The dendritic cell–specific C-type lectin DC-SIGN is a receptor for Schistosoma mansoni egg antigens and recognizes the glycan antigen Lewis x

Irma van Die1,2, Sandra J. van Vliet2, A. Kwame Nyame3, Richard D. Cummings3, Christine M.C. Bank2, Ben Appelmelk4, Teunis B.H. Geijtenbeek2 and Yvette van Kooyk2

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


    Abstract
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Schistosoma mansoni soluble egg antigens (SEAs) are crucially involved in modulating the host immune response to infection by S. mansoni. We report that human dendritic cells bind SEAs through the C-type lectin dendritic cell–specific ICAM-3-grabbing nonintegrin (DC-SIGN). Monoclonal antibodies against the carbohydrate antigens Lewisx (Lex) and GalNAcß1-4(Fuc{alpha}1-3)GlcNAc (LDNF) inhibit binding of DC-SIGN to SEAs, suggesting that these glycan antigens may be critically involved in binding. In a solid-phase adhesion assay, DC-SIGN-Fc binds polyvalent neoglycoconjugates that contain the Lex antigen, whereas no binding was observed to Galß1-4GlcNAc, and binding to neoglycoconjugates containing only {alpha}-fucose or oligosaccharides with a terminal {alpha}1-2-linked fucose is low. These data indicate that binding of DC-SIGN to Lex antigen is fucose-dependent and that adjacent monosaccharides and/or the anomeric linkage of the fucose are important for binding activity. Previous studies have shown that DC-SIGN binds HIV gp120 that contains high-mannose-type N-glycans. Site-directed mutagenesis within the carbohydrate recognition domain (CRD) of DC-SIGN demonstrates that amino acids E324 and E347 are involved in binding to HIV gp120, Lex, and SEAs. By contrast, mutation of amino acid Val351 abrogates binding to SEAs and Lex but not HIV gp120. These data suggest that DC-SIGN recognizes these ligands through different (but overlapping) regions within its CRD. Our data imply that DC-SIGN not only is a pathogen receptor for HIV gp120 but may also function in pathogen recognition by interaction with the carbohydrate antigens Lex and possibly LDNF, which are found on important human pathogens, such as schistosomes and the bacterium Helicobacter pylori.

Key words: adhesion / carbohydrate interaction / dendritic cells / glycosylation / schistosomiasis


    Introduction
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Schistosomiasis is a major tropical parasitic disease, caused by trematode worms of the genus Schistosoma. Pathology results from the host immune response to schistosome eggs and the granulomatous reaction evoked by the soluble egg antigens (SEAs) that are released through ultramicroscopic pores in the eggshell (Ross et al., 2002Go). A major focus of the host immune response to schistosomes are glycoconjugates that abundantly occur on different stages of the parasite (Cummings and Nyame, 1996Go, 1999Go). In particular, fucosylated schistosomal glycoconjugates are prominently involved in the host humoral and cellular immune responses to infection. A strong humoral response has been found against the glycan epitopes GalNAcß1-4(Fuc{alpha}1-3)GlcNAc (LDNF) and GalNAcß1-4(Fuc{alpha}1-2Fuc{alpha}1-3)GlcNAc (LDN-DF) in both infected animals and humans (Nyame et al., 2000Go; Van Remoortere et al., 2001Go; Eberl et al., 2002Go).

Sera of infected hosts also contain antibodies against Galß1-4(Fuc{alpha}1-3)GlcNAc (Lex, CD15), a glycan epitope shared by humans and schistosomes (Nyame et al., 1998Go; Van Remoortere et al., 2001Go; Eberl et al., 2002Go). 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., 1996Go, 1997Go; Van Dam et al., 1996Go). 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., 1992Go; Cummings and Nyame, 1996Go; Van Remoortere et al., 2000Go). 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, 1994Go) and induce the production of IL-10 by peripheral blood mononuclear cells from schistosome-infected individuals (Velupillai et al., 2000Go). In a murine schistosome model, Lex is an effective adjuvant for induction of a Th2 response (Okano et al., 2001Go), 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., 1999Go).

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 cell–antigen contact and cell–cell 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 Th1–Th2 responses, and molecular patterns on the pathogen that are recognized by DCs are crucial for biasing the Th immune response (Jankovic et al., 2002Go). 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., 2001Go). 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, 1999Go). DCs express a variety of C-type lectins that specifically recognize glycan antigens (Figdor et al., 2002Go) and potential C-type lectins that have been described previously to interact with pathogens, including the mannose receptor (MR) (Blackwell et al., 1985Go; Ezekowitz et al., 1990Go) and the C-type lectin dendritic cell–specific 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., 2000cGo) and migration of DCs by binding to endothelial expressed ICAM-2 (Geijtenbeek et al., 2000bGo). 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., 2000aGo). Like other C-type lectins on DC, DC-SIGN captures and internalizes antigen for presentation to T cells (Engering et al., 2002Go). However, binding of HIV-1 to DC-SIGN escapes the antigen presentation route (Geijtenbeek et al., 2000aGo). 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., 2001Go; Feinberg et al., 2001Go). 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.


    Results
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
S. mansoni SEAs bind to human immature DCs through interaction with the C-type lectin DC-SIGN
Because DCs are central in directing Th1–Th2 responses, we searched for a cell-surface receptor expressed on DCs that interacts with S. mansoni SEAs. To detect binding of SEAs to human immature DCs, a fluorescent-bead adhesion assay was developed. Fluorescent beads were precoated with monoclonal antibodies (mAbs) to SEA glycan antigens and then used to capture SEA. The conjugated beads were allowed to interact with DCs, similar to the approach described by Geijtenbeek et al. (1999)Go. SEAs are a mixture of glycoproteins, containing many immunogenic glycan antigens (Khoo et al., 1997Go; Cummings and Nyame, 1999Go). Major glycan antigens present in SEAs and the antiglycan mAbs that specifically recognize them are depicted in Figure 1. To capture SEAs on the fluorescent beads, we used mAbs against the LDN antigen (Figure 1), a glycan epitope that abundantly occurs on many glycoconjugates within SEAs and is absent on DCs (data not shown). The binding of SEA-coated fluorescent beads to DCs was comparable to the binding of HIV-1 gp120 to DCs (Figure 2B). To investigate whether the binding is mediated by a C-type lectin, we investigated whether ethylenediamine tetra-acetic acid (EDTA), which removes Ca2+ ions that are essential for carbohydrate binding, or mannan hapten could inhibit binding of SEAs to the DCs. The interaction of SEAs to DCs was completely blocked by EDTA (data not shown) and mannan (Figure 2B), suggesting that the receptor on a DC is a C-type lectin with mannose specificity. Because the C-type lectins DC-SIGN and MR, which are both expressed by immature DCs (Figure 2A), are potential receptors for the recognition of glycan antigens, we investigated whether antibodies directed against these molecules could inhibit the binding. Interestingly, the binding could be partially blocked by the anti-DC-SIGN mAb AZN-D1, which binds to the CRD of DC-SIGN (Geijtenbeek et al., 2002Go) but not by the anti-MR mAb clone 19 (Figure 2B), indicating that DC-SIGN is a pathogen receptor on DCs for a subset of SEAs.



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Fig. 1. Structures of S. mansoni SEA carbohydrate antigens implicated in immune responses and monoclonal antibodies recognizing these antigens.

 


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Fig. 2. Interaction of SEAs with immature human DCs. (A) Immature human DCs were cultured from monocytes in the presence of granulocyte macrophage-colony stimulating factor and IL4. DCs express high levels of DC-SIGN in addition to expressing MR, CD83, CD86, CD80, and HLA-DR as determined by FACScan analysis. (B) DC-SIGN, expressed by immature DCs, shows a similar binding to SEAs as to HIV-1 gp120, a previously defined ligand of DC-SIGN. The adhesion to both SEAs and HIV-1 gp120 was determined using the fluorescent bead adhesion assay. Mannan and anti-DC-SIGN mAb AZN-D1, but not anti-MR mAb clone 19, block adhesion of SEAs to DCs. The experiment was performed twice in triplicate with DCs derived from a different batch of monocytes. The results shown the mean values of a triplicate assay from one representative experiment. The standard deviation was <5% in all assays.

 
Glycoproteins within SEAs of different schistosome species contain ligands for DC-SIGN
To further analyze the binding properties of DC-SIGN to S. mansoni SEAs, we investigated binding of soluble chimeric DC-SIGN-Fc to SEAs. In a solid phase adhesion assay DC-SIGN-Fc showed efficient binding to wells coated with SEAs (Figure 3A), whereas no binding was observed using another Fc-protein, ICAM3-Fc (data not shown). The interaction was completely inhibited by the anti-DC-SIGN antibody AZN-D1 or EDTA. In contrast, the binding was not affected by a DC-SIGN-specific antiserum directed against a peptide epitope (CCRD) (Engering et al., 2002Go) that is located C-terminal of the CRD (data not shown). These results indicate that binding of DC-SIGN to SEAs is mediated by the CRD of DC-SIGN. Binding of DC-SIGN-Fc to SEAs is likely to be high avidity because binding was observed at very low coating concentrations of SEA (Figure 3B). Because SEAs contain many different glycoproteins, we investigated which glycoproteins within SEAs interact with DC-SIGN-Fc. SEA glycoproteins of S. mansoni, as well as of S. haematobium and S. japonicum, were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and analyzed by western blotting with DC-SIGN-Fc and as a control with ICAM-3-Fc. Several glycoproteins within SEAs of all three schistosome species showed specific interaction with soluble DC-SIGN-Fc (Figure 3C). Silver staining of a SDS–PAGE gel containing similar amounts of SEAs showed that the DC-SIGN binding proteins represent a subfraction of the schistosome SEA.



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Fig. 3. Binding of DC-SIGN to schistosome SEA. (A) DC-SIGN binds to S. mansoni SEAs, as was determined by an ELISA-based assay using soluble DC-SIGN-Fc. Antigens were coated at a concentration of 1 µg/ml, and binding of DC-SIGN-Fc was measured using peroxidase-conjugated anti-human IgG. No binding to SEAs was detected using ICAM3-Fc (data not shown) or using DC-SIGN-Fc in the presence of the anti-DC-SIGN blocking antibody AZN-D1 (20 µg/ml) or EDTA (5 mM). (B) Binding of soluble DC-SIGN-Fc to S. mansoni SEAs, coated to wells in different concentrations, was measured by an anti-IgG-Fc ELISA, similar to that in A. (C) DC-SIGN-Fc binds SEAs of different schistosome species (Sm, S. mansoni; Sj, S. japonicum; Sh, S. haematobium). SEAs (~2 µg each lane) were separated by SDS–PAGE on a 15% PAA gel. The left panel of Figure 3C shows a silver-stained gel. The middle and right panels show western blots incubated with soluble DC-SIGN-Fc and ICAM3-Fc, respectively.

 
DC-SIGN binds to Lex-containing glycans
Because it has been reported previously that SEAs are heavily fucosylated (Khoo et al., 1995Go, 1997Go) and that DC-SIGN may exhibit binding to fucose (Curtis et al., 1992Go; Mitchell et al., 2001Go), we explored whether the binding of DC-SIGN to SEAs is fucose-mediated and can be blocked by mAbs specific for the fucosylated glycans Lex, LDNF, or LDN-DF in a competitive enzyme-linked immunosorbent assay (ELISA). The results show that anti-Lex and anti-LDNF mAbs indeed inhibit the binding between SEAs and DC-SIGN, whereas the binding between DC-SIGN and HIV-1 gp120 was not affected by these antibodies (Figure 4A). A combination of anti-Lex and anti-LDNF mAbs strongly blocked the binding of DC-SIGN to SEA (Figure 4A). These data indicate that both Lex and LDNF glycans, which resemble each other by containing a terminal fucose {alpha}1-3 linked to a GlcNAc residue, contribute to the binding of soluble DC-SIGN-Fc to SEAs. In contrast, no inhibition was found using the anti-LDN-DF mAbs in similar concentration, suggesting that DC-SIGN cannot interact with LDN-DF, in which the {alpha}1-3 fucose is capped with an {alpha}1-2 fucose.



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Fig. 4. DC-SIGN binds to {alpha}1-3-fucosylated glycans (A) Competitive inhibition of the binding of soluble DC-SIGN-Fc to SEAs and HIV-1 gp120 by anti-glycan mAbs in ELISA. Coated antigens were preincubated with antiglycan mAbs before adding DC-SIGN-Fc. Binding of DC-SIGN-Fc was measured by an anti-IgG-Fc ELISA. The experiments were performed twice in duplicate. The results shown represent the mean percentage of binding±SD. (B) DC-SIGN-Fc binds to neoglycoconjugates carrying Lex but poorly to other neoglycoconjugates in ELISA. Neoglycoproteins (1 µg/ml) were coated directly to the microtiter plates in coating buffer, and biotinylated PAA neoglycoconjugates (50 ng/well) were captured on streptavidin-coated microtiter plates. Coated neoglycoconjugates were incubated with soluble DC-SIGN-Fc. The experiments were performed three times in duplicate with essentially similar results. The figure shows the mean values±SD of one representative experiment.

 
Next we analyzed the potential of soluble DC-SIGN-Fc to bind to neoglycoproteins containing different glycan moieties in ELISA. The results in Figure 4B show that DC-SIGN-Fc strongly interacts with a neoglycoprotein carrying Lex. The binding is fucose-dependent, because no binding was observed to Galß1,4GlcNAc (LacNAc). In contrast, binding to similarly presented LDN-DF is poor. In addition, the binding of DC-SIGN-Fc to different oligosaccharides linked to polyacrylamide (PAA) was analyzed. Biotinylated polyvalent PAA-based neoglycoconjugates, carrying a similar density of oligosaccharides, were captured on streptavidin-coated ELISA plates, and binding with soluble DC-SIGN-Fc was detected as described. The results in Figure 4B show that DC-SIGN-Fc binds to Lex-PAA, whereas no or low binding was observed to PAA molecules carrying LacNAc, {alpha}-fucose or Fuc{alpha}1-2Galß1-4GlcNAc (H-type 2). Similar results were obtained when the biotinylated PAA molecules were coated directly to conventional microtiter plates. Our preliminary data indicate that DC-SIGN also binds LDNF containing neoglycoconjugates (data not shown). These data demonstrate that DC-SIGN shows specific, fucose-dependent binding to the trisaccharide antigen Lex and that adjacent monosaccharides, anomeric linkage, and/or position of the fucose residue in the oligosaccharide are important for binding activity.

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 cell–expressed 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., 2000cGo). 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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Fig. 5. Binding of SEAs and Lex to mutant forms of DC-SIGN. (A) SEAs block binding of DC-SIGN, expressed by human immature DCs, to ICAM-3 and HIV-1-gp120. The adhesion of both ICAM-3 and HIV-1 gp120 to DC was determined using the fluorescent bead adhesion assay. Inhibitors were present at a concentration of 20 µg/ml. The experiment was performed twice in triplicate (SD<5%) with DCs derived from different batches of monocytes. The results of both experiments were essentially similar, and the mean values of one experiment are shown. (B) Binding of SEAs and Lex-PAA-coated beads to K562 transfectants expressing wild-type DC-SIGN or the E324A, E347Q, and V351G DC-SIGN mutants was measured using the fluorescent bead adhesion assay. The experiment was performed twice in triplicate (SD<5%) with different cell cultures. The results show the mean values of one representative experiment. (C) Amino acid sequence of part of the CRD of DC-SIGN (AAK20997). The position of the E324A, E347Q, and V351G mutations are indicated. (D) K562 transfectants and different mutant forms of DC-SIGN express similar levels of DC-SIGN as determined by FACScan analysis using AZN-D2 mAb.

 
To further define the interaction of SEA and Lex with the CRD within DC-SIGN, we investigated the binding properties of these antigens to K562 cell transfectants expressing mutant forms of DC-SIGN (Geijtenbeek et al., 2002Go) (Figure 5B–D). Both SEAs and Lex antigens strongly interact with K562 cells expressing DC-SIGN and not with mock-transfected K562 cells (Figure 5B). The C-type lectin domain of DC-SIGN binds two Ca2+ ions (Geijtenbeek et al., 2000cGo), and those amino acid residues in close contact with Ca2+ at site 2 (Glu347, Asn349, Glu354, and Asn365) or with Ca2+ at site 1 (Asp320, Glu324, Asn350, and Asp355) are essential for ligand binding (Geijtenbeek et al., 2002Go). Mutation of either Glu324 to Ala or Glu347 to Gln in DC-SIGN (Figure 5C) resulted in complete loss of interaction with both SEAs and Lex (Figure 5B and data not shown), indicating that binding to these antigens is mediated through the primary ligand-binding site and is Ca2+- dependent. This is similar to results reported for binding of DC-SIGN to HIV-1 gp120 (Geijtenbeek et al., 2002Go).

It was recently demonstrated that a specific mutation in the CRD of DC-SIGN (V351G) allows binding of HIV-1 gp120 (Geijtenbeek et al., 2002Go) 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.


    Discussion
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 Abstract
 Introduction
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 Materials and methods
 References
 
Interactions between the human blood fluke S. mansoni and the host immune system require contacts between parasite antigens and host immune cells. Because schistosome eggs and the antigens they secrete play a major role in the pathogenesis, we explored the molecular interactions between S. mansoni SEAs and host immune cells. In this study we show that S. mansoni SEAs bind to human DCs and that DC-SIGN, a recently identified DC-specific C-type lectin, is primarily involved in this interaction through binding glycan antigens carrying the {alpha}1-3-fucosylated structures Lex and/or LDNF.

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., 1996Go). 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., 1994Go).

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., 2002Go). 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 {alpha}-fucose or oligosaccharides with a terminal {alpha}1-2-linked fucose was poor. The glycan epitopes Lex and LDNF are strongly related; both contain a fucose that is {alpha}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., 1998Go; Haslam et al., 2000Go). 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., 2001Go; Feinberg et al., 2001Go). 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., 2001Go) that may be essential for DC-SIGN binding (Geijtenbeek et al., 2002Go). HIV gp120 contains many high-mannose-type glycans (Geyer et al., 1988Go), 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., 2001Go; Geijtenbeek et al., 2002Go). 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., 2002Go). 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., 1992Go; Van Dam et al., 1994Go; Nyame et al., 2000Go, 2002Go; Van Remoortere et al., 2000Go; Wuhrer et al., 2000Go; Khoo et al., 2001Go). 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)Go. 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., 1991Go; Grzych et al., 1991Go; Velupillai and Harn, 1994Go; Araujo et al., 1996Go; Okano et al., 2001Go; Terrazas et al., 2001Go). Our data may imply that DC-SIGN is involved in this process by interaction with Lex, a hypothesis that is currently under study.


    Materials and methods
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 Abstract
 Introduction
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 Materials and methods
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Materials
Crude S. mansoni SEA extract was kindly provided by Dr. F. Lewis (Biomedical Research Institute, Rockville, MD). The crude extract was centrifuged for 90 min at 100,000xg at 4°C and sterilized by passing through a 0.2-µm filter. Unless otherwise indicated, binding studies were performed with S. mansoni SEAs. The following antibodies were used: anti-MR (clone 19, BD Pharmingen, San Diego, CA), CD11b (bear-1), CD11c (SHCL3), anti-DC-SIGN (AZN-D1) (Geijtenbeek et al., 2000cGo), and the phyco-erythrin–conjugated antibodies CD80, CD83, CD86, and HLA-DR (BD Pharmingen). Several antiglycan mAbs were used: the IgG anti-LDN-DF mAb 114-5B1-A (Van Remoortere et al., 2000Go), the IgM anti-LDN mAb SMLDN1.1 (Nyame et al., 1999Go), the IgG anti-LDNF mAb SMFG4.1, and the IgG anti-Lex Mab F8A1.1. The description and development of the latter two antibodies SMFG4.1 and F8A1.1 will be described elsewhere (Nyame and Cummings, in preparation). The neoglycoprotein Lex-HSA (containing ~20 mol oligosaccharide/mol HSA) was from Isosep AB (Tullinge, Sweden). The neoglycoprotein LDN-DF–bovine serum albumin (BSA), containing on average 12 mol oligosaccharide/mol BSA, was previously synthesized enzymatically as described (Van Remoortere et al., 2000Go). The polyvalent neoglycoconjugates Lex-PAA, {alpha}-L-fucose-PAA, Fuc{alpha}1-2Galß1-4GlcNAc (H-type 2), and LacNAc-PAA, were from Syntesome (Munich, Germany) (saccharide 20% mol, biotin 5% mol). Coating of the neoglycoconjugates was established in all experiments with appropriate lectins and glycan-specific antibodies.

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, 1994Go). 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., 2002Go) 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., 2000aGo).

Fluorescent bead adhesion assay
To demonstrate binding of SEAs to whole cells, a fluorescent bead adhesion assay was used as described (Geijtenbeek et al., 1999Go). Streptavidin was covalently coupled to the TransFluorSpheres (488/645 nm, 1.0 µm; Molecular Probes, Eugene, OR), as described previously (Geijtenbeek et al., 1999Go), 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 Tris–HCl 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., 2000aGo).

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 64–404) fused at the C-terminus to a human IgG1-Fc fragment into the Sig-pIgG1-Fc vector (Geijtenbeek et al., 2002Go). 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., 1992Go) was produced in Chinese hamster ovary cells. The solid phase adhesion assay was performed by coating neoglycoproteins (1 µg/ml) or SEAs (0.03–4 µg/ml) in ELISA plates overnight at 4°C, followed by blocking with 1% BSA in TSM (20 mM Tris–HCl, 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 (0.5–1 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.

SDS–PAGE and western blotting
SEAs (2 µg/lane) were separated by SDS–PAGE 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 phosphatase–conjugated 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).


    Acknowledgements
 
We thank Wietske Schiphorst for technical assistence in part of the experiments. This work was supported by an NIH grant (RO1 AI 47214) to R.D.C.

1 To whom correspondence should be addressed; e-mail: im.van_die.medchem{at}med.vu.nl Back


    Abbreviations
 
BSA, bovine serum albumin; CRD, carbohydrate recognition domain; DC, dendritic cell; DC-SIGN, dendritic cell–specific ICAM-3-grabbing nonintegrin; EDTA, ethylenediamine tetra-acetic acid; ELISA, enzyme-linked immunosorbent assay; HIV, human immunodeficiency virus; Lex, Lewis x; LDN, LacdiNAc; LDNF, GalNAcß1-4 (Fuc{alpha}1-3)GlcNAc; LDN-DF, GalNAcß1-4(Fuc{alpha}1-2Fuc{alpha}1-3)GlcNAc; mAb, monoclonal antibody; MR, mannose receptor; PAA, polyacrylamide; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; SEA, soluble egg antigen.


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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