CD36, a Class B Scavenger Receptor, Is Expressed on Microglia in Alzheimer's Disease Brains and Can Mediate Production of Reactive Oxygen Species in Response to β-Amyloid Fibrils
2002; Elsevier BV; Volume: 160; Issue: 1 Linguagem: Inglês
10.1016/s0002-9440(10)64354-4
ISSN1525-2191
AutoresIndra Sethy Coraci, Jens Husemann, Joan W. Berman, Christine Hulette, Jennifer Dufour, Gabriele K. Campanella, Andrew D. Luster, Samuel C. Silverstein, Joseph El Khoury,
Tópico(s)Peroxisome Proliferator-Activated Receptors
ResumoA pathological hallmark of Alzheimer's disease is the senile plaque, composed of β-amyloid fibrils, microglia, astrocytes, and dystrophic neurites. We reported previously that class A scavenger receptors mediate adhesion of microglia and macrophages to β-amyloid fibrils and oxidized low-density lipoprotein (oxLDL)-coated surfaces. We also showed that CD36, a class B scavenger receptor and an oxLDL receptor, promotes H2O2 secretion by macrophages adherent to oxLDL-coated surfaces. Whether CD36 is expressed on microglia, and whether it plays a role in secretion of H2O2 by microglia interacting with fibrillar β-amyloid is not known. Using fluorescence-activated cell sorting analysis and immunohistochemistry, we found that CD36 is expressed on human fetal microglia, and N9-immortalized mouse microglia. We also found that CD36 is expressed on microglia and on vascular endothelial cells in the brains of Alzheimer's disease patients. Bowes human melanoma cells, which normally do not express CD36, gained the ability to specifically bind to surfaces coated with fibrillar β-amyloid when transfected with a cDNA encoding human CD36, suggesting that CD36 is a receptor for fibrillar β-amyloid. Furthermore, two different monoclonal antibodies to CD36 inhibited H2O2 production by N9 microglia and human macrophages adherent to fibrillar β-amyloid by ∼50%. Our data identify a role for CD36 in fibrillar β-amyloid-induced H2O2 production by microglia, and imply that CD36 can mediate binding to fibrillar β-amyloid. We propose that similar to their role in the interaction of macrophages with oxLDL, class A scavenger receptors and CD36 play complimentary roles in the interactions of microglia with fibrillar β-amyloid. A pathological hallmark of Alzheimer's disease is the senile plaque, composed of β-amyloid fibrils, microglia, astrocytes, and dystrophic neurites. We reported previously that class A scavenger receptors mediate adhesion of microglia and macrophages to β-amyloid fibrils and oxidized low-density lipoprotein (oxLDL)-coated surfaces. We also showed that CD36, a class B scavenger receptor and an oxLDL receptor, promotes H2O2 secretion by macrophages adherent to oxLDL-coated surfaces. Whether CD36 is expressed on microglia, and whether it plays a role in secretion of H2O2 by microglia interacting with fibrillar β-amyloid is not known. Using fluorescence-activated cell sorting analysis and immunohistochemistry, we found that CD36 is expressed on human fetal microglia, and N9-immortalized mouse microglia. We also found that CD36 is expressed on microglia and on vascular endothelial cells in the brains of Alzheimer's disease patients. Bowes human melanoma cells, which normally do not express CD36, gained the ability to specifically bind to surfaces coated with fibrillar β-amyloid when transfected with a cDNA encoding human CD36, suggesting that CD36 is a receptor for fibrillar β-amyloid. Furthermore, two different monoclonal antibodies to CD36 inhibited H2O2 production by N9 microglia and human macrophages adherent to fibrillar β-amyloid by ∼50%. Our data identify a role for CD36 in fibrillar β-amyloid-induced H2O2 production by microglia, and imply that CD36 can mediate binding to fibrillar β-amyloid. We propose that similar to their role in the interaction of macrophages with oxLDL, class A scavenger receptors and CD36 play complimentary roles in the interactions of microglia with fibrillar β-amyloid. Alzheimer's disease (AD) is the most common neurodegenerative disease of adults.1Selkoe DJ The origins of Alzheimer disease: a is for amyloid.JAMA. 2000; 283: 1615-1617Crossref PubMed Scopus (270) Google Scholar It is characterized by the extracellular deposition of insoluble fibrillar β-amyloid protein (fAβ) in the brain parenchyma.2Wisniewski HM Robe A Zigman W Silverman W Neuropathological diagnosis of Alzheimer disease.J Neuropathol Exp Neurol. 1989; 48: 606-609Crossref PubMed Scopus (70) Google Scholar Intraparenchymal deposits of fAβ are composed of Aβ peptides 40 to 42 amino acids in length, surrounded by activated microglia, astrocytes, and dystrophic neurites,1Selkoe DJ The origins of Alzheimer disease: a is for amyloid.JAMA. 2000; 283: 1615-1617Crossref PubMed Scopus (270) Google Scholar, 2Wisniewski HM Robe A Zigman W Silverman W Neuropathological diagnosis of Alzheimer disease.J Neuropathol Exp Neurol. 1989; 48: 606-609Crossref PubMed Scopus (70) Google Scholar all of which constitute the senile plaque. Inflammation, initiated by cellular reactions to these intraparenchymal deposits of fAβ, is thought to play an important role in the pathogenesis of AD.3El Khoury J Hickman SE Thomas CA Loike JD Silverstein SC Microglia, scavenger receptors, and the pathogenesis of Alzheimer's disease.Neurobiol Aging. 1998; 19: S81-S84Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 4Akiyama H Inflammatory response in Alzheimer's disease.Tohoku J Exp Med. 1994; 174: 295-303Crossref PubMed Scopus (79) Google Scholar Microglia, the brain's mononuclear phagocytes, are thought to be the principal cells that mediate this inflammation.3El Khoury J Hickman SE Thomas CA Loike JD Silverstein SC Microglia, scavenger receptors, and the pathogenesis of Alzheimer's disease.Neurobiol Aging. 1998; 19: S81-S84Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 4Akiyama H Inflammatory response in Alzheimer's disease.Tohoku J Exp Med. 1994; 174: 295-303Crossref PubMed Scopus (79) Google Scholar, 5Meda L Cassatella MA Szendrei GI Otvos Jr, L Baron P Villalba M Ferrari D Rossi F Activation of microglial cells by beta-amyloid protein and interferon-gamma.Nature. 1995; 374: 647-650Crossref PubMed Scopus (1255) Google Scholar The interaction of neonatal microglia with fAβ in vitro stimulates these cells to produce proinflammatory and potentially neurotoxic substances such as nitric oxide, tumor necrosis factor-α,5Meda L Cassatella MA Szendrei GI Otvos Jr, L Baron P Villalba M Ferrari D Rossi F Activation of microglial cells by beta-amyloid protein and interferon-gamma.Nature. 1995; 374: 647-650Crossref PubMed Scopus (1255) Google Scholar and reactive oxygen species (ROS).6El Khoury J Hickman SE Thomas CA Cao L Silverstein SC Loike JD Scavenger receptor-mediated adhesion of microglia to beta-amyloid fibrils.Nature. 1996; 382: 716-719Crossref PubMed Scopus (677) Google Scholar, 7Bianca VD Dusi S Bianchini E Dal Pra I Rossi F Beta-amyloid activates the O-2 forming NADPH oxidase in microglia, monocytes, and neutrophils. A possible inflammatory mechanism of neuronal damage in Alzheimer's disease.J Biol Chem. 1999; 274: 15493-15499Crossref PubMed Scopus (276) Google Scholar Removal of microglia from cultures containing mixed brain cells and fAβ almost totally eliminates the toxic effects of fAβ on primary neurons,8Giulian D Haverkamp LJ Yu JH Karshin W Tom D Li J Kirkpatrick J Kuo LM Roher AE Specific domains of beta-amyloid from Alzheimer plaque elicit neuron killing in human microglia.J Neurosci. 1996; 16: 6021-6037Crossref PubMed Google Scholar suggesting that microglia, and/or substances they produce, mediate the neurotoxic effects of fAβ. Microglia express class A scavenger receptors (SR-A).9Bell MD Lopez-Gonzalez R Lawson L Hughes D Fraser I Gordon S Perry VH Upregulation of the macrophage scavenger receptor in response to different forms of injury in the CNS.J Neurocytol. 1994; 23: 605-613Crossref PubMed Scopus (99) Google Scholar In neonatal microglia these receptors promote endocytosis of fAβ in suspension,10Paresce DM Ghosh RN Maxfield FR Microglial cells internalize aggregates of the Alzheimer's disease amyloid beta-protein via a scavenger receptor.Neuron. 1996; 17: 553-565Abstract Full Text Full Text PDF PubMed Scopus (590) Google Scholar and adhesion of microglia to fAβ-containing surfaces.6El Khoury J Hickman SE Thomas CA Cao L Silverstein SC Loike JD Scavenger receptor-mediated adhesion of microglia to beta-amyloid fibrils.Nature. 1996; 382: 716-719Crossref PubMed Scopus (677) Google Scholar SR-A expression is enhanced in microglia in brains of AD patients compared to brains of individuals of similar age who do not have AD,11Christie RH Freeman M Hyman BT Expression of the macrophage scavenger receptor, a multifunctional lipoprotein receptor, in microglia associated with senile plaques in Alzheimer's disease.Am J Pathol. 1996; 148: 399-403PubMed Google Scholar, 12Honda M Akiyama H Yamada Y Kondo H Kawabe Y Takeya M Takahashi K Suzuki H Doi T Sakamoto A Ookawara S Mato M Gough PJ Greaves DR Gordon S Kodama T Matsushita M Immunohistochemical evidence for a macrophage scavenger receptor in Mato cells and reactive microglia of ischemia and Alzheimer's disease.Biochem Biophys Res Commun. 1998; 245: 734-740Crossref PubMed Scopus (48) Google Scholar and in the brains of transgenic mice expressing a mutated form of the human amyloid precursor protein (APP23), which develop AD-like pathology.13Bornemann KD Wiederhold KH Pauli C Ermini F Stalder M Schnell L Sommer B Jucker M Staufenbiel M Aβ-induced inflammatory processes in microglia cells of APP23 transgenic mice.Am J Pathol. 2001; 158: 63-73Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar It is not known whether microglial expression of other scavenger receptors is affected in AD. Like fAβ, oxidized low-density lipoprotein (oxLDL) is a ligand for SR-A. OxLDL is also a ligand for CD36.14Endemann G Stanton LW Madden KS Bryant CM White RT Protter AA CD36 is a receptor for oxidized low density lipoprotein.J Biol Chem. 1993; 268: 11811-11816Abstract Full Text PDF PubMed Google Scholar We have shown that SR-A and CD36 play complementary roles in mediating adhesion of human monocyte-derived macrophages to surfaces coated with oxLDL and in secretion of ROS.15Maxeiner H Husemann J Thomas CA Loike JD El Khoury J Silverstein SC Complementary roles for scavenger receptor A and CD36 of human monocyte-derived macrophages in adhesion to surfaces coated with oxidized low-density lipoproteins and in secretion of H2O2.J Exp Med. 1998; 188: 2257-2265Crossref PubMed Scopus (75) Google Scholar SR-A participates in adhesion of macrophages to oxLDL-coated surfaces, whereas CD36 signals ROS production but is not required for adhesion to these surfaces. The similarities between interactions of microglia with fAβ and of macrophages with oxLDL led us to test expression of CD36 on microglia in vitro and in brains of AD patients and to determine whether it plays a role in fAβ-induced secretion of ROS by microglia. We report here that CD36 is expressed on microglia in vitro and in AD brains and that it cooperates with adhesion-promoting receptors in signaling secretion of ROS by microglia and macrophages in vitro. We used several anti-CD36 monoclonal antibodies (mAbs) obtained as follows: SMϕ (Sigma Chemical Co., St. Louis, MO), NL07 (Pharmingen, San Diego, CA), and FA6-152 (Biodesign International, Kennebunk, ME). Monoclonal antibody anti-CD11b (M1/70) was from Caltag (Burlingame, CA). Rabbit anti-human glial fibrillary acidic protein (GFAP) was a generous gift from Dr. James Goldman, Columbia University, New York, NY.16Goldman JE Chiu FC Growth kinetics, cell shape, and the cytoskeleton of primary astrocyte cultures.J Neurochemistry. 1984; 42: 175-184Crossref PubMed Scopus (78) Google Scholar Biotin-SP-conjugated Affinipure F(ab)2 fragments of rabbit anti-mouse IgG, donkey anti-mouse IgG, and goat anti-human IgG were from Jackson ImmunoResearch (West Grove, PA). Control rat IgG2b was from Zymed (San Francisco, CA), control rabbit anti-serum and monoclonal mouse IgG1 were from Sigma Chemical Co. Control antibody, MOPC 104E was from Organon Teknika Corp. (Cappel Research Products, Durham, NC). Fluorescein isothiocyanate-labeled goat anti-mouse IgG for fluorescence-activated cell sorting (FACS) analysis was from BD Pharmingen (San Diego, CA). Streptavidin-Alexa 568, Amplex Red hydrogen peroxide assay kit, and CyQuant cell proliferation assay kit were from Molecular Probes Inc. (Eugene, OR). Tyramide signal amplification kit (TSA) for immunohistochemistry was from New England Nuclear (Boston, MA). Amyloid-β peptide 25-35 was from Sigma Chemical Co. or from Bachem Bioscience Inc. (King of Prussia, PA). Amyloid-β peptides 1-42, 42-1, and 35-25 (synthetic peptides with the reverse sequences of Aβ 1-42 and 25-35, respectively) were from Bachem Bioscience Inc. or American Peptide Company (Sunnyvale, CA). Thrombospondin-1 (TSP-1) and phorbol-12-myristate acetate were from Sigma Chemical Co. The murine microglial cell line N9, was a generous gift from Dr. P. Ricciardi-Castagnoli (University of Milano, Bicocca, Italy)17Righi M Sassano M Valsasnini P Shammah S Ricciardi-Castagnoli P Activation of the M-CSF gene in mouse macrophages immortalized by retroviruses carrying a v-myc oncogene.Oncogene. 1991; 6: 103-111PubMed Google Scholar and was cultured as previously described13Bornemann KD Wiederhold KH Pauli C Ermini F Stalder M Schnell L Sommer B Jucker M Staufenbiel M Aβ-induced inflammatory processes in microglia cells of APP23 transgenic mice.Am J Pathol. 2001; 158: 63-73Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar in RPMI 1640 medium (Life Technologies, Inc., Long Island, NY) supplemented with 10% heat-inactivated fetal bovine serum and penicillin (100 U/ml) and streptomycin (100 μg/ml). Microglia were isolated from human fetal tissue provided by the Fetal Tissue Bank at Albert Einstein College of Medicine as described.18McManus CM Brosnan CF Berman JW Cytokine induction of MIP-1 alpha and MIP-1 beta in human fetal microglia.J Immunol. 1998; 160: 1449-1455PubMed Google Scholar The cells were maintained in Dulbecco's modified Eagle's medium with 10% heat-inactivated fetal bovine serum, penicillin (100 U/ml), streptomycin (100 μg/ml), and 25 mmol/L Hepes. Human monocyte-derived macrophages were prepared as described.19El Khoury J Thomas CA Loike JD Hickman SE Cao L Silverstein SC Macrophages adhere to glucose-modified basement membrane collagen IV via their scavenger receptors.J Biol Chem. 1994; 269: 10197-10200Abstract Full Text PDF PubMed Google Scholar Bowes human melanoma cells were obtained from American Type Culture Collection (Rockville, MD), and were maintained in Dulbecco's modified Eagle's medium and 10% heat-inactivated fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 μg/ml). The mammalian expression vector for human CD36 (CDM8-CD36) was a generous gift from Dr. Brian Seed (Massachusetts General Hospital, Boston, MA).20Oquendo P Hundt E Lawler J Seed B CD36 directly mediates cytoadherence of Plasmodium falciparum parasitized erythrocytes.Cell. 1989; 58: 95-101Abstract Full Text PDF PubMed Scopus (401) Google Scholar A stable Bowes melanoma cell line expressing human CD36 was generated by co-transfecting CDM8-CD36 with vector pcDNA-neo (Invitrogen, Carlsbad, CA) using the calcium phosphate co-precipitation method and selection in G418 sulfate (Invitrogen) as described previously for SR-A.19El Khoury J Thomas CA Loike JD Hickman SE Cao L Silverstein SC Macrophages adhere to glucose-modified basement membrane collagen IV via their scavenger receptors.J Biol Chem. 1994; 269: 10197-10200Abstract Full Text PDF PubMed Google Scholar Mock-transfected cells were generated by co-transfecting CDM8 (not containing CD36 cDNA) and pcDNA-neo in Bowes cells as described above. Aβ 25-35 and Aβ 35-25 were dissolved in phosphate-buffered saline (PBS) at 1 mg/ml and incubated at 37°C for 1 day.6El Khoury J Hickman SE Thomas CA Cao L Silverstein SC Loike JD Scavenger receptor-mediated adhesion of microglia to beta-amyloid fibrils.Nature. 1996; 382: 716-719Crossref PubMed Scopus (677) Google Scholar Aβ 1-42 and 42-1 were dissolved in either double-distilled H2O (ddH2O) alone at 1 mg/ml, or ddH2O followed by 10× PBS to a final concentration of 1 mg/ml and incubated at 37°C for 3 to 4 days as described.6El Khoury J Hickman SE Thomas CA Cao L Silverstein SC Loike JD Scavenger receptor-mediated adhesion of microglia to beta-amyloid fibrils.Nature. 1996; 382: 716-719Crossref PubMed Scopus (677) Google Scholar Fibril formation was confirmed by several methods (see Results and Figure 1). Fibrils formed by Aβ 1-42 were visualized by transmission electron microscopy as described.21Walsh DM Hartley DM Condron MM Selkoe DJ Teplow DB In vitro studies of β-protein fibril assembly and toxicity provide clues to the aetiology of Flemish variant (Ala692→Gly) Alzheimer's disease.Biochem J. 2001; 355: 869-877Crossref PubMed Scopus (95) Google Scholar Briefly, Formvar-coated nickel grids were incubated on drops of Aβ 1-42 (1 mg/ml) in PBS for 1 minute and then on drops of 2% phosphotungstic acid for 1 minute. The samples were photographed in a Philips CM 10 transmission electron microscope at 80kV. Because of limitations in availability of primary human microglia, we used nitroblue tetrazolium (NBT) reduction22Rook GA Steele J Umar S Dockrell HM A simple method for the solubilisation of reduced NBT, and its use as a calorimetric assay for activation of human macrophages by gamma-interferon.J Immunol Methods. 1985; 82: 161-167Crossref PubMed Scopus (321) Google Scholar to measure ROS production by these cells. Fifty μl of RPMI containing 25 × 103 microglia and 1 mg/ml NBT (Molecular Probes) and 1 mg/ml bovine serum albumin (BSA) was added to each spot of Multispot slides (Shandon-Lipshaw, Philadelphia, PA) coated with the indicated amount and type of Aβ peptide and incubated for 30 minutes at 37°C. The medium on each spot was aspirated and the slides were washed three times in methanol and air-dried. (At this stage, cells can be visualized by light microscopy and the slides can be stored for additional analysis without risk of quenching or loss of signal.) To extract the reduced and precipitated formazan, 30 μl of 2 mol/L KOH was added to each spot followed by 35 μl of dimethyl sulfoxide. The entire KOH-dimethyl sulfoxide mixture was transferred to a 96-well plate and the optical density of each sample was read in a multiwell plate reader at 650 nm. H2O2 secretion by macrophages was assayed using 10-acetyl-3,7-dihydroxy-phenoxazine (Amplex Red) and horseradish peroxidase according to the manufacturer's protocol (Molecular Probes), using a Cytofluor II fluorescence plate reader at excitation 530 nm and emission 590 nm. Where indicated, cells were incubated with 20 μg/ml of anti-CD36-mAb (SMϕ or NLO7), control antibody (MOPC 104E), or 5 μg/ml of TSP-1 in KRBG-A for 30 minutes at room temperature before plating. Human fetal microglia or N9 cells were plated on coverslips and allowed to adhere at 37°C for 0.5 to 12 hours. The coverslips were then rinsed in Hepes-buffered saline (HBS) (125 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L KH2PO4, 10 mmol/L NaHCO3, 20 mmol/L Hepes, 5 mmol/L glucose, 1 mmol/L CaCl2, 1 mmol/L MgCl2), incubated in HBS containing 1% BSA for 30 minutes at 4°C to block nonspecific binding of antibodies. The coverslips were further incubated without fixation at 4°C for 30 minutes with HBS containing 1% BSA and 8 μg/ml mouse monoclonal anti-CD36 antibody FA6-152, or 8 μg/ml of MOPC-31c, an IgG1 isotype-matched control. The cells were washed with HBS on ice, incubated with a 1:200 dilution of Biotin-SP-conjugated Affinipure F(ab)2 fragments of rabbit anti-mouse IgG for 30 minutes, fixed in 3.7% formaldehyde for 10 minutes on ice, washed again with HBS, and incubated at room temperature for 30 minutes with a 1:1000 dilution of Streptavidin-Alexa 568 in HBS. Alexa 568 staining was imaged using a Zeiss LSM 410 confocal microscope at the confocal imaging core facility, Department of Anatomy and Cell Biology and the Herbert Irving Comprehensive Cancer Center at Columbia University. N9 microglia were suspended at 106 cells/ml in PBS containing 1% BSA and 20 μl/ml phycoerythrin-labeled SMϕ (PE-SMϕ) or MOPC (PE-MOPC 104E) antibody, and incubated for 30 minutes at 4°C. The cells were then washed three times in PBS to remove unbound antibodies, and suspended in PBS containing 1% BSA at 106/cells ml. To stain human monocyte-derived macrophages and Bowes melanoma cells transfected with human CD36 cDNA, the cells were incubated with the anti-CD36 antibody FA6-152 (10 μg/ml) for 30 minutes on ice in PBS containing 1% BSA, washed three times, followed by another 30-minute incubation with fluorescein isothiocyanate-labeled rabbit anti-mouse IgG. FACS analysis was performed using a Becton Dickinson FACSCalibur as previously described.19El Khoury J Thomas CA Loike JD Hickman SE Cao L Silverstein SC Macrophages adhere to glucose-modified basement membrane collagen IV via their scavenger receptors.J Biol Chem. 1994; 269: 10197-10200Abstract Full Text PDF PubMed Google Scholar Frontal brain samples from patients with AD, Parkinson's disease, amytropic lateral sclerosis, and other diseases were obtained from the Brain Bank, Department of Pathology, Columbia University; The Alzheimer's Disease and Schizophrenia Brain Bank; Department of Psychiatry, Mount Sinai School of Medicine; and The Kathleen Price Bryan Brain Bank, Duke University Medical Center. Eight-μm-thick brain sections were cut using a Minotome cryostat (International Equipment Co., Needham Heights, MA), collected on Fisherbrand Superfrost/Plus precleaned slides, mounted, dried for 2 hours at room temperature, permeabilized by immersion for 10 minutes in −20°C acetone, dried for 2 hours at room temperature, and immediately frozen at −80°C until needed. Staining of CD36 was performed using monoclonal anti-CD36 antibody FA6-152 and direct tyramide signal amplification using New England Nuclear's TSA direct kit (NEL701) to enhance the intensity of the signal achieved by traditional streptavidin-biotin staining. Tissues were stained according to manufacturer's instructions. Briefly, the tissues were blocked for 30 minutes, incubated for 1 hour with 20 μg/ml of FA6-152, or control IgG1. Slides were then gently washed in PBS, and incubated 30 minutes with a 1:200 dilution of biotin-conjugated donkey or rabbit anti-mouse IgG. Tissues were then washed again in PBS, and incubated with a 1:100 dilution of streptavidin-horseradish peroxidase for 30 minutes then washed with PBS again. The incubation time with tyramide-fluorescein was ∼6 minutes. To visualize astrocytes, some sections were co-stained with a 1:500 dilution of rabbit polyclonal antibody against human GFAP in HBS for 30 minutes at room temperature as described.16Goldman JE Chiu FC Growth kinetics, cell shape, and the cytoskeleton of primary astrocyte cultures.J Neurochemistry. 1984; 42: 175-184Crossref PubMed Scopus (78) Google Scholar To visualize microglia, some sections were co-stained with 1 μg/ml of rat monoclonal antibody M1/70 against mouse and human Mac1 (CD11b/CD18)23Ault KA Springer TA Cross-reaction of a rat-anti-mouse phagocyte-specific monoclonal antibody (anti-Mac-1) with human monocytes and natural killer cells.J Immunol. 1981; 126: 359-364PubMed Google Scholar in HBS for 30 minutes at room temperature. Because microglia also are known to express IgG on their cell surface,24Giulian D Vaca K Inflammatory glia mediate delayed neuronal damage after ischemia in the central nervous system.Stroke. 1993; 24: 184-190Google Scholar identification of microglia in some sections was also verified by staining with goat anti-human IgG (data not shown) as described.24Giulian D Vaca K Inflammatory glia mediate delayed neuronal damage after ischemia in the central nervous system.Stroke. 1993; 24: 184-190Google Scholar All co-staining antibodies were visualized using a 1:3000 dilution of the appropriate Alexa 594-labeled secondary antibody. Identification of endothelial cells was performed using staining with a 1:500 dilution of fluorescein isothiocyanate-labeled Ricinus communis lectin (an established endothelial cell marker)25Nag S Ultrastructural localization of monosaccharide residues on cerebral endothelium.Lab Invest. 1985; 52: 553-558PubMed Google Scholar obtained from Sigma Chemical Co. as described.26Mannoji H Yeger H Becker LE A specific histochemical marker (lectin Ricinus communis agglutinin-1) for normal human microglia, and application to routine histopathology.Acta Neuropathol. 1986; 71: 341-343Crossref PubMed Scopus (187) Google Scholar Slides were mounted in Gel/Mount (Biomeda Corp., Foster City, CA) and coverslips sealed with nail polish before viewing with a Nikon Eclipse E800 microscope with a ×60 objective lens and digital camera (Scientific Instruments, MI) using advanced spot software. Images were assembled in Adobe Photoshop. Cell adhesion assays were performed as described previously using multispot slides.6El Khoury J Hickman SE Thomas CA Cao L Silverstein SC Loike JD Scavenger receptor-mediated adhesion of microglia to beta-amyloid fibrils.Nature. 1996; 382: 716-719Crossref PubMed Scopus (677) Google Scholar, 19El Khoury J Thomas CA Loike JD Hickman SE Cao L Silverstein SC Macrophages adhere to glucose-modified basement membrane collagen IV via their scavenger receptors.J Biol Chem. 1994; 269: 10197-10200Abstract Full Text PDF PubMed Google Scholar Where indicated, the Cyquan cell proliferation assay kit (Molecular Probes) was used to measure the number of adherent cells according to the manufacturer's instructions. We confirmed that Aβ 1-42 peptides prepared as described in Materials and Methods form fibrils in vitro by four different methods. First, we used established methods27Lorenzo A Yankner BA Beta-amyloid neurotoxicity requires fibril formation and is inhibited by Congo red.Proc Natl Acad Sci USA. 1994; 91: 12243-12247Crossref PubMed Scopus (1304) Google Scholar to confirm that fAβ 1-42 was birefringent under polarized light whereas Aβ 42-1 was not. Second, we coated glass slides with fAβ 1-42 or Aβ 42-1, incubated them with Thioflavin S and examined them by fluorescence microscopy as described.28Caputo CB Fraser PE Sobel IE Kirschner DA Amyloid-like properties of a synthetic peptide corresponding to the carboxy terminus of beta-amyloid protein precursor.Arch Biochem Biophys. 1992; 292: 199-205Crossref PubMed Scopus (26) Google Scholar Glass surfaces coated with Aβ 42-1 did not fluoresce after staining with Thioflavin S (Figure 1A), whereas those coated with fAβ 1-42 did (Figure 1B). Third, using transmission electron microscopy, we found that Aβ 1-42 formed thin elongated fibrils (Figure 1C) similar to those described by others.21Walsh DM Hartley DM Condron MM Selkoe DJ Teplow DB In vitro studies of β-protein fibril assembly and toxicity provide clues to the aetiology of Flemish variant (Ala692→Gly) Alzheimer's disease.Biochem J. 2001; 355: 869-877Crossref PubMed Scopus (95) Google Scholar Fourth, analysis of fAβ 1-42 by nonreducing SDS-PAGE and Coomassie-blue staining showed two bands, an ≅4.2-kd band (the monomeric form of the peptide), and a second band of ≅21 kd (likely to be pentamers of Aβ 1-42) (Figure 1D). As expected, Aβ 42-1 peptides ran at ≅4.2 kd indicating that they did not oligomerize (Figure 1D). Using more sensitive Western blotting and silver staining analyses other investigators have described up to four different bands under denaturing nonreducing conditions.21Walsh DM Hartley DM Condron MM Selkoe DJ Teplow DB In vitro studies of β-protein fibril assembly and toxicity provide clues to the aetiology of Flemish variant (Ala692→Gly) Alzheimer's disease.Biochem J. 2001; 355: 869-877Crossref PubMed Scopus (95) Google Scholar The data presented in Figure 1D suggests that the ≅21-kd species is the dominant oligomer after denaturation of fAβ 1-42 under nonreducing conditions. Taken together, the data presented in Figure 1 confirm that Aβ 1-42 used in the experiments reported here was fibrillar, whereas Aβ 42-1 was not. Murine microglia express scavenger receptor activity as evidenced by their ability to endocytose DiI-AcLDL24Giulian D Vaca K Inflammatory glia mediate delayed neuronal damage after ischemia in the central nervous system.Stroke. 1993; 24: 184-190Google Scholar and adhere to fAβ-containing surfaces.6El Khoury J Hickman SE Thomas CA Cao L Silverstein SC Loike JD Scavenger receptor-mediated adhesion of microglia to beta-amyloid fibrils.Nature. 1996; 382: 716-719Crossref PubMed Scopus (677) Google Scholar SR-A is expressed by microglia in normal brains11Christie RH Freeman M Hyman BT Expression of the macrophage scavenger receptor, a multifunctional lipoprotein receptor, in microglia associated with senile plaques in Alzheimer's disease.Am J Pathol. 1996; 148: 399-403PubMed Google Scholar and in increased amounts by microglia in the brains of patients with AD,11Christie RH Freeman M Hyman BT Expression of the macrophage scavenger receptor, a multifunctional lipoprotein receptor, in microglia associated with senile plaques in Alzheimer's disease.Am J Pathol. 1996; 148: 399-403PubMed Google Scholar, 12Honda M Akiyama H Yamada Y Kondo H Kawabe Y Takeya M Takahashi K Suzuki H Doi T Sakamoto A Ookawara S Mato M Gough PJ Greaves DR Gordon S Kodama T Matsushita M Immunohistochemical evidence for a macrophage scavenger receptor in Mato cells and reactive microglia of ischemia and Alzheimer's disease.Biochem Biophys Res Commun. 1998; 245: 734-740Crossref PubMed Scopus (48) Google Scholar and in the brains of transgenic APP23 mice with AD-like pathology.13Bornemann KD Wiederhold KH Pauli C Ermini F Stalder M Schnell L Sommer B Jucker M Staufenbiel M Aβ-induced inflammatory processes in microglia cells of APP23 transgenic mice.Am J Pathol. 2001; 158: 63-73Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar To test whether CD36 is expressed on microglia, we incubated N9 microglia with anti-CD36 monoclonal antibody PE-SMϕ or with an isotype-matched PE-labeled control IgM (PE-MOPC 104E) and analyzed the cells by FACS. N9 cells incubated with PE-SMϕ anti-CD36 were approximately four times more fluorescent than N9 cells incubated wi
Referência(s)