CD163 Identifies Perivascular Macrophages in Normal and Viral Encephalitic Brains and Potential Precursors to Perivascular Macrophages in Blood
2006; Elsevier BV; Volume: 168; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2006.050215
ISSN1525-2191
AutoresWoong‐Ki Kim, Xavier Álvarez, Jeanne Y. Fisher, Benjamin Bronfin, Susan V. Westmoreland, JoAnne McLaurin, Kenneth C. Williams,
Tópico(s)HIV Research and Treatment
ResumoPerivascular macrophages are uniquely situated at the intersection between the nervous and immune systems. Although combined myeloid marker detection differentiates perivascular from resident brain macrophages (parenchymal microglia), no single marker distinguishes perivascular macrophages in humans and mice. Here, we present the macrophage scavenger receptor CD163 as a marker for perivascular macrophages in humans, monkeys, and mice. CD163 was primarily confined to perivascular macrophages and populations of meningeal and choroid plexus macrophages in normal brains and in brains of humans and monkeys with human immunodeficiency virus or simian immunodeficiency virus (SIV) encephalitis. Scattered microglia in SIV encephalitis lesions and multinucleated giant cells were also CD163 positive. Consistent with prior findings that perivascular macrophages are primary targets of human immunodeficiency virus and SIV, all SIV-infected cells in the brain were CD163 positive. Using fluorescent dyes that definitively and selectively label perivascular macrophages in vivo, we confirmed that dye-labeled simian perivascular macrophages were CD163 positive and able to repopulate the central nervous system within 24 hours. Flow cytometric studies demonstrated a subset of monocytes (CD163+CD14+CD16+) that were immunophenotypically similar to brain perivascular macrophages. These findings recognize CD163+ blood monocytes/macrophages as a source of brain perivascular macrophages and underscore the utility of this molecule in studying the biology of perivascular macrophages and their precursors in humans, monkeys, and mice. Perivascular macrophages are uniquely situated at the intersection between the nervous and immune systems. Although combined myeloid marker detection differentiates perivascular from resident brain macrophages (parenchymal microglia), no single marker distinguishes perivascular macrophages in humans and mice. Here, we present the macrophage scavenger receptor CD163 as a marker for perivascular macrophages in humans, monkeys, and mice. CD163 was primarily confined to perivascular macrophages and populations of meningeal and choroid plexus macrophages in normal brains and in brains of humans and monkeys with human immunodeficiency virus or simian immunodeficiency virus (SIV) encephalitis. Scattered microglia in SIV encephalitis lesions and multinucleated giant cells were also CD163 positive. Consistent with prior findings that perivascular macrophages are primary targets of human immunodeficiency virus and SIV, all SIV-infected cells in the brain were CD163 positive. Using fluorescent dyes that definitively and selectively label perivascular macrophages in vivo, we confirmed that dye-labeled simian perivascular macrophages were CD163 positive and able to repopulate the central nervous system within 24 hours. Flow cytometric studies demonstrated a subset of monocytes (CD163+CD14+CD16+) that were immunophenotypically similar to brain perivascular macrophages. These findings recognize CD163+ blood monocytes/macrophages as a source of brain perivascular macrophages and underscore the utility of this molecule in studying the biology of perivascular macrophages and their precursors in humans, monkeys, and mice. Brain macrophages are normal constituents of the central nervous system (CNS) and central players in many CNS pathologies.1Gonzalez-Scarano F Baltuch G Microglia as mediators of inflammatory and degenerative diseases.Annu Rev Neurosci. 1999; 22: 219-240Crossref PubMed Scopus (906) Google Scholar, 2Stoll G Jander S The role of microglia and macrophages in the pathophysiology of the CNS.Prog Neurobiol. 1999; 58: 233-247Crossref PubMed Scopus (605) Google Scholar They are heterogeneous in their location, turnover, and function, although they are all of common developmental origin. 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The rat macrophage-associated antigen ED2 selectively identifies perivascular macrophages, but no such marker exists in humans.14Dijkstra CD Dopp EA Joling P Kraal G The heterogeneity of mononuclear phagocytes in lymphoid organs: distinct macrophage subpopulations in rat recognized by monoclonal antibodies ED1, ED2 and ED3.Adv Exp Med Biol. 1985; 186: 409-419PubMed Google Scholar, 15Graeber MB Streit WJ Kreutzberg GW Identity of ED2-positive perivascular cells in rat brain.J Neurosci Res. 1989; 22: 103-106Crossref PubMed Scopus (273) Google Scholar, 16Honda H Kimura H Silvers WK Rostami A Perivascular location and phenotypic heterogeneity of microglial cells in the rat brain.J Neuroimmunol. 1990; 29: 183-191Abstract Full Text PDF PubMed Scopus (23) Google Scholar, 17Kida S Steart PV Zhang ET Weller RO Perivascular cells act as scavengers in the cerebral perivascular spaces and remain distinct from pericytes, microglia and macrophages.Acta Neuropathol. 1993; 85: 646-652Crossref PubMed Scopus (156) Google Scholar In addition, a mannose receptor specific for perivascular macrophages in mouse brain was recently reported,18Galea I Palin K Newman TA Van Rooijen N Perry VH Boche D Mannose receptor expression specifically reveals perivascular macrophages in normal, injured, and diseased mouse brain.Glia. 2005; 49: 375-384Crossref PubMed Scopus (145) Google Scholar but a human equivalent has yet to be identified. At present, no single phenotypic marker, equivalent or comparable with ED2, that solely identifies perivascular macrophages is available in humans. In humans and monkeys, perivascular macrophages can be distinguished from parenchymal microglia based on levels of CD14 and CD45.8Fischer-Smith T Croul S Sverstiuk AE Capini C L'Heureux D Regulier EG Richardson MW Amini S Morgello S Khalili K Rappaport J CNS invasion by CD14+/CD16+ peripheral blood-derived monocytes in HIV dementia: perivascular accumulation and reservoir of HIV infection.J Neurovirol. 2001; 7: 528-541Crossref PubMed Scopus (292) Google Scholar, 10Williams KC Corey S Westmoreland SV Pauley D Knight H deBakker C Alvarez X Lackner AA Perivascular macrophages are the primary cell type productively infected by simian immunodeficiency virus in the brains of macaques: implications for the neuropathogenesis of AIDS.J Exp Med. 2001; 193: 905-915Crossref PubMed Scopus (332) Google Scholar, 19Becher B Antel J Comparison of phenotypic and functional properties of immediately ex vivo and cultured human adult microglia.Glia. 1996; 18: 1-10Crossref PubMed Scopus (198) Google Scholar, 20Dick AD Pell M Brew BJ Foulcher E Sedgwick JD Direct ex vivo flow cytometric analysis of human microglial cell CD4 expression: examination of central nervous system biopsy specimens from HIV-seropositive patients and patients with other neurological disease.AIDS. 1997; 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66: 858-866PubMed Google Scholar This protein (primarily molecular weight 130 kd) is expressed exclusively on all circulating monocytes and on subpopulations of tissue macrophages.30Lau SK Chu PG Weiss LM CD163: a specific marker of macrophages in paraffin-embedded tissue samples.Am J Clin Pathol. 2004; 122: 794-801Crossref PubMed Google Scholar, 31Fabriek BO Dijkstra CD van den Berg TK The macrophage scavenger receptor CD163.Immunobiology. 2005; 210: 153-160Crossref PubMed Scopus (315) Google Scholar CD163 mediates removal of hemoglobin-heptaglobin complexes by monocytes/macrophages, and its soluble form regulates inflammation.32Kristiansen M Graversen JH Jacobsen C Sonne O Hoffman HJ Law SK Moestrup SK Identification of the haemoglobin scavenger receptor.Nature. 2001; 409: 198-201Crossref PubMed Scopus (1334) Google Scholar Unlike other monocyte-associated markers such as CD14, CD16, and CD64 that label other leukocytes, CD163 is reportedly expressed specifically on human monocytes in which expression is highest on a CD14+CD16+ subset.33Buechler C Ritter M Orso E Langmann T Klucken J Schmitz G Regulation of scavenger receptor CD163 expression in human monocytes and macrophages by pro- and antiinflammatory stimuli.J Leukoc Biol. 2000; 67: 97-103PubMed Google Scholar Recently, Dijkstra and colleagues34van den Berg TK Puklavec MJ Barclay AN Dijkstra CD Monoclonal antibodies against rat leukocyte surface antigens.Immunol Rev. 2001; 184: 109-116Crossref PubMed Scopus (21) Google Scholar suggested that ED2 may be a rat homolog of human CD163; however, definitive data for this has yet to be published. Although it has been demonstrated that CD163 identifies perivascular macrophages in human CNS tissues,35Rezaie P Male D Microglia in fetal and adult human brain can be distinguished from other mononuclear phagocytes through their lack of CD163 expression.Neuroembryology. 2003; 2: 130-133Crossref Scopus (15) Google Scholar, 36Fabriek BO Van Haastert ES Galea I Polfliet MM Dopp ED Van Den Heuvel MM Van Den Berg TK De Groot CJ Van Der Valk P Dijkstra CD CD163-positive perivascular macrophages in the human CNS express molecules for antigen recognition and presentation.Glia. 2005; 51: 297-305Crossref PubMed Scopus (172) Google Scholar whether CD163 is a marker of perivascular macrophages remains open. In the present study, we examined CD163 expression on perivascular macrophages in normal human, monkey, and mouse brains and in human and monkey brains infected with HIVE and SIVE. We report that CD163 is a marker for perivascular macrophages in humans, monkeys, and mice in the normal CNS and that CD163+ perivascular macrophages are a prime target of SIV infection in monkeys. Using dextran amine dyes injected into the cerebrospinal fluid (CSF) of live monkeys, we show that perivascular macrophages selectively in-corporate dye and are all CD163 positive. Correspondingly, we observed a subset of blood monocytes (CD163+CD14+CD16+) that are immunophenotypically similar to the HIV- or SIV-infected perivascular macrophages found in encephalitic brains. This subset may represent precursors to specific macrophage lineage subpopulations in the CNS that are CD163 positive. Formalin-fixed, paraffin-embedded sections of frontal white and gray matter and pons were obtained from the Manhattan HIV Brain Bank.8Fischer-Smith T Croul S Sverstiuk AE Capini C L'Heureux D Regulier EG Richardson MW Amini S Morgello S Khalili K Rappaport J CNS invasion by CD14+/CD16+ peripheral blood-derived monocytes in HIV dementia: perivascular accumulation and reservoir of HIV infection.J Neurovirol. 2001; 7: 528-541Crossref PubMed Scopus (292) Google Scholar A total of four HIVE cases, two HIV-1-positive cases without encephalitis, and two seronegative controls were analyzed. CNS pathology was diagnosed by neuropathological examination initially performed by S. Morgello. Necropsy specimens of brain and lymph node from 24 adult rhesus macaques (Macaca mulatta) were used in the present study. Twenty-one of these animals were infected with SIVmac251 (20 ng of SIV p27) by intravenous injection and killed at peak viremia (n = 5) or when they developed AIDS (n = 16). Nine of the monkeys with AIDS had evidence of SIVE, defined by the presence of SIV in the brain and the accumulation of macrophages and multinucleated giant cells (MNGCs).37Budka H Multinucleated giant cells in brain: a hallmark of the acquired immune deficiency syndrome (AIDS).Acta Neuropathol. 1986; 69: 253-258Crossref PubMed Scopus (198) Google Scholar, 38Lackner AA Smith MO Munn RJ Martfeld DJ Gardner MB Marx PA Dandekar S Localization of simian immunodeficiency virus in the central nervous system of rhesus monkeys.Am J Pathol. 1991; 139: 609-621PubMed Google Scholar Three normal, age-matched, uninfected animals were used as controls. All animals were anesthetized with ketamine-HCl, euthanized by intravenous pentobarbital overdose, and exsanguinated. Tissues were either collected in 10% neutral buffered formalin and embedded in paraffin or blocked in optimum cutting temperature compound (Miles Scientific, Elkhart, IN) and snap-frozen. Formalin-fixed, paraffin-embedded tissues were cut into 6-μm-thick sections, and frozen tissues were sectioned at 10 μm. Brain and spleen were obtained from two 13-week-old C3H mice. Whole-brain tissue was frozen in optimum cutting temperature compound (Miles Scientific) and stored at −80°C until use. Sagittal sections of 10-μm thickness were cut, dried, and acetone fixed for 10 minutes on ice. Hydrophilic fluorescent dyes injected into the lateral cerebral ventricle selectively label perivascular macrophages.39Bechmann I Kwidzinski E Kovac AD Simburger E Horvath T Gimsa U Dirnagl U Priller J Nitsch R Turnover of rat brain perivascular cells.Exp Neurol. 2001; 168: 242-249Crossref PubMed Scopus (104) Google Scholar We used the same approach to rhesus monkeys to visualize perivascular macrophages. A total of four normal uninfected rhesus macaques were used: three animals received either fluorescein-conjugated dextran (Fluoro-Emerald; Molecular Probes, Eugene, OR), rhodamine-conjugated dextran (Fluoro-Ruby; Molecular Probes), or biotinylated dextran amine (BDA-10,000; Molecular Probes); one received Fluoro-Emerald and BDA-10,000 sequentially. Before injections, animals were initially tranquilized with ketamine or telazol and anesthetized with sodium pentobarbital. One milliliter of 5% dextran amines dissolved in 0.9% NaCl saline was injected into the cerebellomedullary cistern. Injections were performed over a 5-minute period using a stereotaxic apparatus. These animals were killed 1 day after Fluoro-Emerald, Fluoro-Ruby, or BDA-10,000 injection. To follow the sequential traffic of new macrophages into perivascular spaces, one animal was injected with a second dye BDA-10,000 7 days after the first Fluoro-Emerald injection and killed 24 hours later. Excess fluorescent dye that was not taken up by perivascular macrophages was completely cleared from the CSF within 24 hours. Because of this, newly immigrated perivascular macrophages can be identified by the second dye label. Primary antibodies used are listed in Table 1. To examine CD163 expression in paraffin-embedded human brain tissue, several monoclonal antibodies (mAbs) directed against human CD163 antigen were initially tested with or without antigen retrieval pretreatments (microwave or heat treatment in 0.01 mol/L sodium citrate buffer, pH 6.0). Two clones, EDHu-1 (Serotec, Oxford, UK) and 10D6 (Novocastra, Newcastle on Tyne, UK), were selected as most effective in formalin-fixed, paraffin-embedded human tissues.29Van den Heuvel MM Tensen CP van As JH Van den Berg TK Fluitsma DM Dijkstra CD Dopp EA Droste A Van Gaalen FA Sorg C Hogger P Beelen RH Regulation of CD 163 on human macrophages: cross-linking of CD163 induces signaling and activation.J Leukoc Biol. 1999; 66: 858-866PubMed Google Scholar, 40Roberts ES Zandonatti MA Watry DD Madden LJ Henriksen SJ Taffe MA Fox HS Induction of pathogenic sets of genes in macrophages and neurons in NeuroAIDS.Am J Pathol. 2003; 162: 2041-2057Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar Clone 10D6 was effective on paraffin-embedded tissues only after pretreatment; however, clone EDHu-1 was effective on paraffin-embedded tissues without pretreatment and also functioned on frozen tissues. Because of this, EDHu-1 was the primary antibody used for immunohistochemical and fluorescence studies. The cross-species reactivity of these anti-human CD163 mAbs with rhesus macaque tissue was evaluated by immunohistochemistry and immunoblot. To verify results obtained with paraffin-embedded tissues after antigen retrieval, which can result in the generation of neo-antigens, we used EDHu-1 and additional anti-human CD163 clones GHI/61 (PharMingen, San Diego, CA) and 5C6-FAT (BMA Biomedicals, Augst, Switzerland), which function on frozen tissues.28Pulford K Micklem K McCarthy S Cordell J Jones M Mason DY A monocyte/macrophage antigen recognized by the four antibodies GHI/61, Ber-MAC3, Ki-M8 and SM4.Immunology. 1992; 75: 588-595PubMed Google Scholar Two polyclonal antibodies, one against the N-terminal region of mouse CD163 (G-17; Santa Cruz Biotechnology; Santa Cruz, CA) and another against its internal region (K-18; Santa Cruz Biotechnology), which had not been previously assessed by immunohistochemistry, were also tested on mouse CNS tissues. Clones MAC 2-158 (Trillium Diagnostics, Scarborough, ME), EDHu-1, GHI/61, and 5C6-FAT were used for flow cytometry studies of CD163 expression on monocytes and monocyte-derived macrophages as described below.Table 1Antibodies Used in the Present StudyAntigenCloneIsotypeReactivityManufacturerApplicationCD14AML-2-23Mouse IgG2bHu, MkMedarexIHC(F), IF(F)CD147Mouse IgG2aHuNovocastraIHC(P), IF(P)CD14RMO52Mouse IgG2aHu, MkImmunotechFCCD162H7Mouse IgG2aHu, MkNovocastraIHC(P), IF(P)CD163G8Mouse IgG1Hu, MkPharMingenFCCD163EDHu-1Mouse IgG1Hu, MkSerotecIHC(F,P), IF(F,P), FCCD16310D6Mouse IgG1Hu, MkNovocastraIHC(P), IF(P)CD163GHI/61Mouse IgG1Hu, MkPharMingenIHC(F), FCCD1635C6-FATMouse IgG1Hu, MkBMAIHC(F), FCCD163MAC 2-158Mouse IgG1Hu, MkTrilliumFCCD163Polyclonal G-17Goat IgGMsSanta CruzIHC(F), IF(F)CD163Polyclonal K-18Goat IgGMsSanta CruzIHC(F), IF(F)Glut-1PolyclonalRabbit IgGHu, Mk, RtChemiconIHC(F,P), IF(F,P)HLA-DRCD3/43Mouse IgG1Hu, MkDAKOIHC(F,P)HLA-DRL243Mouse IgG2aHu, MkBecton DickinsonFCHu, human; Mk, monkey; Ms, mouse; Rt, rat; IHC, immunohistochemistry; IF, immunofluorescence; FC, flow cytometry; F, frozen; P, paraffin. Open table in a new tab Hu, human; Mk, monkey; Ms, mouse; Rt, rat; IHC, immunohistochemistry; IF, immunofluorescence; FC, flow cytometry; F, frozen; P, paraffin. Deparaffinized sections and acetone-fixed frozen sections were assessed by immunohistochemistry for CD163. Immunohistochemistry was performed using avidin-biotin peroxidase complex (Vectastain kit; Vector Laboratories, Burlingame, CA) or peroxidase-conjugated polymer reagent (SuperPicTure kit; Zymed, San Francisco, CA) according to the manufacturers' instructions. Before primary antibody incubation, serum or nonserum protein block was applied for 2 hours at room temperature or overnight at 4°C. The color reaction product was developed using 3,3′-diaminobenzidine tetrahydrochloride (DAB; DAKO) as the chromogenic substrate for horseradish peroxidase. The sections were counterstained with hematoxylin and then dehydrated and mounted. Controls consisted of omission of the primary antibody or addition of isotype-matched immunoglobulin. To detect CD163+ cells and Glut-1+ endothelial cells in human and monkey brains, double-label immunohistochemistry was performed using the DAKO Double Stain System, according to the manufacturer's instructions. The color reaction product was developed using DAB and Fast Red (DAKO) for CD163 and Glut-1 staining, respectively. The sections were mounted using Faramout aqueous mounting medium (DAKO). In situ hybridization for SIV RNA was performed using digoxigenin-labeled antisense riboprobes obtained from Lofstrand Labs (Gaithersburg, MD; with permission from Dr. V. Hirsch and C. Brown [National Institute of Health, Rockville, MD]). In situ hybridization was performed with a modification of previously published procedures.41Williams K Schwartz A Corey S Orandle M Kennedy W Thompson B Alvarez X Brown C Gartner S Lackner AA Proliferating cellular nuclear antigen expression as a marker of perivascular macrophages in simian immunodeficiency virus encephalitis.Am J Pathol. 2002; 161: 575-585Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar For simultaneous detection of viral nucleic acid and CD163 in the same tissue section, in situ hybridization for SIV RNA was followed by immunohistochemistry or immunofluorescence for CD163, using previously described methods10Williams KC Corey S Westmoreland SV Pauley D Knight H deBakker C Alvarez X Lackner AA Perivascular macrophages are the primary cell type productively infected by simian immunodeficiency virus in the brains of macaques: implications for the neuropathogenesis of AIDS.J Exp Med. 2001; 193: 905-915Crossref PubMed Scopus (332) Google Scholar and as described below. Immunofluorescence confocal microscopy was used to identify CD163+ cells in brain, to examine whether CD163+ cells were SIV RNA positive, and to determine whether dextran dye-labeled perivascular macrophages were CD163 positive. Deparaffinized sections or acetone-fixed frozen sections were washed with phosphate-buffered saline containing 0.2% fish skin gelatin (PBS/FSG) at room temperature and blocked with 10% normal goat serum diluted in PBS/FSG (2% normal rabbit serum was used for goat polyclonal anti-mouse CD163 antibody). Primary antibodies were diluted in blocking solution and incubated for 1 hour at room temperature or overnight at 4°C. CD163 immunofluorescence was revealed using goat anti-mouse or rabbit anti-goat secondary antibodies labeled with Alexa Fluor 488 or 568 (Molecular Probes). Glut-1 was followed by goat anti-rabbit Alexa Fluor 633 (Molecular Probes). Sections were washed with PBS/FSG before the addition of the next primary or secondary antibody. Secondary antibodies were also diluted in blocking solution and incubated for 30 minutes. Triple-label immunofluorescence was performed to colocalize CD163 with CD14 or CD16 on perivascular macrophages with Glut-1+ CNS microvessels. After incubation with primary mAbs to CD14 or CD16 and biotinylated isotype-specific secondary antibodies (PharMingen), sections were visualized with streptavidin-conjugated Alexa Fluor 568 (Molecular Probes). Confocal microscopy was performed using a Leica TCS SP laser scanning microscope equipped with three lasers. Individual optical slices represent 0.2 μm, and 32 to 62 optical slices were collected at 512 × 512 pixel resolution. The fluorescence of individual fluorochromes was captured separately in sequential mode after optimization to reduce bleed-through between channels (photomultiplier tubes) using Leica software. NIH Image version 1.62 and Adobe Photoshop version 7 software were used to assign correct colors of up to four channels collected, including the fluorochromes Alexa Fluor 488 (green), Alexa Fluor 568 (red), and Alexa Fluor 633 (blue) and the differential interference contrast image (gray scale). Some images were rendered with Volocity 3.6 Software (Improvision, Lexington, MA). Colocalization of antigens is indicated by the addition of colors as indicated in the figure legends. To examine the expression of CD163 on circulating monocytes, EDTA-treated fresh whole venous blood from normal uninfected humans and monkeys was tested by four-color flow cytometry. Prior studies using anti-CD163 mAbs GHI/61, EDHu-1, and MAC 2-158 reported extreme inconsistencies in the level of expression per cell or percentage of circulating monocytes expressing CD163 (reported percentage of positive cells ranged from 0 to 90% depending on the clones and methods).28Pulford K Micklem K McCarthy S Cordell J Jones M Mason DY A monocyte/macrophage antigen recognized by the four antibodies GHI/61, Ber-MAC3, Ki-M8 and SM4.Immunology. 1992; 75: 588-595PubMed Google Scholar, 29Van den Heuvel MM Tensen CP van As JH Van den Berg TK Fluitsma DM Dijkstra CD Dopp EA Droste A Van Gaalen FA Sorg C Hogger P Beelen RH Regulation of CD 163 on human macrophages: cross-linking of CD163 induces signaling and activation.J Leukoc Biol. 1999; 66: 858-866PubMed Google Scholar, 42Sulahian TH Hogger P Wahner AE Wardwell K Goulding NJ Sorg C Droste A Stehling M Wallace PK Morganelli PM Guyre PM Human monocytes express CD163, which is upregulated by IL-10 and identical to p155.Cytokine. 2000; 12: 1312-1321Crossref PubMed Scopus (245) Google Scholar Initially, we also found great variability among blood samples and mAbs when using heparinized blood. However, using EDTA as a coagulant, we found that more than 90% of peripheral blood monocytes expressed CD163 regardless of the clone used. Fo
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