2 Department of Molecular Cell Biology, VU University Medical Center, van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands
3 Department of Biochemistry and Molecular Biology, the Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA
4 Medical Microbiology, VU University Medical Center, van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands
Received on December 2, 2002; revised on January 28, 2003; accepted on January 28, 2003
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Abstract |
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Key words: adhesion / carbohydrate interaction / dendritic cells / glycosylation / schistosomiasis
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Introduction |
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Sera of infected hosts also contain antibodies against Galß1-4(Fuc1-3)GlcNAc (Lex, CD15), a glycan epitope shared by humans and schistosomes (Nyame et al., 1998
; Van Remoortere et al., 2001
; Eberl et al., 2002
). These Lex antibodies may induce autoimmune reactions, as was shown by their ability to mediate complement-dependent cytolysis of myeloid cells and granulocytes (Nyame et al., 1996
, 1997
; Van Dam et al., 1996
). Lex as well as many other glycan epitopes occur on several stages of the parasite, for instance, cercariae, eggs, schistosomula, or adult worms (Srivatsan et al., 1992
; Cummings and Nyame, 1996
; Van Remoortere et al., 2000
). Importantly, Lex-containing glycoconjugates trigger cellular immune responses. They induce proliferation of B cells from infected animals, which secrete IL-10 and PGE2 (Velupillai and Harn, 1994
) and induce the production of IL-10 by peripheral blood mononuclear cells from schistosome-infected individuals (Velupillai et al., 2000
). In a murine schistosome model, Lex is an effective adjuvant for induction of a Th2 response (Okano et al., 2001
), and it has been demonstrated that sensitization with Lex results in an increased cellular response toward SEA-coupled beads implanted in the liver and to the formation of granulomas (Jacobs et al., 1999
).
It is not clear yet how schistosome glycans trigger host immune responses and which cellular receptors are involved. Obviously, the immune responsiveness of T-lymphocytes to antigens and their effector functions in response to inflammation require cellantigen contact and cellcell adhesion. Antigen-presenting cells, such as dendritic cells (DCs), are expected to play an important role in recognition of the schistosome antigens. DCs are central in directing Th1Th2 responses, and molecular patterns on the pathogen that are recognized by DCs are crucial for biasing the Th immune response (Jankovic et al., 2002). In mouse models, DCs pulsed with SEAs potently stimulate Th2 responses both in vivo and in vitro while failing to undergo a conventional maturation process (MacDonald et al., 2001
). Therefore we hypothesized that receptors on DCs, possibly C-type lectins that recognize glycans, modulate DC functions on interaction with Schistosoma mansoni SEA.
C-type lectins bind sugars in a Ca2+-dependent manner using conserved carbohydrate recognition domains (CRDs) (Drickamer, 1999). DCs express a variety of C-type lectins that specifically recognize glycan antigens (Figdor et al., 2002
) and potential C-type lectins that have been described previously to interact with pathogens, including the mannose receptor (MR) (Blackwell et al., 1985
; Ezekowitz et al., 1990
) and the C-type lectin dendritic cellspecific ICAM-3-grabbing nonintegrin (DC-SIGN). DC-SIGN was originally defined as a receptor that supports DC-mediated T cell clustering by interaction with ICAM-3 (Geijtenbeek et al., 2000c
) and migration of DCs by binding to endothelial expressed ICAM-2 (Geijtenbeek et al., 2000b
). In addition, DC-SIGN binds human immunodeficiency virus type 1 (HIV-1) gp120 to facilitate transport of HIV from mucosal sites to draining lymph nodes to infect T cells (Geijtenbeek et al., 2000a
). Like other C-type lectins on DC, DC-SIGN captures and internalizes antigen for presentation to T cells (Engering et al., 2002
). However, binding of HIV-1 to DC-SIGN escapes the antigen presentation route (Geijtenbeek et al., 2000a
). Elucidation of the crystal structure of DC-SIGN in combination with binding studies suggests that high-mannose glycans are recognized by DC-SIGN (Mitchell et al., 2001
; Feinberg et al., 2001
). However, the glycan structures present on ICAM-2, ICAM-3, or HIV-1 gp120 that regulate the binding of DC-SIGN to these ligands have not yet been identified.
Here we demonstrate that DC-SIGN interacts with SEAs from S. mansoni, which may have important implications for its function as a receptor for schistosomes.
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Results |
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The interaction of SEAs with DC-SIGN differs from that of HIV-1 gp120
DC-SIGN recognizes both ligands derived from the pathogens HIV and schistosomes as well as ligands from self-molecules. To increase our understanding of how DC-SIGN interacts with these different ligands, we investigated in a DC-binding assay whether SEAs inhibit the interaction of dendritic cellexpressed DC-SIGN to HIV-1 gp120 and to ICAM-3, a T cell ligand that has been shown previously to interact with DC-SIGN (Geijtenbeek et al., 2000c). The results demonstrate that SEAs can inhibit the binding of both ICAM-3 and HIV gp120 to DC (Figure 5A), suggesting that SEAs interact with both the ICAM-3 and HIV gp120 ligand binding sites of DC-expressed DC-SIGN.
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It was recently demonstrated that a specific mutation in the CRD of DC-SIGN (V351G) allows binding of HIV-1 gp120 (Geijtenbeek et al., 2002) but abrogates binding to ICAM-3. Our results indicate that the DC-SIGN V351G mutant does not bind to either SEA or Lex, whereas binding to HIV-1 was still observed, indicating that Val351 is essential for binding to both SEAs and Lex (Figure 5B). These results demonstrate that the SEA binding site on DC-SIGN may resemble the ICAM-3 binding site and partly overlaps the binding site for HIV-1 gp120.
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Discussion |
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In schistosomiasis, SEA glycoconjugates are a major focus of the immune responses generated in infected hosts. Among the SEA glycans, the Lex epitope is of particular interest because it has been shown to be involved in both humoral and cellular immune responses and is crucially involved in granuloma formation. Despite the accumulating data about the importance of glycans in modulating the host immune response, not much is known about the host receptors that interact with SEA or other schistosome glycans. Schistosome eggs have been demonstrated to interact with soluble L-selectin, but the egg glycan ligands involved were not identified (Elridi et al., 1996). In addition, it has been shown that E-selectin interacts with low affinity with the LDNF antigen on human protein C, which is also present on schistosome glycans, but direct interaction between schistosome antigens and E-selectin has not been demonstrated (Grinnell et al., 1994
).
DCs are the first immune cells that encounter invading pathogens, and they express a variety of C-type lectins that can interact with carbohydrates on pathogen-derived antigens (Figdor et al., 2002). Our data show that DCs interact with SEAs and that a major part of this binding activity could be blocked by antibodies against the DC-specific antigen receptor DC-SIGN. This suggests that DC-SIGN is a receptor for SEAs on DCs, although it cannot be excluded that other DC receptors contribute to the binding of SEAs. Because binding of DC-SIGN-Fc to SEAs is inhibited by antibodies recognizing Lex and LDNF carbohydrate antigens but not by antibodies against the difucosylated LDN-DF antigen, we propose that specific presentation and anomeric linkage of the fucose residue in an oligosaccharide, such as in Lex and LDNF, is important for binding. This is supported by our findings that DC-SIGN specifically binds to polyvalent neoglycoconjugates carrying Lex, whereas binding to similar polyvalent neoglycoconjugates carrying the monosaccharide
-fucose or oligosaccharides with a terminal
1-2-linked fucose was poor. The glycan epitopes Lex and LDNF are strongly related; both contain a fucose that is
1-3-linked to GlcNAc, and both contain a nonreducing sugar ß1-4-linked to GlcNAc, which is galactose in Lex and N-acetylgalactosamine in LDNF. Their occurrence differs though, because as far as it is known, expression of Lex in invertebrates is unique for schistosomes and the nematode Dictyocaulus viviparus, whereas LDNF is a common glycan epitope in many helminths (Nyame et al., 1998
; Haslam et al., 2000
). Future studies that involve isolation and characterization of the SEA glycoproteins that bind DC-SIGN will be required to establish the identity and structural properties of the SEA ligands in more detail.
The binding of DC-SIGN to the Lex antigen is remarkable because previous studies showed that DC-SIGN interacts with mannose-containing structures, especially when presented in multivalent form (Mitchell et al., 2001; Feinberg et al., 2001
). So far, the glycan moieties on HIV gp120 and the self-glycoproteins ICAM-2 and ICAM-3 that are recognized by DC-SIGN have not been characterized. ICAM-3 contains a small amount of N-linked high- mannose-type oligosaccharides (Funatsu et al., 2001
) that may be essential for DC-SIGN binding (Geijtenbeek et al., 2002
). HIV gp120 contains many high-mannose-type glycans (Geyer et al., 1988
), which may account for its high-affinity binding to DC-SIGN. Because our data show that the carbohydrate recognition profile of DC-SIGN is broader than originally thought, future studies are required to establish which carbohydrate structures are recognized by DC-SIGN on the different ligands.
The primary binding site of DC-SIGN is situated in the C-type lectin domain and the Ca2+ at site 2 and the amino acid residues Glu347, Asn349, Glu354, and Asn365 form the core of this site (Feinberg et al., 2001; Geijtenbeek et al., 2002
). Here, we demonstrate that SEAs and Lex specifically bind to this site, similar to the other DC-SIGN ligands ICAM-3 and HIV-1 gp120. The ligand binding site of DC-SIGN forms a hydrophobic surface resembling a shallow trough, with the amino acid residue Val351 forming the edge of the binding pocket. This Val351 is essential for the interaction of DC-SIGN with ICAM-3, whereas HIV-1 gp120 still binds a mutant DC-SIGN in which Val351 is converted to Gly (Geijtenbeek et al., 2002
). We show that binding of both SEAs and Lex to DC-SIGN requires Val351, demonstrating that their binding sites on DC-SIGN differ from that of HIV-1 gp120. By binding to different carbohydrate ligands, DC-SIGN may trigger distinct effector functions in DC, allowing them to induce pathogen-specific immune responses. Such a differential binding capacity may also allow DC-SIGN to discriminate between the self-antigens (i.e., ICAM-2 and ICAM-3) and pathogens, interactions that require the induction of different DC effector functions.
The binding of DC-SIGN to SEA and Lex and/or LDNF containing glycoconjugates suggests that DC-SIGN may function in the host immune response to schistosomes through recognition of these glycan determinants. The occurrence of Lex and LDNF carbohydrate antigens on different schistosome stages and species has been described (Srivatsan et al., 1992; Van Dam et al., 1994
; Nyame et al., 2000
, 2002
; Van Remoortere et al., 2000
; Wuhrer et al., 2000
; Khoo et al., 2001
). Thus, the recognition of these glycan antigens by DC-SIGN may allow an early and continuous interaction with DCs, which may result in antigen presentation and explain the specific antiglycan humoral immune responses found in schistosome-infected hosts. It may also be possible that interaction between DC-SIGN and Lex early in infection results in priming the host for vigourous granuloma formation when challenged with egg antigens, as was proposed by Jacobs et al. (1999)
. In addition, SEAs induce a predominant Th2-type immune response in murine as well as human schistosomiasis, and a role for Lex antigens in this process has been proposed (Pearce et al., 1991
; Grzych et al., 1991
; Velupillai and Harn, 1994
; Araujo et al., 1996
; Okano et al., 2001
; Terrazas et al., 2001
). Our data may imply that DC-SIGN is involved in this process by interaction with Lex, a hypothesis that is currently under study.
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Materials and methods |
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Cells
Immature DCs were cultured from monocytes in the presence of IL4 and granulocyte macrophage-colony stimulating factor (500 and 800 U/ml, respectively; Schering-Plough, Brussels, Belgium) (Sallusto and Lanzavecchia, 1994). At day 7 the phenotype of the cultured DCs was confirmed by flow cytometric analysis. The DCs express high levels of MHC class I and II, CD11b, CD11c, and ICAM-1; moderate levels of LFA-1 and CD80; and low levels of CD14. K562 transfectants expressing wild-type DC-SIGN or mutant DC-SIGN (Geijtenbeek et al., 2002
) were generated by transfection of K562 cells with 10 µg pRc/CMV-DC-SIGN or mutant DC-SIGN plasmid by electroporation as described previously (Geijtenbeek et al., 2000a
).
Fluorescent bead adhesion assay
To demonstrate binding of SEAs to whole cells, a fluorescent bead adhesion assay was used as described (Geijtenbeek et al., 1999). Streptavidin was covalently coupled to the TransFluorSpheres (488/645 nm, 1.0 µm; Molecular Probes, Eugene, OR), as described previously (Geijtenbeek et al., 1999
), and the streptavidin-coated beads were incubated with biotinylated F(ab')2 fragment of goat anti-mouse IgG (6 µg/ml; Jackson Immunoresearch Laboratories Inc., West Grove, PA), followed by an overnight incubation at 4°C with anti-LDN mAb. The beads were washed and incubated with 1 µg/ml SEAs overnight at 4°C. Essentially, 50x103 cells were preincubated in adhesion buffer (20 mM TrisHCl pH 8.0, 150 mM NaCl, 1 mM CaCl2, 2 mM MgCl2, 0.5% BSA) with or without blocking mAbs (20 µg/ml) or mannan (50 µg/ml) for 10 min at room temperature. Ligand-coated fluorescent beads (20 beads/cell) were added to the cells, and the suspension was incubated for 45 min at 37°C. Cells were washed, and adhesion was determined using flow cytometry (FACScan; Becton Dickinson, Oxnard, CA) by measuring the percentage of cells that had bound fluorescent beads. HIV-1 gp120 fluorescent beads were prepared as described previously (Geijtenbeek et al., 2000a
).
Solid-phase DC-SIGN-Fc adhesion assay (ELISA) and inhibition by antiglycan mAbs
DC-SIGN-Fc consists of the extracellular portion of DC-SIGN (amino acid residues 64404) fused at the C-terminus to a human IgG1-Fc fragment into the Sig-pIgG1-Fc vector (Geijtenbeek et al., 2002). DC-SIGN-Fc was produced in Chinese hamster ovary K1 cells by cotransfection of DC-SIGN-Sig-pIgG1 Fc (20 µg) and pEE14 (5 µg) vector. DC-SIGN-Fc concentrations in the supernatant were determined by an anti-IgG1 Fc ELISA. Recombinant human ICAM3-Fc (Fawcett et al., 1992
) was produced in Chinese hamster ovary cells. The solid phase adhesion assay was performed by coating neoglycoproteins (1 µg/ml) or SEAs (0.034 µg/ml) in ELISA plates overnight at 4°C, followed by blocking with 1% BSA in TSM (20 mM TrisHCl, pH 7.4, containing 150 mM NaCl, 2 mM CaCl2, and 2 mM MgCl2) for 30 min at room temperature. Alternatively, biotinylated PAA-based neoglycoconjugates in TSM (1 µg/ml) were incubated for 1 h in preblocked streptavidin-coated ELISA-plates (Pierce, Rockford, IL) or coated directly in conventional ELISA plates similarly to the neoglycoproteins. After washing, soluble DC-SIGN-Fc (0.51 µg/ml in TSM buffer) or, when indicated, ICAM3-Fc (1 µg/ml in TSM buffer) as a negative control was added and the adhesion was performed for 60 min at room temperature. Unbound Fc-protein was washed away, and binding was determined by an anti-IgG1 Fc ELISA using a peroxidase conjugate of goat anti-human IgG1-Fc. DC-SIGN specificity was determined in the presence of either 20 µg/ml anti-DC-SIGN mAb (AZN-D1) or 5 mM EDTA. When indicated, antiglycan mAbs were used as competitive inhibitors. In those experiments SEAs were coated at 0.5 µg/ml. After blocking, coated SEAs were preincubated with antiglycan mAbs (at a concentration of 0.1 mg/ml of total mAb) for 30 min at room temperature before adding DC-SIGN-Fc.
SDSPAGE and western blotting
SEAs (2 µg/lane) were separated by SDSPAGE under reducing conditions on a 15% PAA gel, using the Mini-Protean II system (BioRad, Hercules, CA) and blotted onto a nitrocellulose membrane. The membrane was blocked in a solution of 5% BSA in TSM for 2 h, followed by incubation with DC-SIGN-Fc or ICAM3-Fc (both 1 µg/ml in TSM buffer containing 1% BSA) for 1 h. After washing (in TSM containing 0.1% Tween) and incubation with alkalic phosphataseconjugated goat anti-human IgG, bound antibodies were detected using x-phosphate/5-bromo-4-chloro-3-inodyl-phosphate (Boehringer GmbH, Mannheim, Germany) and 4-nitrobluetetrazoliumchloride (Boehringer Mannheim).
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Acknowledgements |
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1 To whom correspondence should be addressed; e-mail: im.van_die.medchem{at}med.vu.nl
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Abbreviations |
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References |
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