Expression of DC-SIGN and DC-SIGNR on Human Sinusoidal Endothelium
2006; Elsevier BV; Volume: 169; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2006.051191
ISSN1525-2191
AutoresWai Kwan Lai, Phoebe Jun Sun, Jie Zhang, Adam Jennings, Patricia F. Lalor, Stefan G. Hübscher, Jane A. McKeating, David H. Adams,
Tópico(s)Protein Tyrosine Phosphatases
ResumoHepatic sinusoidal endothelial cells are unique among endothelial cells in their ability to internalize and process a diverse range of antigens. DC-SIGNR, a type 2 C-type lectin expressed on liver sinusoids, has been shown to bind with high affinity to hepatitis C virus (HCV) E2 glycoprotein. DC-SIGN is a closely related homologue reported to be expressed only on dendritic cells and a subset of macrophages and has similar binding affinity to HCV E2 glycoprotein. These receptors function as adhesion and antigen presentation molecules. We report distinct patterns of DC-SIGNR and DC-SIGN expression in human liver tissue and show for the first time that both C-type lectins are expressed on sinusoidal endothelial cells. We confirmed that these receptors are functional by demonstrating their ability to bind HCV E2 glycoproteins. Although these lectins on primary sinusoidal cells support HCV E2 binding, they are unable to support HCV entry. These data support a model where DC-SIGN and DC-SIGNR on sinusoidal endothelium provide a mechanism for high affinity binding of circulating HCV within the liver sinusoids allowing subsequent transfer of the virus to underlying hepatocytes, in a manner analogous to DC-SIGN presentation of human immunodeficiency virus on dendritic cells. Hepatic sinusoidal endothelial cells are unique among endothelial cells in their ability to internalize and process a diverse range of antigens. DC-SIGNR, a type 2 C-type lectin expressed on liver sinusoids, has been shown to bind with high affinity to hepatitis C virus (HCV) E2 glycoprotein. DC-SIGN is a closely related homologue reported to be expressed only on dendritic cells and a subset of macrophages and has similar binding affinity to HCV E2 glycoprotein. These receptors function as adhesion and antigen presentation molecules. We report distinct patterns of DC-SIGNR and DC-SIGN expression in human liver tissue and show for the first time that both C-type lectins are expressed on sinusoidal endothelial cells. We confirmed that these receptors are functional by demonstrating their ability to bind HCV E2 glycoproteins. Although these lectins on primary sinusoidal cells support HCV E2 binding, they are unable to support HCV entry. These data support a model where DC-SIGN and DC-SIGNR on sinusoidal endothelium provide a mechanism for high affinity binding of circulating HCV within the liver sinusoids allowing subsequent transfer of the virus to underlying hepatocytes, in a manner analogous to DC-SIGN presentation of human immunodeficiency virus on dendritic cells. Collectins comprise a family of calcium-dependent pattern-recognition lectins that bind oligosaccharide structures on the surface of microorganisms to facilitate clearance through aggregation, complement lysis, and opsonization. Two members of this family, DC-SIGN (CD209) and the related molecule DC-SIGNR (L-SIGN, CD209L), have been extensively studied for their ability to bind a variety of viral pathogens.1Alvarez CP Lasala F Carrillo J Muniz O Corbi AL Delgado R C-type lectins DC-SIGN and L-SIGN mediate cellular entry by Ebola virus in cis and in trans.J Virol. 2002; 76: 6841-6844Crossref PubMed Scopus (526) Google Scholar, 2Gardner JP Durso RJ Arrigale RR Donovan GP Maddon PJ Dragic T Olson WC L-SIGN (CD 209L) is a liver-specific capture receptor for hepatitis C virus.Proc Natl Acad Sci USA. 2003; 100: 4498-4503Crossref PubMed Scopus (260) Google Scholar, 3Klimstra WB Nangle EM Smith MS Yurochko AD Ryman KD DC-SIGN and L-SIGN can act as attachment receptors for alphaviruses and distinguish between mosquito cell- and mammalian cell-derived viruses.J Virol. 2003; 77: 12022-12032Crossref PubMed Scopus (186) Google Scholar, 4Lozach PY Amara A Bartosch B Virelizier JL Arenzana-Seisdedos F Cosset FL Altmeyer R C-type lectins L-SIGN and DC-SIGN capture and transmit infectious hepatitis C virus pseudotype particles.J Biol Chem. 2004; 279: 32035-32045Crossref PubMed Scopus (173) Google Scholar, 5Lozach PY Lortat-Jacob H de Lacroix d Staropoli I Foung S Amara A Houles C Fieschi F Schwartz O Virelizier JL Arenzana-Seisdedos F Altmeyer R DC-SIGN and L-SIGN are high affinity binding receptors for hepatitis C virus glycoprotein E2.J Biol Chem. 2003; 278: 20358-20366Crossref PubMed Scopus (331) Google Scholar, 6Pohlmann S Zhang J Baribaud F Chen Z Leslie GJ Lin G Granelli-Piperno A Doms RW Rice CM McKeating JA Hepatitis C virus glycoproteins interact with DC-SIGN and DC-SIGNR.J Virol. 2003; 77: 4070-4080Crossref PubMed Scopus (341) Google Scholar, 7Tassaneetrithep B Burgess TH Granelli-Piperno A Trumpfheller C Finke J Sun W Eller MA Pattanapanyasat K Sarasombath S Birx DL Steinman RM Schlesinger S Marovich MA DC-SIGN (CD209) mediates dengue virus infection of human dendritic cells.J Exp Med. 2003; 197: 823-829Crossref PubMed Scopus (687) Google Scholar, 8Halary F Amara A Lortat-Jacob H Messerle M Delaunay T Houles C Fieschi F Arenzana-Seisdedos F Moreau JF Dechanet-Merville J Human cytomegalovirus binding to DC-SIGN is required for dendritic cell infection and target cell trans-infection.Immunity. 2002; 17: 653-664Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, 9Marzi A Gramberg T Simmons G Moller P Rennekamp AJ Krumbiegel M Geier M Eisemann J Turza N Saunier B Steinkasserer A Becker S Bates P Hofmann H Pohlmann S DC-SIGN and DC-SIGNR interact with the glycoprotein of Marburg virus and the S protein of severe acute respiratory syndrome coronavirus.J Virol. 2004; 78: 12090-12095Crossref PubMed Scopus (293) Google Scholar, 10Yang ZY Huang Y Ganesh L Leung K Kong WP Schwartz O Subbarao K Nabel GJ pH-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through DC-SIGN.J Virol. 2004; 78: 5642-5650Crossref PubMed Scopus (391) Google Scholar Indeed, these molecules are often referred to as ‘viral attachment factors' and can potentiate infectivity of some viruses.11Bashirova AA Geijtenbeek TB van Duijnhoven GC van Vliet SJ Eilering JB Martin MP Wu L Martin TD Viebig N Knolle PA KewalRamani VN van Kooyk Y Carrington M A dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN)-related protein is highly expressed on human liver sinusoidal endothelial cells and promotes HIV-1 infection.J Exp Med. 2001; 193: 671-678Crossref PubMed Scopus (316) Google Scholar DC-SIGN is reported to be expressed on a subset of macrophages and dendritic cells,12Krutzik SR Tan B Li H Ochoa MT Liu PT Sharfstein SE Graeber TG Sieling PA Liu YJ Rea TH Bloom BR Modlin RL TLR activation triggers the rapid differentiation of monocytes into macrophages and dendritic cells.Nat Med. 2005; 11: 653-660Crossref PubMed Scopus (332) Google Scholar, 13McCully ML Chau TA Luke P Blake PG Madrenas J Characterization of human peritoneal dendritic cell precursors and their involvement in peritonitis.Clin Exp Immunol. 2005; 139: 513-525Crossref PubMed Scopus (14) Google Scholar, 14Jameson B Baribaud F Pohlmann S Ghavimi D Mortari F Doms RW Iwasaki A Expression of DC-SIGN by dendritic cells of intestinal and genital mucosae in humans and rhesus macaques.J Virol. 2002; 76: 1866-1875Crossref PubMed Scopus (228) Google Scholar, 15Turville SG Cameron PU Handley A Lin G Pohlmann S Doms RW Cunningham AL Diversity of receptors binding HIV on dendritic cell subsets.Nat Immunol. 2002; 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98: 2670-2675Crossref PubMed Scopus (289) Google Scholar DC-SIGN promotes cellular uptake and presentation of antigen and potentiates the interaction of DC-SIGN-expressing cells with leukocytes via an interaction with intercellular adhesion molecule-3.18Engering A Geijtenbeek TB van Vliet SJ Wijers M van Liempt E Demaurex N Lanzavecchia A Fransen J Figdor CG Piguet V van Kooyk Y The dendritic cell-specific adhesion receptor DC-SIGN internalizes antigen for presentation to T cells.J Immunol. 2002; 168: 2118-2126PubMed Google Scholar Recent biochemical and structural studies suggest that DC-SIGN and DC-SIGNR have different physiological functions and distinct ligand-binding properties.19Guo Y Feinberg H Conroy E Mitchell DA Alvarez R Blixt O Taylor ME Weis WI Drickamer K Structural basis for distinct ligand-binding and targeting properties of the receptors DC-SIGN and DC-SIGNR.Nat Struct Mol Biol. 2004; 11: 591-598Crossref PubMed Scopus (480) Google Scholar Hepatitis C virus (HCV)3Klimstra WB Nangle EM Smith MS Yurochko AD Ryman KD DC-SIGN and L-SIGN can act as attachment receptors for alphaviruses and distinguish between mosquito cell- and mammalian cell-derived viruses.J Virol. 2003; 77: 12022-12032Crossref PubMed Scopus (186) Google Scholar is an enveloped positive-stranded RNA virus and the sole member of the Hepacivirus genus, within the family Flaviviridae. Approximately 170 million individuals are infected worldwide, and the majority are at risk of developing progressive liver disease. Cellular and humoral immune responses are generated during HCV infection, but in the majority of individuals, humoral immune responses are inefficient to effect viral clearance, with ∼80% of new infections becoming chronic. The liver is thought to be the primary reservoir supporting HCV replication, although studies on HCV cell entry and tropism have been limited due to technical difficulties in propagating infectious HCV in cell culture. However, the recent development of infectious retroviral pseudotypes bearing HCV glycoproteins (HCVpp)20Bartosch B Vitelli A Granier C Goujon C Dubuisson J Pascale S Scarselli E Cortese R Nicosia A Cosset FL Cell entry of hepatitis C virus requires a set of co-receptors that include the CD81 tetraspanin and the SR-B1 scavenger receptor.J Biol Chem. 2003; 278: 41624-41630Crossref PubMed Scopus (507) Google Scholar, 21Hsu M Zhang J Flint M Logvinoff C Cheng-Mayer C Rice CM McKeating JA Hepatitis C virus glycoproteins mediate pH-dependent cell entry of pseudotyped retroviral particles.Proc Natl Acad Sci USA. 2003; 100: 7271-7276Crossref PubMed Scopus (687) Google Scholar, 22Zhang J Randall G Higginbottom A Monk P Rice CM McKeating JA CD81 is required for hepatitis C virus glycoprotein-mediated viral infection.J Virol. 2004; 78: 1448-1455Crossref PubMed Scopus (311) Google Scholar and the robust replication of HCV strain JFH in cell culture (HCVcc)8Halary F Amara A Lortat-Jacob H Messerle M Delaunay T Houles C Fieschi F Arenzana-Seisdedos F Moreau JF Dechanet-Merville J Human cytomegalovirus binding to DC-SIGN is required for dendritic cell infection and target cell trans-infection.Immunity. 2002; 17: 653-664Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, 23Lindenbach BD Rice CM Unravelling hepatitis C virus replication from genome to function.Nature. 2005; 436: 933-938Crossref PubMed Scopus (676) Google Scholar, 24Zhong J Gastaminza P Cheng G Kapadia S Kato T Burton DR Wieland SF Uprichard SL Wakita T Chisari FV Robust hepatitis C virus infection in vitro.Proc Natl Acad Sci USA. 2005; 102: 9294-9299Crossref PubMed Scopus (1510) Google Scholar, 25Wakita T Pietschmann T Kato T Date T Miyamoto M Zhao Z Murthy K Habermann A Krausslich HG Mizokami M Bartenschlager R Liang TJ Production of infectious hepatitis C virus in tissue culture from a cloned viral genome.Nat Med. 2005; 11: 791-796Crossref PubMed Scopus (2404) Google Scholar have enabled studies on HCV cell entry. HCVpp and HCVcc show a restricted tropism for human liver cell lines, and infection is dependent on CD81 expression.20Bartosch B Vitelli A Granier C Goujon C Dubuisson J Pascale S Scarselli E Cortese R Nicosia A Cosset FL Cell entry of hepatitis C virus requires a set of co-receptors that include the CD81 tetraspanin and the SR-B1 scavenger receptor.J Biol Chem. 2003; 278: 41624-41630Crossref PubMed Scopus (507) Google Scholar, 21Hsu M Zhang J Flint M Logvinoff C Cheng-Mayer C Rice CM McKeating JA Hepatitis C virus glycoproteins mediate pH-dependent cell entry of pseudotyped retroviral particles.Proc Natl Acad Sci USA. 2003; 100: 7271-7276Crossref PubMed Scopus (687) Google Scholar, 22Zhang J Randall G Higginbottom A Monk P Rice CM McKeating JA CD81 is required for hepatitis C virus glycoprotein-mediated viral infection.J Virol. 2004; 78: 1448-1455Crossref PubMed Scopus (311) Google Scholar, 24Zhong J Gastaminza P Cheng G Kapadia S Kato T Burton DR Wieland SF Uprichard SL Wakita T Chisari FV Robust hepatitis C virus infection in vitro.Proc Natl Acad Sci USA. 2005; 102: 9294-9299Crossref PubMed Scopus (1510) Google Scholar, 25Wakita T Pietschmann T Kato T Date T Miyamoto M Zhao Z Murthy K Habermann A Krausslich HG Mizokami M Bartenschlager R Liang TJ Production of infectious hepatitis C virus in tissue culture from a cloned viral genome.Nat Med. 2005; 11: 791-796Crossref PubMed Scopus (2404) Google Scholar, 26Lindenbach BD Evans MJ Syder AJ Wolk B Tellinghuisen TL Liu CC Maruyama T Hynes RO Burton DR McKeating JA Rice CM Complete replication of hepatitis C virus in cell culture.Science. 2005; 309: 623-626Crossref PubMed Scopus (1944) Google Scholar We6Pohlmann S Zhang J Baribaud F Chen Z Leslie GJ Lin G Granelli-Piperno A Doms RW Rice CM McKeating JA Hepatitis C virus glycoproteins interact with DC-SIGN and DC-SIGNR.J Virol. 2003; 77: 4070-4080Crossref PubMed Scopus (341) Google Scholar and others2Gardner JP Durso RJ Arrigale RR Donovan GP Maddon PJ Dragic T Olson WC L-SIGN (CD 209L) is a liver-specific capture receptor for hepatitis C virus.Proc Natl Acad Sci USA. 2003; 100: 4498-4503Crossref PubMed Scopus (260) Google Scholar, 4Lozach PY Amara A Bartosch B Virelizier JL Arenzana-Seisdedos F Cosset FL Altmeyer R C-type lectins L-SIGN and DC-SIGN capture and transmit infectious hepatitis C virus pseudotype particles.J Biol Chem. 2004; 279: 32035-32045Crossref PubMed Scopus (173) Google Scholar, 5Lozach PY Lortat-Jacob H de Lacroix d Staropoli I Foung S Amara A Houles C Fieschi F Schwartz O Virelizier JL Arenzana-Seisdedos F Altmeyer R DC-SIGN and L-SIGN are high affinity binding receptors for hepatitis C virus glycoprotein E2.J Biol Chem. 2003; 278: 20358-20366Crossref PubMed Scopus (331) Google Scholar, 27Cormier EG Tsamis F Kajumo F Durso RJ Gardner JP Dragic T CD81 is an entry coreceptor for hepatitis C virus.Proc Natl Acad Sci USA. 2004; 101: 7270-7274Crossref PubMed Scopus (247) Google Scholar demonstrated that HCVpp can interact with DC-SIGN- and DC-SIGNR-expressing cells; however, the physiological relevance of these virus-lectin interactions for HCV infection of the liver remains unclear. Hepatic sinusoidal endothelial cells (HSECs) are unique among endothelial cells in their ability to internalize and process a diverse range of antigens.28Seternes T Sorensen K Smedsrod B Scavenger endothelial cells of vertebrates: a nonperipheral leukocyte system for high-capacity elimination of waste macromolecules.Proc Natl Acad Sci USA. 2002; 99: 7594-7597Crossref PubMed Scopus (114) Google Scholar In contrast to most other endothelial cells, HSECs can process and present antigen to naive CD4 T cells and cross prime CD8 T cells. This latter response may lead to antigen-specific tolerance rather than immunity, suggesting the HSECs may contribute to the tolerogenic properties of the liver.29Crispe IN Hepatic T cells and liver tolerance.Nat Rev Immunol. 2003; 3: 51-62Crossref PubMed Scopus (563) Google Scholar, 30Knolle PA Schmitt E Jin S Germann T Duchmann R Hegenbarth S Gerken G Lohse AW Induction of cytokine production in naive CD4(+) T cells by antigen-presenting murine liver sinusoidal endothelial cells but failure to induce differentiation toward Th1 cells.Gastroenterology. 1999; 116: 1428-1440Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar This is of particular relevance in the setting of HCV infection if one considers its chronic nature and the apparent ineffectiveness of the cellular immune responses. A single report of DC-SIGN expression in brain microvascular endothelial cells31Mukhtar M Harley S Chen P BouHamdan M Patel C Acheampong E Pomerantz RJ Primary isolated human brain microvascular endothelial cells express diverse HIV/SIV-associated chemokine coreceptors and DC-SIGN and L-SIGN.Virology. 2002; 297: 78-88Crossref PubMed Scopus (58) Google Scholar supports a model where DC-SIGN may be expressed by HSECs and contribute to their unique antigen-presenting capabilities and regulation of immune responses to pathogens entering the liver. Here, we report distinct patterns of DC-SIGNR and DC-SIGN expression in human liver tissue and show that both C-type lectins are expressed on HSECs. We confirm that these receptors are functional by demonstrating their ability to bind HCV E2 protein and show that stimulation of isolated HSECs with interleukin-4 (IL-4) increases expression of both DC-SIGN and DC-SIGNR, promoting HCV E2 binding. However, isolated HSECs do not support HCVpp or HCVcc infection, suggesting that expression of these receptors is not sufficient to render these cells permissive for HCV infection. Expression of DC-SIGN on HSECs may allow internalization of antigens, including HCV particles for subsequent processing and presentation to naïve T cells. If these interactions result in ineffective T-cell activation or tolerance, they may contribute to the failed immune response against HCV infection. Ethics approval for the study was given by the South Birmingham Local Research Ethics Committee (Queen Elizabeth Hospital, Birmingham, UK) and the University Hospital Birmingham Trust (Queen Elizabeth Hospital). All liver tissue was collected with informed consent. Liver tissue from nondiseased liver was used for immunohistochemical studies. Nondiseased liver was obtained either from patients undergoing hemi-hepatectomy to remove liver metastases or from organ donors in whom the liver tissue was not used for transplantation. Liver endothelial cells were isolated from human liver tissue (surplus to surgical requirements) as previously described using a modified collagenase digestion technique.32Lalor PF Edwards S McNab G Salmi M Jalkanen S Adams DH Vascular adhesion protein-1 mediates adhesion and transmigration of lymphocytes on human hepatic endothelial cells.J Immunol. 2002; 169: 983-992PubMed Google Scholar Briefly, nonparenchymal cells were separated by density gradient centrifugation over metrizamide (Sigma-Aldrich, St. Louis, MO), and endothelial cells were isolated from the resultant heterogeneous cell mixture by positive immunomagnetic selection using antibodies against CD31 (M823, 10 μg/ml; Dako, Ely, UK) and Dynabeads conjugated with goat-anti-mouse monoclonal antibody (Dynal Biotech, Wirral, UK) according to the manufacturer's protocol. Following isolation, HSECs were cultured in complete medium composed of human endothelial basal growth medium (Invitrogen, Carlsbad, CA), 10% AB human serum (HD Supplies, Glasgow, UK), and 10 ng/ml vascular endothelial growth factor and 10 ng/ml hepatocyte growth factor (R&D Systems, Minneapolis, MN). The cells were propagated in collagen-coated culture flasks and maintained at 37°C in a humidified 5% CO2 incubator until cells were confluent. The following primary antibodies were used: DC-SIGN (MAB161, IgG2b) and DC-SIGNR (MAB162, IgG2b) from R&D Systems, LYVE-1 (8C, IgG1; a gift from David Jackson, University of Oxford), CD68 (EBM11, IgG1; from Abcam, Cambridge, UK), and mannose receptor (MCA2155, IgG1; from Serotec Ltd., Oxford, UK). 5-μm cryostat sections derived from normal liver were fixed in acetone for 10 minutes and stained using a standard alkaline phosphatase anti-alkaline phosphatase technique. Briefly, primary antibody was followed by rabbit anti-mouse monoclonal and mouse monoclonal alkaline phosphatase anti-alkaline phosphatase (Dako). The stain was developed with fast red and naphthol AS-MX phosphate substrate (Sigma-Aldrich). Sections for dual immunofluorescence were prewetted with staining buffer (phosphate-buffered saline containing 10% fetal calf serum and 0.1% sodium azide) for 10 minutes. Slides were incubated with primary antibodies diluted in staining buffer for 1 hour in a humidified chamber. Control sections were incubated with isotype-matched IgG2b or IgG1 (R&D Systems). Sections were stained with goat anti-mouse IgG2b Alexa Fluor (Molecular Probes, Eugene, OR) and goat anti-mouse IgG1 fluorescein isothiocyanate (Serotec). Immunofluorescence was assessed using AxioVision software (Carl Zeiss MicroImaging, Inc., Jena, Germany). HSECs were plated at 1 × 105 ml−1 and grown to confluence in collagen-coated 96-well flat bottom plates (BD Falcon, Oxford, UK). Cells were left under basal conditions or stimulated with cytokines for 24 hours (10 ng/ml recombinant tumor necrosis factor-α (TNF-α), IL-17, IL-10, or 100 ng/ml recombinant IL-4, all from PeproTech, London, UK). Following stimulation the cell monolayers were fixed in methanol. Nonspecific binding of monoclonal antibody (mAb) was inhibited by preincubation of cells for 1 hour at 37°C with 4% goat serum (Sigma) before the addition of mouse-anti-human mAbs (DC-SIGN: MAB161; DC-SIGNR: MAB162 (5 μg/ml; R&D Systems); E-Selectin: M7105, 1.6 μg/ml; CD31: M0823, 2.25 μg/ml (Dako). The cells were then washed thoroughly before incubation with peroxidase-conjugated goat-anti-mouse secondary Ab (P0447 1/5000; Dako). An enzyme-linked immunosorbent assay was developed using O-phenylenediamine substrate (S2045; Dako) according to the manufacturer's instructions, and the enzymatic reaction was stopped by using 0.5 mol/L H2SO4 (Fisher Scientific, Leicestershire, UK). Colorimetric analysis was performed by measuring absorbance values at 490 nm using an MRX plate reader (Dynatech Laboratories, Sussex, UK). All treatments were performed in triplicate for each experiment. 293-T cells were transiently transfected with plasmids expressing HC-J4 E2661 or vector alone (control mock antigen) with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Tissue culture supernatants containing HC-J4 E2661 were harvested 48 hours after transfection, and the amount of HC-J4 E2 antigen was quantified by enzyme immunoassay, as described previously.33Flint M Maidens C Loomis-Price LD Shotton C Dubuisson J Monk P Higginbottom A Levy S McKeating JA Characterization of hepatitis C virus E2 glycoprotein interaction with a putative cellular receptor, CD81.J Virol. 1999; 73: 6235-6244Crossref PubMed Google Scholar Normal liver sections were preincubated with isotype-matched IgG2b (control block), DC-SIGN, DC-SIGNR (5 μg/ml; both from R&D Systems), or mannan (20 μg/ml; Sigma-Aldrich) for 1 hour, washed and then incubated with mock antigen or HC-J4 E2661 at a saturating concentration in phosphate-buffered saline/1% fetal bovine serum/0.05% sodium azide/1 mmol/L CaCl2 for 1 hour at room temperature, washed, and labeled with rat anti-E2 mAb 9/75, which recognizes the CD81-binding site on E2 and hence fails to interact with E2-CD81 complexes.33Flint M Maidens C Loomis-Price LD Shotton C Dubuisson J Monk P Higginbottom A Levy S McKeating JA Characterization of hepatitis C virus E2 glycoprotein interaction with a putative cellular receptor, CD81.J Virol. 1999; 73: 6235-6244Crossref PubMed Google Scholar Detection of binding on normal liver section was as described above using immunofluorescence with goat anti-rat fluorescein isothiocyanate (Serotec). Immunofluorescence was assessed using AxioVision software (Carl Zeiss MicroImaging, Inc.). HSECs were dissociated from the flask using trypsin (Invitrogen), washed, and preincubated in human immunoglobulins to block Fc receptors. The HCV E2 binding and blocking assay was as described above. The cells were fixed in paraformaldehyde (Sigma-Aldrich), washed, and resuspended in phosphate-buffered saline/10% fetal calf serum before analysis on an Epics XL flow cytometer (Beckman Coulter, Fullerton, CA) using Summit software (DakoCytomation, Glostrup, Denmark). HCV pseudoparticles (HCVpp) were generated as previously described.21Hsu M Zhang J Flint M Logvinoff C Cheng-Mayer C Rice CM McKeating JA Hepatitis C virus glycoproteins mediate pH-dependent cell entry of pseudotyped retroviral particles.Proc Natl Acad Sci USA. 2003; 100: 7271-7276Crossref PubMed Scopus (687) Google Scholar Briefly, 293-T cells were cotransfected with pNL4–3.Luc.R-.E- and a plasmid expressing strain H and HCJ4 E1E2 and vesicular stomatitis virus G protein using Lipofectamine 2000 (Invitrogen). Medium was replaced with Dulbecco's modified Eagle's medium/3% fetal bovine serum after 6 hours. Pseudoparticle-containing supernatants were collected after 48 hours and clarified by low speed centrifugation for 10 minutes. For infection experiments target cells were plated into 96-well plates at 1.2 × 104 per well 24 hours before infection. Pseudoparticle-containing supernatants were diluted in Dulbecco's modified Eagle's medium/3% fetal bovine serum with 4 μg/ml Polybrene with or without the desired lipoprotein species. The mixture was incubated at 37°C for 1 hour before being applied to the target cells for 4 to 6 hours. Pseudoparticles were removed, and the cells were incubated for a further 72 hours. Cells were lysed with 40 μl of Cell Culture Lysis Reagent (Promega, Madison, WI), and infection was measured by quantifying the luciferase reporter gene by the addition of 50 μl of luciferase substrate (Promega) using a Centro LB960 luminometer (Berthold Technologies, Bad Wildbad, Germany). Huh 7.5 cells were trypsinized, harvested by centrifugation (500 × g, 5 minutes), washed twice, and resuspended in ice-cold phosphate-buffered saline at 1.5 × 107 cells/ml. One microgram of FL-J6/JFH RNA was mixed with 0.4 ml of cells in a 0.2-cm gap cuvette and pulsed using a BTX ElectroSquarePorator (Harvard Apparatus, Hollistion, MA) as described previously.26Lindenbach BD Evans MJ Syder AJ Wolk B Tellinghuisen TL Liu CC Maruyama T Hynes RO Burton DR McKeating JA Rice CM Complete replication of hepatitis C virus in cell culture.Science. 2005; 309: 623-626Crossref PubMed Scopus (1944) Google Scholar Electroporated cells were allowed to recover for 10 minutes at room temperature before the addition of complete media and plated into a 150-mm diameter cell culture dish. After 72 hours, virus containing supernatant was harvested and viral stocks stored at −80°C. Naïve Huh-7.5 cells were infected with J6/JFH at a multiplicity of infection of 0.01, and the cells were maintained in culture for 3 days. The frequency of HCV-infected cells in the culture was investigated by staining cells for the expression of NS5A as previously described.26Lindenbach BD Evans MJ Syder AJ Wolk B Tellinghuisen TL Liu CC Maruyama T Hynes RO Burton DR McKeating JA Rice CM Complete replication of hepatitis C virus in cell culture.Science. 2005; 309: 623-626Crossref PubMed Scopus (1944) Google Scholar The persistently infected Huh-7.5 culture was mixed 1:4 with primary HSECs, cultured for 48 hours, and stained for HCV NS5A expression.26Lindenbach BD Evans MJ Syder AJ Wolk B Tellinghuisen TL Liu CC Maruyama T Hynes RO Burton DR McKeating JA Rice CM Complete replication of hepatitis C virus in cell culture.Science. 2005; 309: 623-626Crossref PubMed Scopus (1944) Google Scholar HSECs were isolated from human liver tissue, propagated in cell culture, and shown to internalize acetylated low density lipoprotein as previously described.32Lalor PF Edwards S McNab G Salmi M Jalkanen S Adams DH Vascular adhesion protein-1 mediates adhesion and transmigration of lymphocytes on human hepatic endothelial cells.J Immunol. 2002; 169: 983-992PubMed Google Scholar Monoclonal antibodies specific for DC-SIGN and DC-SIGNR bound the cultured cells (Figure 1, A and B). Appropriate isotype-matched controls were negative in staining (not shown). DC-SIGN expression during monocyte differentiation is dependent on the cytokine microenvironment.34Puig-Kroger A Serrano-Gomez D Caparros E Dominguez-Soto A Relloso M Colmenares M Martinez-Munoz L Longo N Sanchez-Sanchez N Rincon M Rivas L Sanchez-Mateos P Fernandez-Ruiz E Corbi AL Regulated expression of the pathogen receptor dendritic cell-specific intercellular adhesion molecule 3 (ICAM-3)-grabbing nonintegrin in THP-1 human leukemic cells, monocytes, and macrophages.J Biol Chem. 2004; 279: 25680-25688Crossref PubMed Scopus (87) Google Scholar, 35Relloso M Puig-Kroger A Pello OM Rodriguez-Fernandez JL de la RG Longo N Navarro J Munoz-Fernandez MA Sanchez-Mateos P Corbi AL DC-SIGN (CD209) expression is IL-4 dependent and is negatively regulated by IFN, TGF-beta, and anti-inflammatory agents.J Immunol. 2002; 168: 2634-2643PubMed Google Scholar To determine whether cytokines similarly regulate DC-SIGN and DC-SIGNR expression in HSECs, the cells were treated with various cytokines for 24 hours, and lectin expressio
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