Glycobiology Advance Access originally published online on November 10, 2005
Glycobiology 2006 16(3):197-209; doi:10.1093/glycob/cwj057
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CEACAM1, an adhesion molecule of human granulocytes, is fucosylated by fucosyltransferase IX and interacts with DC-SIGN of dendritic cells via Lewis x residues
2 Institute of Clinical Chemistry, University Clinic Hamburg-Eppendorf, Martinistr, 52, 20251 Hamburg, Germany; and 3 Institute of Biochemistry and Molecular Biology, Charité Campus Benjamin Franklin, Arnimallee 22, 14195 Berlin, Germany
1 To whom correspondence should be addressed; e-mail: wagener{at}uke.uni-hamburg.de
Received on August 18, 2005; revised on November 4, 2005; accepted on November 6, 2005
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Abstract |
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The CEA-related cell adhesion molecule 1, CEACAM1, is a glycoprotein expressed on the surface of human granulocytes and lymphocytes, endothelia, and many epithelia. CEACAM1 is involved in the regulation of important biological processes, such as tumor growth, angiogenesis, and modulation of the immune response. CEACAM1, a member of the immunoglobulin superfamily carries several Lewis x (Lex) structures as we recently demonstrated by mass spectrometry of native CEACAM1 from human granulocytes. Since Lex residues of pathogens bind to the C-type lectin dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN) expressed on human DCs, we hypothesized that Lex glycans of CEACAM1 are recognized by DC-SIGN. Here, we demonstrate that CEACAM1, the major carrier of Lex residues in human granulocytes, is specifically recognized by DC-SIGN via Lex residues mediating the internalization of CEACAM1 into immature DCs. Expression studies with CEACAM1 in combination with different fucosyltransferases (FUTs) revealed that FUTIX plays a key role in the synthesis of Lex groups of CEACAM1. As Lex groups on CEACAM1 are selectively attached and specifically interact with DC-SIGN, our findings suggest that CEACAM1 participates in immune regulation in physiological conditions and in pathological conditions, such as inflammation, autoimmune disease, and cancer.
Key words: carcinoembryonic antigen / CEACAM1 / DC-SIGN / fucosyltransferase / Lewis x
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Introduction |
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The carcinoembryonic antigen (CEA) family comprises human glycoproteins belonging to the immunoglobulin superfamily. The founding member of this family, CEA, is a normal constituent of the glycocalix of the colonic mucosa (Frangsmyr et al., 1995
). Among the members of the CEA family, the CEA-related cell adhesion molecule 1 (CEACAM1) has attracted particular interest since it is involved in several important cellular activities, such as tumor progression, angiogenesis, and immune modulation. In addition, CEACAM1 is a receptor of bacteria, such as Neisseria, Haemophilus influenzae, and Moraxella catarrhalis. CEACAM1 is expressed by neutrophilic polymorphonuclear leukocytes (PMNs) where it has been assigned to the CD66 cluster. In previous studies, we and others provided evidence that CEACAM1 is a carrier of Lewis x (Lex) residues on human granulocytes (Kuijpers et al., 1993; Stocks and Kerr, 1993). By a combination of enzymatic digestion and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, at least seven Lex expressing carbohydrate side chains were identified (Lucka et al., 2005).
The Lex carbohydrate epitope has been detected as stage-specific embryonal antigen-1 (SSEA-1) at the morula stage of the mouse embryo (Gooi et al., 1981). In human leukocytes, the epitope has been assigned to the CD15 cluster. It is expressed preferentially on monocytes, mature neutrophils, and all myeloid cells from the promyelocyte stage onward (Nakayama et al., 2001). In addition to leukocytes, Lex is expressed in the nervous system, in the digestive and urinary tracts, and in the cervix (Fox et al., 1983). Though it is well established that glycoproteins and glycolipids may be decorated with Lex groups in humans and animals, the functional significance of these groups remained obscure, because no specific lectin ligands were known. Only recently, it was reported that a dendritic C-type lectin binds Lex residues present on the eggs of Schistosoma mansonii (van Die et al., 2003) and on Helicobacter pylori (Appelmelk et al., 2003). The lectin is known as dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN) based on the observation that it binds ICAM-3. So far, the carbohydrate structures on ICAM-3 involved in binding to DC-SIGN have not been identified. However, it has been shown that DC-SIGN binds high-mannose residues in addition to Lex and related Lewis glycans. The lectin does not bind sialyl Lex (Guo et al., 2004).
The cellular synthesis of Lex groups is guided by 
1,3-fucosyltransferases (FUTs). The main FUTs responsible for the synthesis the Lex structure are FUTIV and FUTIX (de Vries et al., 2001). FUTIX preferentially fucosylates the distal GlcNAc residue of polylactosamine chains, whereas FUTIV preferentially fucosylates the inner GlcNAc residue. Thus, FUTIX exhibits more efficient activity for the synthesis of the Lex carbohydrate epitope (Nishihara et al., 1999). Recently, a detailed analysis of the acceptor specificity of FUTIX has been published (Toivonen et al., 2002). In mature granulocytes, the expression of the CD15 epitope is directed by FUTIX, whereas it is determined in promyelocytes and monocytes by FUTIV (Nakayama et al., 2001).
Since, on the one hand, our structural data prove the presence of Lex residues on CEACAM1, and, on the other hand, DC-SIGN has been reported to bind Lex residues, we asked if the Lex glycans of CEACAM1 and other members of the CEA family are interacting with DC-SIGN. Here we provide evidence that native CEACAM1 from human granulocytes is recognized by DC-SIGN. Binding of CEACAM1 to DC-SIGN is dependent on Lex residues leading to the uptake of CEACAM1 by immature DCs. In appropriate eukaryotic expression systems, the co-expression of FUTIX with CEACAM1 is essential for the binding of DC-SIGN. Linking granulocytes to DCs, our findings imply that CEACAM1 and possibly other members of the CEACAM family may be important mediators between the innate and adaptive immune system.
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CEACAM1 expressed on human granulocytes is a major carrier of Lex groups
To identify CEACAM1 in western blots of whole cellular extracts of human granulocytes, three monoclonal antibodies (mAbs) were applied (Figure 1A). MAb T84.1 binds CEACAM6 in addition to CEACAM1. MAbs 12140–4 and 4D1C2 recognize only CEACAM1 among the CEACAMs expressed in human granulocytes. Applying mAb T84.1, CEACAM1 and CEACAM6 are detected as broad bands typical for glycoproteins with an apparent molecular weight of
160 and 90 kD, respectively. MAb 4D1C2 and mAb 12140–4 specifically recognize CEACAM1 confirming the identity and expression of CEACAM1 in human granulocytes. To investigate the surface expression of CEACAM1, fluorescence-activated cell sorter (FACS) analyses were performed with freshly prepared human granulocytes (Figure 1B). Binding of mAb T84.1 indicates that CEACAM1 and/or CEACAM6 are expressed on the cell surface of human granulocytes. To specifically detect CEACAM1, mAbs 12140–4 and 4D1C2 were applied. The strong and specific binding of mAb 12140–4 demonstrates that CEACAM1 is expressed at high levels on the cell surface of human granulocytes. The lower intensity obtained by mAb 4D1C2 most probably is explained by its lower affinity for CEACAM1. In addition, prominent membranous staining of CEACAM1 on the cell surface of granulocytes was observed by confocal microscopy analyzing freshly drawn and immediately fixed capillary blood cells. This observation suggests that CEACAM1 is already expressed in the native state on the cell surface of human granulocytes and not artificially activated and transported on the cell surface during purification or other steps of experimental manipulation (Figure 1C).
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By mass spectrometry, we recently demonstrated that CEACAM1 isolated form human granulocytes carries several Lex residues (Lucka et al., 2005). Western blot analysis with the Lex-specific mAb L5 revealed that CEACAM1 is a major carrier of Lex among the glycoproteins of human granulocytes (Figure 1D). In whole cellular extracts of human granulocytes, strong binding of mAb L5 was observed to a band of 160 kD well comparable with CEACAM1. In contrast, only weak binding of mAb L5 occurred to proteins of 50, 80, and 90 kD. The identity of the 80 and 90 kD proteins is presently unknown. The 50-kD band was also detectable by the secondary Ab indicating unspecific binding (data not shown). To confirm that the Lex-carrying 160 kD protein observed in whole cellular lysates of granulocytes represents CEACAM1, immunoprecipitation (IP) by mAb T84.1 was performed. Strong binding of mAb L5 to CEACAM1 was observed after precipitation which was completely abrogated after the digestion of CEACAM1 by (1–3,4) fucosidase (Fuc) specifically removing Lex residues (Figure 1D). Interestingly, Lex epitopes were undetectable on CEACAM6 indicating that the attachment of Lex residues is specific to CEACAM1. Taken together, these data demonstrate that CEACAM1 is the major carrier of Lex epitopes on human granulocytes.
CEACAM1 carrying Lex is the major ligand of DC-SIGN
Recently, DC-SIGN expressed on DCs has been identified as the major receptor for fucose-containing structures like Lex and other Lewis glycans (Guo et al., 2004). We therefore hypothesized that CEACAM1-carrying Lex residues is a ligand of DC-SIGN. To investigate binding of DC-SIGN to CEACAM1, the extracellular domain of human DC-SIGN carrying a Fc-tag (human IgG1) at the N-terminus for detection was expressed in human embryonic kidney 293 (HEK293) cells. After the purification of cell-culture supernatants, recombinant soluble Fc-DC-SIGN was applied as a probe in blot overlay assays. In whole cellular extracts of human granulocytes, strong and selective binding of Fc-DC-SIGN was observed to a protein of 160 kD well comparable with CEACAM1 (Figure 2A). The additional band of 55 kD corresponds to human IgG present in the granulocyte preparation and was also detectable by the anti-human Fc-antiserum applied for detection (data not shown). Immunoprecipitation of whole cellular extracts of granulocytes by mAb T84.1 confirmed that the 160-kD protein represents CEACAM1 as strong binding of Fc-DC-SIGN to CEACAM1 occurred after precipitation. As the interaction between DC-SIGN and carbohydrates is dependent on calcium and magnesium, binding of DC-SIGN to CEACAM1 was completely abrogated in the presence of ethylenediaminetetraacetic acid (EDTA) (data not shown). Treatment of precipitates by Fuc further confirmed that the binding of DC-SIGN was solely dependent on Lex residues on CEACAM1 (Figure 2A). Most importantly, no binding of Fc-DC-SIGN to whole cellular extracts of granulocytes was observed in the supernatant after precipitation clearly demonstrating that Lex-carrying CEACAM1 is the major binding partner of DC-SIGN in extracts of human granulocytes.
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In addition to western blot analysis, binding of Fc-DC-SIGN to Lex residues on CEACAM1 was studied by FACS analyses. Double staining by the fluorescein isothiocyanate (FITC)-labeled mAb 12140–4 and the phycoerythin (PE)-labeled mAb 120507 specific for DC-SIGN revealed that most granulocytes (>95%) were positive for CEACAM1 and DC-SIGN indicating strong binding of DC-SIGN to the surface of human granulocytes involving CEACAM1 (Figure 2B). To proof the specificity of interaction, Lex residues on granulocytes were blocked before FACS analysis by the Lex-specific mAb L5 or by a polyclonal antiserum directed against CEACAM. In accordance to our western blot data, binding of Fc-DC-SIGN to CEACAM1 was inhibited after blocking demonstrating that Lex residues on CEACAM1 are specifically recognized by DC-SIGN on the surface on human granulocytes (Figure 2C). With respect to mAb L5, incomplete inhibition of DC-SIGN binding may be explained by the differences in binding affinities or concentration of inhibiting Abs. Regarding the CEA antiserum, incomplete coverage of Lex residues on CEACAM1 or other ligands should also be considered. In both instances, DC-SIGN may interact with membrane structures, which are not recognized by the Abs. Lex residues present on other glycoproteins or on glycolipids may bind DC-SIGN, but not mAb L5. Alternatively, DC-SIGN may bind to high-mannose residues. It should be noted, however, that the binding of DC-SIGN was not inhibited by the high–mannose-specific mAb L3.
1–3 FUTIX specifically transfers Lex residues to CEACAM1
To gain insights into the mechanisms of terminal fucosylation of CEACAM1, the human FUTs FUTIII, -IV, -VII, and -IX, respectively, involved in terminal modifications of glycoproteins were co-expressed with CEACAM1 in HEK293 cells. As demonstrated by western blot analysis applying the Lex-specific mAb L5, Lex residues were detectable on CEACAM1 when co-expressed with FUTIX (Figure 3A). In contrast, Lex groups were not present when CEACAM1 was co-expressed with FUTIII and FUTVII and only weakly detectable in the presence of FUTIV. The preferential fucosylation of CEACAM1 by FUTIX as compared with FUTIV is further supported by flow cytometry of HEK 293 transfectants using mAb L5 (Figure 3B). As FUTIX is expressed at high levels in mature granulocytes transferring fucose to GlcNAc residues at distal lactosamine units (Nakayama et al., 2001), our findings indicate that FUTIX plays a central role in the formation of Lex epitopes on CEACAM1 in human granulocytes.
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Having identified FUTIX as the major enzyme transferring Lex residues to CEACAM1, we tested the binding of DC-SIGN to CEACAM1 from HEK293 and Chinese hamster ovary (CHO) cells transfected by FUTIX (Figure 4). Unexpectedly, strong binding of Fc-DC-SIGN to CEACAM1 was observed in HEK293 cells independent of the expression of FUTIX (Figure 4A). In contrast, in CHO cells Lex epitopes on CEACAM1 were only present when CEACAM1 was co-expressed with FUTIX (Figure 4A). In accordance to our observations with human granulocytes, treatment of CEACAM1 from FUTIX-transfected CHO cells by Fuc resulted in abrogation of DC-SIGN binding confirming that binding of DC-SIGN to CEACAM1 is solely dependent on Lex residues (Figure 4C). To further characterize the glycostructures of CEACAM1 in HEK293 cells recognized by DC-SIGN but lacking Lex residues, blot overlay assays were performed with Galanthus nivalis agglutinin (GNA), a lectin binding to high-mannose structures (Figure 4B). Strong binding of GNA to CEACAM1 expressed in HEK293 was observed, whereas high-mannose structures were not detectable on CEACAM1 expressed in CHO cells. DC-SIGN is known to bind high-mannose structures in addition to Lewis glycans (Guo et al., 2004). Thus, the different behavior of binding of DC-SIGN to CEACAM1 from HEK293 and CHO cells, respectively, is explained by the presence of high-mannose groups in HEK 293 cells. To exclude that high-mannose groups are involved in the binding of CEACAM1 by DC-SIGN in granulocytes, the binding of GNA to CEACAM1 extracted from human granulocytes was tested. No binding of GNA to CEACAM1 was detectable confirming that in human granulocytes, not high-mannose but Lex groups are central for the recognition of CEACAM1 by DC-SIGN (data not shown). Taken together, these data demonstrate that terminal glycosylation of CEACAM1 occurs in a cell–type-dependent manner requiring appropriate and well-characterized cellular systems when interactions of carbohydrates and their ligands are studied in vitro.
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Lex groups on CEACAM1 specifically mediate the binding to DC-SIGN on DCs
Having identified CEACAM1 as a specific ligand of DC-SIGN and the major carrier of Lex epitopes on human granulocytes, we investigated the interaction between CEACAM1 and DC-SIGN on the cellular level. For this purpose, monocytes were isolated from peripheral human blood and differentiated by interleukin-4 (IL-4) and GM-CSF to immature DCs marked by high levels of DC-SIGN expression (data not shown). Binding studies were performed with CHO cells stably expressing CEACAM1 in the absence or presence of expression of FUTIX. CHO cells were fluorescently labeled, were incubated with immature DCs, and bound cells were quantified by fluorescence microscopy. Compared with transfectomas expressing CEACAM1 only, the number of CEACAM1/FUTIX-transfectomas bound to immature DCs was 3-fold increased (Figure 5). Attachment of CEACAM1/FUTIX-transfectomas was substantially decreased to background levels when Lex epitopes on CEACAM1 were blocked by the Lex-specific mAb L5. Likewise, binding of the CEACAM1/FUTIX-transfectomas to immature DCs was inhibited by the anti-CEACAM antiserum or by mAb DC28 blocking the binding of DC-SIGN to its ligands. Because CHO cells expressing CEACAM1 and FUTIX bind to DC-SIGN of DCs in a Lex-dependent manner, the role of CEACAM1 and DC-SIGN in the adhesion of freshly prepared vital human granulocytes to immature DCs was tested. The adhesive interactions were not significantly blocked by Abs against DC-SIGN, Lex (mAb L5), or CEACAM1 (data not shown). Thus, in our experimental system, primary adhesion between granulocytes and DCs appears to be mediated by other adhesion molecules, possibly integrins. Taken together, these data suggest that Lex residues of CEACAM1 can mediate cellular interactions with immature DCs. The actual contribution of CEACAM1 appears to depend on the expression level of CEACAM1 and on other adhesion molecules expressed in particular cell types.
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Lex groups mediate the binding and internalization of CEACAM1 in DCs
To elucidate the functional role of the interaction of Lex carrying CEACAM1 and DC-SIGN on DCs, native CEACAM1 was isolated by affinity chromatography from extracts of human granulocytes and covalently coupled to fluorescent microbeads. Lex–bovine serum albumin (Lex–BSA) coupled beads were used as a positive control, and fluorescent beads coated by BSA served to determine background binding. Beads were incubated with immature DCs, and bound beads were visualized and quantified by fluorescence microscopy. Strong increase in binding of CEACAM1 and Lex–BSA-coated beads was observed compared with background binding of the BSA beads (Figure 6). Binding of CEACAM1 and Lex–BSA-coupled beads was significantly decreased when DCs were preincubated with the DC-SIGN-blocking Ab DC28. Likewise, binding of CEACAM1-coated beads was inhibited by over 50% when CEACAM1 beads were pretreated by Fuc confirming that the interaction between DC-SIGN and CEACAM1 is mediated by Lex residues present on native CEACAM1.
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Studies by confocal microscopy further revealed that CEACAM1-coupled beads are localized in the cytoplasm of DCs (Figure 7A). Large numbers of beads were clustered near the cell nucleus suggesting the internalization of CEACAM1-coated microbeads into the cytoplasm of immature DCs. As expected from our previous binding studies, uptake was drastically reduced when DC-SIGN on DCs was blocked by the DC-SIGN-blocking Ab DC28 indicating that binding and uptake of CEACAM1 is specifically mediated by DC-SIGN (Figure 7B). Parallel staining of the lysosomal-associated membrane protein 1 (LAMP1) showed that the fluorescent beads coated by native CEACAM1 are co-localized with LAMP1 demonstrating that most beads are internalized and subsequently transferred to lysosomal compartments (Figure 7C).
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DC-SIGN, a C-type lectin expressed on DCs, binds a variety of pathogens and is assumed to play an important role in the modulation of the immune response (Figdor et al., 2002
; Cambi et al., 2005). In addition, DC-SIGN has been reported to mediate the interaction of DCs with T cells via ICAM-3 (Geijtenbeek et al., 2000b) and with endothelia via ICAM-2 (Geijtenbeek et al., 2000a). Most of the interactions described so far are based on the binding of oligomannosidic residues to the carbohydrate-recognition domain of DC-SIGN (Feinberg et al., 2001). Recently, it has been reported that DC-SIGN binds Lex residues expressed by H. pylori (Appelmelk et al., 2003) and on the eggs of S. mansonii (van Die et al., 2003). The binding of both oligomannosidic and Lex residues has been confirmed by structural studies (Guo et al., 2004).
MAbs have been used to localize the Lex (CD15) epitope in a variety of normal and malignant human cells and tissues (Fox et al., 1983; Itzkowitz et al., 1986). It is well established that the Lex epitope is present on glycolipids (Gooi et al., 1981; Kannagi et al., 1982). So far, however little structural information is available on Lex expressing carbohydrate side chains of glycoproteins. Recently, we performed a detailed structural analysis of Lex residues of the adhesion molecule CEACAM1 isolated from human granulocytes. By a combination of enzymatic digestion and mass spectrometry, at least seven Lex residues were identified (Lucka et al., 2005). As DC-SIGN binds Lex residues and CEACAM1 carries Lex structures, we investigated whether CEACAM1 is a specific ligand of DC-SIGN.
Here, we demonstrate that CEACAM1 purified from the membrane fraction of human granulocytes is a major glycoprotein ligand of DC-SIGN and that the binding to DC-SIGN is mediated by Lex residues. In crude membrane extracts from human granulocytes, a single band corresponding to the Mr of CEACAM1 was bound by recombinant DC-SIGN. No binding of DC-SIGN was observed in the supernatant after CEACAM1 was immunoprecipitated by a CEACAM1-reactive Ab. The immunoprecipitated CEACAM1 showed strong binding to DC-SIGN. Binding was completely abolished after treatment by Fuc. Since Fuc cleaves fucose residues from the Lex carbohydrate structure, we conclude that native CEACAM1 expressed on the surface of human granulocytes binds DC-SIGN via its Lex residues.
In mature human granulocytes, the expression of the Lex (CD15) epitope is directed by FUTIX (Nakayama et al., 2001). Since our mass spectrometric analysis revealed a high number of Lex groups on CEACAM1, we reasoned that CEACAM1 may be a substrate of FUTIX and that the transfer of fucose to CEACAM1 by FUTIX may be a prerequisite for the interaction by DC-SIGN. Our co-transfection studies performed in CHO cells are in full agreement with this assumption. Recombinant CEACAM1 expressed in CHO cells did not bind DC-SIGN or a monoclonal Lex Ab. In contrast, when CEACAM1 and FUTIX were co-expressed, a strong reactivity with DC-SIGN and the Lex mAb was observed. The binding was completely abolished after treatment of CEACAM1 by Fuc. These findings suggest that, in human granulocytes, CEACAM1 is a substrate of FUTIX and that the FUTIX-dependent transfer of fucose residues to CEACAM1 is essential for the binding of DC-SIGN. The fucosylation of CEACAM1 appears to be rather specific because co-transfection studies by FUTIX revealed that other members of the immunoglobulin superfamily family such as the platelet-derived growth factor (PDGF) or fibroblast growth factor (FGF) receptors were not decorated by Lex residues (unpublished data). Moreover, in human granulocytes, Lex residues were not detectable on the closely related CEACAM family member CEACAM6 suggesting that CEACAM1 and CEACAM6 differ in certain structural characteristics required for specific fucosylation. Though CEACAM1 is fucosylated preferentially by FUTIX, we would not exclude that other potential ligands of DC-SIGN such as the Mac-1 integrin (van Gisbergen K.P., Sanchez-Hernandez M., et al., 2005) or glycolipids are also fucosylated by FUTIV, which is active at earlier stages of granulocytopoiesis (Nakayama et al., 2001).
To prove that the Lex groups on CEACAM1 mediate the binding to cellular DC-SIGN, binding studies with immature DCs and CHO cells stably expressing either CEACAM1 or CEACAM1 in combination with FUTIX were performed. In contrast to the CEACAM1 transfectomas, enhanced attachment of the CEACAM1/FUTIX transfectants to immature DCs was observed. Attachment was inhibited by the Lex Ab, the DC-SIGN-blocking Ab, and by the polyclonal CEACAM antiserum. These findings indicate that increased attachment between the CEACAM1/FUTIX transfectomas and the immature DCs is directly dependent on the interaction between DC-SIGN and Lex carrying CEACAM1. However, when the cellular interactions between immature DCs and freshly prepared, vital human granulocytes were tested, attachment of granulocytes was not significantly blocked by either CEACAM1 or DC-SIGN Abs. These findings indicate that Lex residues of CEACAM1 may contribute to cell–cell attachment depending on the cell system investigated and the absence or presence of other cell adhesion molecules involved in cell adhesion. As native CEACAM1 attached to microbeads was specifically bound by immature DCs, it could well be that fucosylated CEACAM1 on intact human granulocytes is involved in recognition and signaling via DC-SIGN once granulocytes are bound to DCs, though binding is primarily directed by other cell adhesion molecules. Considering the internalization of CEACAM1-coated beads by DCs, CEACAM1 could also mediate the uptake of damaged granulocytes or cell debris in inflammatory situations such as bacterial infections. In both instances, the interaction between Lex groups of CEACAM1 and DC-SIGN may have substantial impact on the regulation of self- and nonself-recognition thereby modulating the immune response. Related assumptions may also apply to malignant tumors such as melanomas, in which the expression of CEACAM1 has been correlated with an unfavorable prognosis of the patients (Thies et al., 2002).
Very recently, it has been published that the m chain of the Mac-1 integrin is a ligand of DC-SIGN in human granulocytes (van Gisbergen K.P., Sanchez-Hernandez M., et al., 2005). Since the molecular weights of both the CEACAM1 and the m integrin are in the range of 160 kD, one could argue that the native CEACAM1 preparation used in this study may be contaminated by m integrin. To exclude this possibility, the binding of three different monoclonal Mac-1 Abs to our CEACAM1 preparation was investigated. No binding was observed. The absence of the m chain of Mac-1 was further confirmed by peptide N-glycosidase F (PNGase F) treatment. The PNGase F cleavage product shifted to the estimated molecular weight of the CEACAM1 polypeptide chains, which bound the monoclonal CEACAM1 Ab (Obrink, 1997). No additional bands were observed by a protein stain (data not shown). These findings exclude that the preparations of native CEACAM1 used in this investigation are contaminated by the m chain of Mac-1. The specific interaction of DC-SIGN with the Lex groups of CEACAM1 is further corroborated by the fact that recombinant CEACAM1 expressed in CHO cells binds DC-SIGN only after co-transfection with FUTIX. Considering that m integrin binds DC-SIGN in addition to CEACAM1, the integrin may mediate initial cell–cell contacts between granulocytes and DCs. Once firm binding between granulocytes and DCs is established, the interaction between fucosylated CEACAM1 and DC-SIGN may participate in recognition and signaling affecting the immune response, as discussed above.
It is well established that DC-SIGN binds to oligomannosidic residues in addition to Lex and related structures. Here, we show that CEACAM1 expressed in HEK293 cells bound DC-SIGN in the absence of FUTIX. Because the monoclonal Lex Ab did not bind to recombinant CEACAM1 from HEK293 cells, an interaction via Lex residues can be excluded. Considering the binding specificity of DC-SIGN, binding of the high–mannose-specific lectin of Galanthus nivalis (GNA) to CEACAM1 was tested. Indeed, GNA was strongly binding to the recombinant CEACAM1 preparation of HEK293 cells. This finding indicates that recombinant CEACAM1 expressed in HEK293 cells contains high-mannose residues and that these residues bind DC-SIGN. This conclusion is supported by our unpublished MALDI-TOF-MS/MS data, which revealed a high proportion of high-mannose residues in recombinant CEACAM1 preparations isolated from HEK293 cells. Since native CEACAM1 binds DC-SIGN via Lex residues, the binding of DC-SIGN to recombinant CEACAM1 from HEK293 cells must be regarded as a cell-culture artifact. From these results, it becomes apparent that, with respect to the structure and binding function of glycans expressed in recombinant proteins, one should be cautious to relate in vitro findings to the in vivo situation. It is safe to base conclusions primarily on the structural analysis of glycans of native glycoproteins.
Besides PMNs, a high-expression level of the Lex epitope has been observed in the colonic mucosa. Among members of the CEACAM family, CEA (CEACAM5) is highly expressed in human colon (Prall et al., 1996). In analogy to CEACAM1 in granulocytes, we hypothesized that CEA may represent a major carrier of Lex residues in the colon mucosa. Indeed, we recently showed that native CEA purified of colon cancer metastases carry Lex residues (Lucka et al., 2005). Transfection studies supported our hypothesis as CEA binds the monoclonal Lex Ab only when co-expressed with FUTIX. More detailed studies revealed that CEA immunoprecipitated from colon carcinomas carries Lex groups and is bound by DC-SIGN. While our studies were in progress, similar results were published (van Gisbergen K.P., Aarnoudse C.A., et al., 2005) supporting the view that comparable with CEACAM1 in human granulocytes, CEA decorated by Lex groups may play an important role in the regulation of the immune response in normal colon and colon carcinomas.
In conclusion, our results demonstrate that Lex groups on CEACAM1 from human granulocytes mediate the binding to DC-SIGN of DCs. The expression of Lex groups is not restricted to CEACAM1 because CEA in the human colonic mucosa similarly carries Lex residues. These findings indicate that CEACAM1, CEA, and possibly other members of the CEACAM family are important mediators between the innate and adaptive immune system. The interaction of members of the CEACAM family with DCs may have important implications in inflammatory diseases, such as polyarthritis (Honig et al., 1999) and ulcerative colitis (Iijima et al., 2004).
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Abs and reagents
CEACAM1-specific mAb 4D1C2 and mAb 12140–4 and the pan-specific mAb T84.1 were purified form hybridoma supernatants, as previously described (Stoffel et al., 1993
). Abs were directly labeled by a FITC-labeling kit, as recommended by the supplier (Calbiochem, Darmstadt, Germany). The PE-labeled DC-SIGN-specific mAb 120507, the DC-SIGN-blocking Ab DC28, mAb anti-CD83, and mAb anti-CD86 were purchased from R & D Systems Inc. (Minneapolis, MN). Rat monoclonal IgM Abs L3 and L5 directed against high-mannose structures and Lex, respectively, were a kind gift of M. Schachner, ZMNH, Hamburg, Germany. The PE-labeled LAMP1-specific mAb H5G11, the myc-tag-specific mAb 9E10, and rabbit polyclonal hemagglutinin (HA) antiserum were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The mouse polyclonal CEACAM antiserum was obtained from Dianova (Hamburg, Germany). Digoxigenin-labeled GNA and the horseradish peroxidase (HRP)-labeled anti-digoxigenin mAb were purchased from Roche (Mannheim, Germany). Lex–BSA conjugate and Fuc were obtained from Calbiochem. All other reagents were obtained at p.a. quality from Merck (Darmstadt, Germany).
Cell lines and tissue culture
CHO and HEK293 cells were obtained from the American Type Culture Collection and cultured in Dulbeco’s modified Eagle’s medium and RPMI 1640, respectively, containing 10% fetal calf serum, 100 U/mL penicillin, and 10 mg/mL streptomycin. All culture reagents were purchased from PAA Laboratories (Cölbe, Germany). Stable CHO transfectants expressing CEACAM1 or CEACAM1/FUTIX were generated, as described before (Ebrahimnejad et al., 2004), and cultivated in the presence of G418 and Zeocine, respectively, at a final concentration of 0.5 mg/mL. Expression of comparable levels of CEACAM1 on the surface of both transfectomas was confirmed by FACS analysis applying T84.1 (data not shown).
Transfection and generation of soluble Fc-DC-SIGN
The entire coding regions of the FUTs, FUTIII, IV, VII and IX, and the extracellular part of DC-SIGN were amplified from human cDNA by polymerase chain reaction (PCR) and cloned into a pEF-BOS based expression vector, as described previously (Ebrahimnejad et al., 2004). For precipitation and detection, a myc-tag was attached to the C-terminus of CEACAM1, and a human IgG1 Fc-fragment was attached to the N-terminus of DC-SIGN. The identity of all expression constructs was confirmed by DNA sequencing. Transient transfection of CHO and HEK293 cells was performed with 5 µg of DNA applying Lipofectamine 2000, as recommended by the supplier (Invitrogen, Karlsruhe, Germany). Cells were cultivated for 48 h and subsequently lysed in Triton extraction buffer (10mM Tris [ph 7.4], 150mM NaCl, 5mM EDTA, 10% [vol/vol] glycerol, 1% triton X-100, 1mM phenylmethylsulfonyl fluoride, aprotinin [15 ng/ml], 1mM dithiothreitol, 10mM sodium pyrophosphate, 10mM glycerolphosphate, 1mM sodium orthovanadate, 0.1mM sodium pervanadate, 10mM sodium fluoride), as previously described (Nollau and Mayer, 2001). For the generation of soluble Fc-DC-SIGN, HEK293 cells were transiently transfected as described above and cultivated for 48 h in serum-free medium (SFMII, Invitrogen). Cell-culture supernatants were harvested, concentrated by ultrafiltration (Amicon 10; Millipore Cooperation, Bedford, MA), exchanged against phosphate-buffered saline (PBS) by PD10 columns (Amersham Bioscience, Freiburg, Germany), and protein concentration was determined by Bradford assay (BioRad, München, Germany).
Isolation of granulocytes and generation of DCs
Granulocytes were isolated from fresh buffy coats of normal human blood donors. Blood was layered on Ficoll-Paque (d = 1.077 g/cm3; Pharmacia, Freiburg, Germany), spun at 2000 rpm at 4°C for 20 min, the upper phase was carefully removed, and granulocytes were collected. To remove contaminating erythrocytes, cells were resuspended in erythrocyte lysis buffer (155 mM ammonium chloride, 10 mM potassium bicarbonate, 0.1 mM EDTA, pH 8.0) and incubated for 20 min at room temperature. After centrifugation (1500 rpm, 5 min), the supernatant was removed, and granulocytes were washed three times with PBS and immediately analyzed. Purity of granulocyte preparations was confirmed by FACS applying anti-human CD15 mAb CBL144 (Chemicon, Hampshire, UK; data not shown).
For the generation of immature DCs, mononuclear cells from normal human blood donors were isolated from the interphase after density centrifugation, as described above. Monocytes were isolated by anti-CD14 magnetic microbeads following the protocol of the supplier (Miltenyi Biotec, Bergisch-Gladbach, Germany). Monocytes were cultivated for 5–7 days in RPMI 1640/10% fetal calf serum (FCS) in the presence of IL-4 (500 U/mL) and GM-CSF (800 U/mL), both purchased from PromoKine (Heidelberg, Germany). Differentiation of monocytes to immature DCs was confirmed by FACS analysis using DC-SIGN-specific mAb 120507 and Abs directed against the surface markers CD14, CD83, and CD86, respectively, demonstrating high levels of DC-SIGN expression, low to moderate levels of expression of CD83 and CD86, respectively, and lack of CD14 expression typical for immature DCs (data not shown).
Sodium dodecyl sulfate polyacrylamide gel electrophoresis, western blot analysis, digestion, and IP
After determination of the total protein amount by Bradford assay, 50 µg of whole cellular protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Immobilon; Millipore, Schwalbach, Germany). After blocking overnight at 4°C in 1% blocking reagent (Roche), membranes were incubated with primary Abs (1 µg/mL) in Tris-buffered saline with Tween (TBST: 150 mM NaCl, 10 mM Tris, pH 8.0, 0.05% Tween 20) for 1 h at room temperature, washed three times with TBST and incubated at a dilution of 1:25,000 with the appropriate secondary Ab for 1 h at room temperature. As secondary Abs, HRP-conjugated goat anti-mouse IgG, goat anti-rabbit IgG, or rat anti-IgM Abs were applied all purchased from Dianova. After washing for 3 h at room temperature in TBST, signals were detected by chemiluminescence (ECL reagent, Amersham Bioscience). Defucosylation of CEACAM1 was carried out overnight at 37°C with 25 mU/mL of Fuc in 50 mM sodium phosphate buffer (pH 5.0). For IP, 200 µL of whole cellular lysate were cleared with 10 µL of protein G-PLUS agarose (Santa Cruz Biotechnology, Santa Cruz, CA), incubated with 2 µg of the appropriate mAb at 4°C for 2 h, subsequently 10 µL of protein G-PLUS agarose were added, and the reaction was incubated for further 2 h at 4°C. After washing by lysis buffer, beads were boiled for 3 min at 95°C in 5x Laemmli buffer, and precipitates were separated by SDS–PAGE.
FACS analysis
Cells were incubated with the appropriate Ab or Fc-DC-SIGN for 30 min at 4°C in FACS buffer (1 mM CaCl2, 2 mM MgCl2, 1% FCS in PBS). Before Ab incubation, potential binding of Fc-DC-SIGN or Abs by Fc receptors was blocked by preincubation of granulocytes and DCs by mouse or human polyclonal IgG (Sigma, Taufkirchen, Germany) at a final concentration of 200 µg/mL for 30 min at 4°C. After washing, cells were fixed in 1% paraformaldehyde/PBS, and FACS analysis was performed on a FACS Calibur flow cytometer (Becton Dickinson Immunocytometry System, Manisfield, MA). For blocking experiments, cells were preincubated by the appropriate Abs at a final concentration of 20 µL for monoclonal and at 100 µl for polyclonal Abs for 30 min at 4°C. Histograms were generated by the CELLQUEST software.
DC-SIGN overlay assay
Whole cellular extracts or immunoprecipitates were separated by SDS–PAGE and transferred to PVDF membranes, as described above. After blocking, membranes were incubated with recombinant soluble Fc-DC-SIGN (5 µg/mL) in TSM buffer (20 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM CaCl2, 2 mM MgCl2) for 2 h at 4°C. After washing in TSM buffer, detection was performed by HRP-labeled rabbit anti-human IgG followed by ECL.
Cell- and bead-binding assays
Plasma membranes of CHO cells were fluorescently labeled by FM 1–43FX, as recommended by the supplier (Molecular Probes, Eugene, OR). Cells were washed in PBS, resuspended in cell-culture medium containing 1 mM CaCl2 and 2 mM MgCl2, respectively, and incubated at a ratio of 1:1 with 1 x 105 immature DCs cultivated on Lab Tek glass chamber slides (surface area: 1.8 cm2; Nunc, Wiesbaden, Germany). After incubation for 30 min at 37°C, slides were washed five times in prewarmed PBS, cells were fixed in 4% paraformaldehyde/PBS, and binding of CHO cells was visualized by fluorescence microscopy (Leitz DMRB fluorescence microscope, Leitz, Wetzlar, Germany) and photographically documented by a digital camera. Cell numbers per microscopic field were determined from digital images applying the cell-counting tool of the NIH image software package (version 1.63; NIH, Bethesda, MR). For blocking studies, immature DCs were preincubated by mAb DC28 (20 µg/mL) for 30 min at 37°C. Levels of statistical significance were calculated by the Student’s t-test.
For the bead-binding assay, BSA, Lex–BSA, and native CEACAM1 from human granulocytes were covalently coupled in the presence of 1-ethyl-3-(dimethylaminopropyl)-carbodiimide (EDAC) to carboxylate-modified FluoSpheres (diameter: 1 µm) at 4°C overnight, as recommended by the supplier (Molecular Probes). Native CEACAM1 was purified from human granulocytes by immunoaffinity chromatography and by gel chromatography, as described previously (Lucka et al., 2005). Beads were washed in PBS, shortly sonicated to disperse particles, and kept in 1% BSA/PBS at 4°C until further use. About 1 x 105 immature DCs cultivated on glass chamber slides were incubated with 1 x 107 beads and further processed under identical conditions, as described for the cell binding assay. After fixation and photographic documentation, the number of beads per microscopic field was determined by the particle analysis tool of the NIH image software package. For confocal microscopy, the TCS SP2 confocal microscope from Leica Microsystems GmbH (Heidelberg, Germany) was used, and digital images were further processed by the Adobe Photoshop 6.0 software. Nuclear chromatin was stained by TOTO3 after the fixation of cells, as recommended by the manufacturer (Molecular Probes).
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Supplementary data |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataAcknowledgmentsReferences |
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Supplementary data are available at Glycobiology online (http://glycob.oxfordjournals.org/).
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataAcknowledgmentsReferences |
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We thank C. Fischer for the construction of Fc-DC-SIGN, H. Sander and K. Dierck for cloning the different fucosyltransferases, M. Schweitzer (ZMNH, Hamburg, Germany) for access and help with the confocal microscope, M. Schachner (ZMNH, Hamburg, Germany) for providing mAb L3 and mAb L5, and O. Børmer for providing mAb12140–4. This work was supported by the Deutsche Forschungsgemeinschaft (SFB470-C5 to C.W.).
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Abbreviations |
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Ab, antibody; BSA, bovine serum albumin; CD, cluster of differentiation; CEA, carcinoembryonic antigen; CEACAM, CEA-related cell adhesion molecule; CHO, Chinese hamster ovary; DC, dendritic cell; DC-SIGN, DC-specific ICAM-3 grabbing nonintegrin; FACS, fluorescence-activated cell sorter; FITC, fluorescein isothiocyanate; Fuc,
(1–3,4) fucosidase; FUT, fucosyltransferase; GNA, Galanthus nivalis agglutinin; HA, hemagglutinin; HEK293, human embryonic kidney 293; HRP, horseradish peroxidase; IP, immunoprecipitation; kD, kilodalton; LAMP1, lysosomal-associated membrane protein 1; Lex, Lewis x; mAb, monoclonal Ab; PBS, phosphate-buffered saline; PE, phycoerythin; PVDF, polyvinylidene fluoride; SDS–PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; TBST, Tris-buffered saline with Tween |
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