify the pathophysiology of Aβ clearance in AD, we examined Aβ clearance using human brain tissues and human macrophages, and observed that the AD innate immune system is defective in proper disposal of Aβ in the brain.
Materials and methods
Antibodies and reagents
We stained macrophages using mouse anti-human CD68 (Dako, Carpinteria, CA) and goat anti-human CD68 (Santa Cruz Biotech, Santa Cruz, CA, USA). Neurons were stained with mouse anti-human neuronal nuclei (NeuN, Chemicon, Temecula, CA, USA); mouse anti-human microtubule associated protein 2 (MAP2, Sigma, St Louis, MO, USA); and rabbit anti-human neuron specific enolase (Immunostar, Hudson, WI, USA). To visualize Aβ in brain tissue and in macrophages, we utilized rabbit anti-Aβ 1-42 (COOH-terminal epitope) (Millipore, Billerica, MA, USA); mouse biotinylated anti-Aβ 1-42 (COOH-terminal epitope) (Signet); rabbit anti-oligomer A11 (Biosource, Carlsbad, CA, USA), which recognizes Aβ-42 and Aβ-40 pre-fibrillar oligomers ; and rabbit anti-fibrillar OC, which stains Aβ fibrils, as well as α-synuclein fibrils and islet amyloid polypeptide fibrils . To stain apoptotic markers, we used anti-caspase-6, -7, and -8 antibodies, which were raised in rabbits using catalytic subunits of the relevant autoprocessed recombinant caspases as immunogens (Burnham Institute, La Jolla, CA, USA) . Secondary antibodies were anti-mouse, anti-rabbit, and anti-goat IgG’s conjugated to Alexa Fluor 488, 555, and 647 (Invitrogen, Carlsbad, CA, USA). The reagents were monocyte chemotactic protein-1 (MCP-1) (PeproTech, Rocky Hill, NJ, USA); fluorescein isothiocyanate (FITC)-conjugated Aβ (Anaspec, San Jose, CA, USA); fibrillar FITC-Aβ and protofibrillar Aβ prepared by M. Inayathullah; and 14C-labeled Aβ from C. Glabe, UCI. Aβ was used at 2 μg/mL in most experiments.
Patients and controls
A total of ten patients [mean age 76.9 ± 5.8 years, mean Mini-Mental State Exam (MMSE) score of 21.7 ± 5.1] with a diagnosis of probable AD established by the National Institute of Neurological and Communication Disorders and Stroke/Alzheimer’s Disease and Related Disorders Association criteria  were recruited into the study since 2004 through the University of California, Los Angeles (UCLA), Alzheimer’s Disease Research Center under a UCLA Institutional Review Board-approved protocol. In addition, eight aged-matched control subjects (mean age 77.5 + 6.0 years) and three young control subjects (ages 20, 21, 44) were recruited from UCLA personnel and families of patients.
Isolation of PBMC’s
Peripheral blood mononuclear cells (PBMC’s) were isolated from venous blood of the AD patients and control subjects by Ficoll–Hypaque gradient centrifugation as previously described . Monocytes were purified using RosetteSep Monocyte Enrichment Cocktail (StemCell Technologies, Vancouver, BC, Canada) from PBMC’s of normal subjects (“normal monocytes” or “normal macrophages”) and AD patients (“AD monocytes” or AD macrophages”).
Preparation of fibrils and protofibrils of Aβ (1-42)
Aβ fibrils were prepared by dissolving 1 mg of Aβ (1-42) peptide in 100 μL of 10 mM NaOH. The solution was diluted to a volume of 0.5 mL with milliQ water followed by addition of 0.5 mL of 10 mM (2×) phosphate buffer (pH 7.4). The resulting solution was then centrifuged at 16,000×g for 10 min. One-half milliliter of the resulting supernatant was transferred into a new tube and incubated at 37°C for 7 days. The fibrils were pelleted out by centrifugation for 10 min and the supernatant was transferred to another tube for protofibril isolation. The fibril pellet was washed thrice with MilliQ water and the resulting pellet thereafter resuspended in MilliQ water after each wash.
Protofibrils were isolated from the supernatant by filtering through a Centricon filter (molecular weight cutoff of 35 kDa) to remove any small oligomers and monomers and collecting the filtrate. The pellet was washed three times with MilliQ and then reconstituted and diluted with MilliQ water. A small aliquot of each sample was analyzed by amino acid analysis to determine the protein concentration. The samples were characterized by size exclusion chromatography and electron microscopy (Fig. 2). Fibrillar and protofibrillar Aβ also was prepared in smaller amounts from FITC-Aβ.
Monocyte migration across a human blood-brain barrier model
A human blood-brain barrier model (BBB) model with primary human brain microvascular endothelial cells (BMVEC’s) was constructed in a 24-well plate as described previously [8, 12]. In the model, 50,000 BMVEC’s in passages 4–8 coated either the upper surface (“regular model”) or the lower surface of a porous membrane insert (Collaborative Biomedical Products, Bedford, MA, USA) (“reverse model”) which rests above a well. Both the well and the membrane insert contained RPMI medium with 10% fetal bovine serum or 10% autologous serum.
In migration experiments, 250,000 monocytes from two control subjects (ages 74 and 78) and two AD patients (ages 80 and 84 with MMSE scores 20 and 23, respectively) were allowed to migrate across the BBB for 17 h. The number of transmigrated cells was determined by triplicate cell counting in eight sections of a hemocytometer chamber. The inserts were washed gently with a buffer containing 0.1 M sodium cacodylate plus 0.2 M sucrose X2, then fixed with 3% glutaraldehyde in 0.1 M sodium cacodylate buffer for 1 h at 25°C . The cells were stored in this buffer at 4°C, post-fixed with 1% osmium tetroxide at 4°C, dehydrated in an ethanol series and embedded in plastic . One-micron sections were stained with toluidine blue and examined by bright field and transmission electron microscopy.
Aβ phagocytosis and apoptosis by macrophages
Macrophages of four controls (ages 74, 74, 81, 90) and four AD patients (ages 70, 77, 82, 86 with MMSE scores 15, 27, 19, 27, respectively) were prepared in 8-well chamber slides as described . The cultures were incubated with fibrillar or protofibrillar Aβ for 3 days. Macrophage apoptosis was determined using the FLICA VAD-FMK poly-caspases assay kit (Immunohistochemistry Technologies, Bloomington, MN, USA). This assay utilizes a membrane permeant, sulforhodamine B (SR)-labeled inhibitor targeted to all active caspases to covalently label apoptotic cells. Macrophage cultures were incubated with the FLICA apoptosis detection probe for 1 h at 37°C and then washed to remove any non-covalently bound probe from non-apoptotic cells. Cells were examined with an Olympus Bmax fluorescence microscope with 100× objective. Fluorescence density was determined by Image-Pro Plus 4.1 (Media Cybernetics, Silver Spring, MD). We examined the middle strip of each well for 6–9 consecutive fields with macrophages, analyzing the integrated optical density (IOD) of Aβ (green) and FLICA (red) per macrophage in three experiments for each group.
In a set of related experiments, control and AD macrophages were incubated with soluble FITC-Aβ for 1 h, or 3, 5, or 7, and apoptosis was detected using the FLICA assay. In some experiments, macrophages were incubated with soluble unconjugated Aβ for 1 h, washed twice, then incubated for 2, 4, or 6 days. Apoptosis was determined by the FLICA assay, and Aβ fibrils and oligomers were detected by the indirect technique using OC and A11 antibodies, respectively.
ELISA assay of Aβ clearance from monocytes
Purified monocytes (300,000 per sample) were incubated with soluble Aβ (2 μg/mL) for 2 h, washed four times, re-incubated for the indicated number of days, and the amount of intracellular Aβ remaining at each time point was determined (0, 1, 2, 3, 5, and 7 days). To measure the amount of intracellular Aβ, the cells were harvested into an ELISA lysis buffer (Invitrogen), supplemented with a protease inhibitor cocktail (Sigma) and assayed using the Aβ 1-42 ELISA kit (Invitrogen) by spectrophotometry. Monocytes from four control subjects and three AD patients were tested.
Frozen sections from the frontal lobe of two control subjects with no neuropathology (ages 23 and 37) and four AD patients were provided by the UCLA Brain Bank. The AD brain sections were from: (1) a 62-year-old patient with Binswanger encephalopathy and scattered senile plaques in the entorhinal cortex and hippocampus; (2) a 64-year-old Braak stage VI patient; (3) a 69-year-old Braak stage VI patient; (4) an 82-year-old Braak stage VI patient with Lewy body disease.
Co-incubation of PBMC’s with brain tissues
Frozen sections of post-mortem brain tissues of four AD patients and two controls were co-incubated with 500,000 PBMC’s (from seven normal donors, ages 20, 21, 72, 74, 74, 79, and 85 years old) for 1 to 6 days in Dulbecco’s Minimum Essential Medium (DMEM) with 10% fetal calf serum, washed with PBS, fixed with 4% paraformaldehyde, and processed for immunofluorescence.
In some experiments, PBMC’s were first pre-labeled with Qtracker 525, which distributes green Qdot nanocrystals in cytoplasmic vesicles, or CellTracker CMFDA (Invitrogen, Carlsbad, CA, USA), which undergoes an esterase reaction to produce a green fluorescent product in the cytoplasm. After isolation of PBMC’s from blood, cells were incubated with Qtracker or CellTracker according to the manufacturer’s recommendations, pelleted and washed twice with DMEM, and then incubated with tissues as above.
Immunofluorescence and confocal microscopy of brain tissues
Fixed tissues were permeabilized with 1% Triton X-100 and blocked with 1% bovine serum albumin (BSA) at 37°C for 30 min each. Brain sections were then incubated with various primary antibodies for 48 h at 4°C, washed with PBS, and incubated with appropriate secondary antibodies labeled with Alexa 488, Alexa 555, and Alexa 647 fluorophores for 1 h at 37°C. A mixture consisting of 0.2% Triton X-100 and 1% BSA was used as the diluent of both primary and secondary antibodies. As control staining, the sections were stained by secondary antibodies without primary antibodies. The preparations were examined using a Zeiss 510 Meta multiphoton confocal microscope or a Bio-Rad Laboratories MRC-1024 Es laser scanning confocal system attached to a Nikon E800 fluorescent microscope.
The data on monocyte migration and 14C-Αβ transport were analyzed by t test and Mann–Whitney and Kruskall–Wallis tests.
Aβ uptake by monocytes inhibits monocyte emigration and Aβ export across BBB
Aβ uptake by monocytes in the brain chamber inhibits monocyte migration and Aβ export into the blood chamber
77,250 ± 9,032
1,960 ± 198
13,750 ± 3,774
1,712 ± 94
29,600 ± 8,998
1,456 ± 168
5,375 ± 411
Normal monocytes bind and clear more Aβ in comparison to AD monocytes
Fibrillar, protofibrillar, and soluble Aβ induce apoptosis of AD macrophages
Το explore macrophage clearance of Aβ from AD brain, we first examined the distribution of Aβ assemblies in AD brain sections.
Soluble and oligomeric Aβ are present in neurons and macrophages, and fibrillar Aβ in plaques and congophilic microvessels
Monocytes upload soluble and oligomeric Aβ in neurons
In transgenic animal models of AD, Aβ-induced pathology seems to be upstream of tau and is considered the primary mechanism . We have attempted to glean the mechanisms responsible for clearance of Aβ from human brain by comparing the results of morphologic and experimental investigations. Although emigration of macrophages across the BBB has been infrequently investigated, macrophage immigration is well known in HIV-1 encephalitis and is becoming accepted in AD . Our results in the model BBB exclude the emigration of Aβ-engorged macrophages as a mechanism of Aβ clearance since these adhered to the endothelium and were inhibited in emigration. Thus the fate of Aβ in aging brain seems to depend upon its handling and degradation inside the BBB.
Our observations suggest that oligomeric Aβ in perivascular macrophages may contribute to the fibrillar Aβ in congophilic vessels. AD macrophages are defective in uptake of Aβ (Fig. 3), yet are particularly susceptible to apoptosis from all assembly states of Aβ (Figs. 4, 5), contrasting with the ability of control macrophages to phagocytize Aβ  and to resist apoptosis (Figs. 4, 5). The phagocytic propensity of AD macrophages is compounded by their low clearance of Aβ (Fig. 2). These deficits might be related to downregulation of transcription of certain genes, including MGAT-3 and Toll-like receptors .
In AD brain, macrophages are apoptotic through activation of caspases -6, -7, and -8 (Fig. 6e, f, g), and they abut and infiltrate congophilic microvessels from the outside  (Fig. 6d), in agreement with the assembly of Αβ fibrils outside of the basal lamina . Immunohistochemical studies of cerebral amyloid angiopathy (CAA) revealed its association with a significant increase and activation of macrophages in leptomeningeal and cortical vessels—implicating a central role for macrophages in amyloid angiopathy . Furthermore, the BBB in AD brain is impaired  by immigrating macrophages (Fig. 1d), thus allowing leakage of plasma proteins into the brain and further aggravation of angiopathy. Taken together, AD macrophages loaded with oligomeric Aβ seem to suffer apoptosis, disintegrate and release oligomeric and fibrillar Aβ into the wall of congophilic vessels. Still, it is important to recognize that although phagocytosis of Αβ is generally greater in control monocytes compared to AD monocytes, this is a multifactorial and heterogeneous effect, and monocytes of some aged control subjects occasionally demonstrate inferior innate immunity  and phagocytosis of Aβ (SI Fig. 1).
Proposed mechanisms for the pathogenesis of CAA have included production of Aβ by myocytes in vessel walls , derivation of Aβ from blood or cerebrospinal fluid, and, more recently, deposition of Aβ from interstitial fluid being drained from the central nervous system . In APP-transgenic mice, amyloid angiopathy developed specifically in areas of the brain that over-produce Aβ, suggesting that brain, as opposed to blood, is the major source of Aβ . In mice, tracers were injected intracerebrally and found to be distributed, similarly to Aβ, within the basement membranes of intracerebral capillaries and arteries, and in leptomeningeal arteries . Interestingly, within 24 h of injection the tracers were located in perivascular macrophages and these cells remained in brain microvessels, experiencing limited migration. Thus both drainage of the interstitial fluid and shuttling of Aβ by macrophages seem to lead to entrapment of Aβ in perivascular macrophages.
The results of the experimental co-culture of normal subjects’ monocytes with AD brain tissues show intrusion by these monocyte/macrophages into neurons and uploading of oligomeric Aβ, suggesting a protective neuronal mechanism by the normal innate immune system. However, AD monocyte/macrophages seem to be defective in such protection of neurons . Although we have not investigated this mechanism by electron microscopy of AD brain tissues, we have observed that macrophages and monocytes exposed to Aβ in vitro develop prolific microvilli and larger dendrites, which could reach into neurons and upload Aβ .
In animal models of amyloidosis, microglia were first noted for their proinflammatory role . Microglia of APOE4 transgenic mice produced more nitric oxide compared to APOE3 transgenic mice . Subsequently, microglia were found to provide clearance of Aβ and were assisted in clearing Aβ deposits by antibodies  and by complement opsonization [16, 45, 46]. Clearance of Aβ was recognized to include two phases: the first mediated by antibodies and the second by microglia and antibodies . However, in triple transgenic APP-thymidine kinase mice, bone marrow-derived rather than resident microglia restricted amyloid deposits as shown by ganciclovir destruction of microglia .
However, human brain disorders involve both microglial activation and macrophage recruitment . In AD brain, the initial inflammatory signal is induced by microglia  and neuronal chemokine RANTES. These stimuli attract large macrophages (clearly not resembling ramified microglia) to migrate across brain microvessels and invade neuritic plaques in AD brain . Enzyme studies mitigate against the role of microglia in Aβ clearance since, in comparison to macrophages, degradation of Aβ by microglia is poor due to defective lysosomal enzymes .
We thank J. Sayre for statistical analysis, S. Krajewski (Burnham Institute) for antibodies to activated caspases, B. Carter for nursing assistance with collection of blood specimens from patients and control subjects, B. Magrys for cutting sections of the blood-brain barrier model, C. Glabe for providing 14C-Αβ and A11 and OC antibodies, and C. Serhan for lipoxin A4. The UCLA Brain Bank provided the brain tissues for immunostaining.
Conflict of interest statement
This work was supported by the Alzheimer’s Association, MPBio Ltd (MF) and NIH grant AG027818 (DBT).
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