Alzheimer disease macrophages shuttle amyloid-beta from neurons to vessels, contributing to amyloid angiopathy.

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 [ 22 ]; and rabbit anti-fibrillar OC, which stains Aβ fibrils, as well as α-synuclein fibrils and islet amyloid polypeptide fibrils [ 23 ]. 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) [ 24 ]. 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 14 C-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 [ 29 ] 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 [ 13 ]. 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 [ 26 ]. 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 [ 27 ]. 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 [ 13 ]. The cultures were incubated with fibrillar or protofibrillar Aβ for 3 days. Macrophage apoptosis was determined using the FLICA VAD-FMK polycaspases 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. Brain tissues 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.

Statistical analysis

The data on monocyte migration and 14 C-Aβ transport were analyzed by t test and Mann–Whitney and Kruskall–Wallis tests.