Macrophage C-type lectin on bone marrow–derived immature dendritic cells is involved in the internalization of glycosylated antigens

Kaori Denda-Nagai, Nobuyoshi Kubota, Makoto Tsuiji, Mika Kamata and Tatsuro Irimura1

Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

Received on February 4, 2002; revised on April 15, 2002; accepted on April 15, 2002


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Bone marrow–derived dendritic cells (DCs) were examined for the expression of the murine macrophage C-type lectin specific for galactose and N-acetylgalactosamine (mMGL). Flow cytometric analysis after double staining for MHC class II and mMGL with specific monoclonal antibodies indicated that mMGL was expressed on immature DCs with low to moderate levels of MHC class II and down-regulated during maturation. Immature DCs bound and internalized {alpha}-N-acetylgalactosaminides conjugated to soluble polyacrylamide ({alpha}-GalNAc polymers), whereas mature DCs and bone marrow cells did not. The two-color flow cytometric profiles indicated that the degree of {alpha}-GalNAc polymer bindings exactly coincided with the intensity of the binding of a mMGL-specific monoclonal antibody LOM-14. The internalized {alpha}-GalNAc polymers seemed to be transported to MHC class II compartments. Thus, mMGL is transiently expressed on bone marrow–derived DCs during their development and maturation and suggested to be involved in the uptake of glycosylated antigens for presentation.

Key words: antigen uptake/C-type lectin/dendritic cells/glycosylated antigen


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Dendritic cells (DCs) are known as antigen-presenting cells that play a key role in the immune system. They capture foreign antigens in peripheral tissues, process them into peptides that bind to class II major histocompatibility complex (MHC) antigens and migrate to lymphoid organs, where they activate naive T cells by presenting their MHC–peptide complexes on their cell surfaces (Banchereau and Steinman, 1998Go). During this process, DCs differentiate from an immature state to a mature state as judged by their morphology, biological functions, and cell surface phenotypes.

DCs are known to incorporate antigens by phagocytosis, macropinocytosis, and receptor-mediated endocytosis through a variety of putative antigen receptors having C-type lectin domains. At least two such receptors, DEC-205 and the macrophage mannose receptor (MMR), type I Ca2+-dependent C-type multilectins, appear to be involved in antigen uptake and processing (Jiang et al., 1995Go; Mahnke et al., 2000Go; Sallusto et al., 1995Go). Recently, many novel C-type lectins, such as DCIR (Bates et al., 1999Go), Langerin (Valladeau et al., 2000Go), DC-SIGN (Geijtenbeek et al., 2000Go), Dectin-1 (Ariizumi et al., 2000bGo), and Dectin-2 (Ariizumi et al., 2000aGo), all type II transmembrane proteins, have also been reported to be expressed on DCs and Langerhans cells. Their contributions to antigen recognition and processing are not clear, although Langerin was shown to be an endocytic receptor (Valladeau et al., 2000Go). Among them, MMR, DC-SIGN (Mitchell et al., 2001Go), and Langerin were previously shown to be specific for mannose. Other C-type lectins expressed on DCs do not seem to recognize carbohydrates.

Mannose-specific recognition should be effective to incorporate and to process glycoproteins with high-mannose type carbohydrate chains expressed on pathogens and truncated or unprocessed N-glycans expressed by defective and apoptotic cells. However, none of these C-type lectins recognize galactose or N-acetylgalactosamine, which also seem to be important pathogenic substances. The macrophage galactose-type C-type lectin (MGL) has a binding capacity to galactose and N-acetylgalactosamine as monosaccharides and is a type II transmembrane glycoprotein that contains a single carbohydrate recognition domain. Murine MGL (mMGL) was originally detected on tumoricidal peritoneal macrophages (Sato et al., 1992Go) and human MGL (hMGL) was cloned from IL-2-treated peripheral blood monocytes (Suzuki et al., 1996Go). The Mgl gene consists of ten exons and has been mapped to mouse chromosome 11 (Tsuiji et al., 1999Go), and the MGL gene has been mapped to human chromosome 17p13.2. The chromosome localization of MGL is distinct from that of many C-type lectins, MMR, DEC-205, DCIR, langerin, DC-SIGN, and Dectin-1, previously claimed to be specific for DCs (Bates et al., 1999Go; Kato et al., 1998Go; Soilleux et al., 2000Go; Valladeau et al., 2000Go). mMGL is expressed on histiocytic macrophages but not on Langerhans cells (Imai et al., 1995Go; Mizuochi et al., 1997Go). However, it has not previously been examined whether or not DCs express MGL.

We have examined the expression of mMGL on bone marrow (BM)–derived DCs by flow cytometry using mMGL-specific monoclonal antibody (mAb) LOM-14. We found that only immature DCs expressed high levels of mMGL and that such immature cells were also able to bind and uptake soluble polyacrylamides with clusters of GalNAc residues. We hypothesize that mMGL expressed on BM-derived immature DCs can function as a recognition and internalization molecule with distinct carbohydrate specificity from other DC lectins and potentially participates in the presentation of antigens bearing clusters of GalNAc.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Surface phenotypes of BM-DCs
Surface phenotypes of BM-derived cells were examined by two-color flow cytometry. BM-derived nonadherent cells could be divided into three subpopulations according to their expression levels of MHC class II, namely, MHC class II, MHC class IIlow/moderate, and MHC class IIhigh. The proportions of these subpopulations in a given cell preparation were indicated by staining with phycoerythrin (PE)-conjugated anti-MHC class II antibody alone (Figure 1), and these proportions differed with the duration of BM cell culture. On day 6, most cells (42.1%) were MHC class II, whereas 33.2% was MHC class IIlow/moderate, and 10.5% was MHC class IIhigh (Figure 1A). On day 8 in the absence of lipopolysaccharide (LPS), there was a higher percentage (28.2%) of MHC class IIhigh cells than on day 6 (Figure 1B), while if the cells were treated with LPS, 70.8% of the cells was MHC class IIhigh on day 8 (Figure 1C). In all cases, MHC class IIhigh cells coexpressed CD11c, DEC-205, CD40, and CD86 and were thus considered to be mature DCs. MHC class IIlow/moderate cells also expressed CD11c but not DEC-205, CD40, or CD86. Judging from the time-dependent shift and the effect of LPS, it is reasonable to speculate that the MHC class IIlow/moderate population consists of immature DCs.



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Fig. 1. Surface phenotypes of BM-derived DCs at various stages of development and maturation. Nonadherent cells cultured with GM-CSF were collected and analyzed by flow cytometry on day 6 (A), day 8 without LPS treatment (B), and day 8 after 24 h LPS treatment (C). Two-color flow cytometric analysis was performed using antibodies tagged with FITC indicated below each pattern together with PE-labeled anti-mouse MHC class II (see Materials and methods). The control was the staining pattern of PE-labeled anti-mouse MHC class II alone. When control rat or hamster antibodies were used, patterns were almost the same (data not shown). Cells could be divided into three subpopulations according to MHC class II expression levels, and the percentage of each subpopulation is indicated in the panels showing control staining patterns.

 
Expression of mMGL on DCs
The expression of mMGL on the DC surface was examined by flow cytometry using the specific mAb LOM-14 (Figure 1). mMGL was not expressed on BM cells before incubation with granulocyte-macrophage colony-stimulating factor (GM-CSF) (data not shown). mAb LOM-14 binding was very low on the majority of MHC class IIhigh cells (Figure 1). In contrast, mMGL was detected by mAb LOM-14 on almost all MHC class IIlow/moderate cells at significant levels (Figure 1). To assess whether the surface expression of mMGL was regulated at the level of transcription, the expression of mMGL transcripts was examined by reverse transcriptase polymerase chain reaction (RT-PCR) (Fig. 2). In contrast to the absence of mMGL on BM cell surfaces, these cells expressed mMGL mRNA. The cell surface expressions and mRNA levels of mMGL were elevated at day 6. Immature DCs sorted for cell surface mMGL and MHC class II from day 6 DCs showed high levels of mMGL mRNA. Day 8 mature DCs sorted from day 8 LPS-treated DCs for high levels of MHC class II exhibited low mMGL mRNA.



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Fig. 2. The expression of mMGL mRNA analyzed by RT-PCR. Lane 1, BM cells; 2, day 6 DCs; 3, day 8 DCs without LPS treatment; 4, day 8 DCs after LPS treatment for 24 h; 5, immature DCs sorted from day 6 DCs; 6, mature DCs sorted from day 8 DCs after LPS treatment for 24 h.

 
Binding of GalNAc polymers to DCs
To assess whether mMGL on DCs acts as a lectin, we performed carbohydrate binding assays that were measured by flow cytometry. {alpha}-GalNAc polymers, but not ß-GlcNAc polymers, bound to immature DCs but not to mature DCs and BM cells in a Ca2+-dependent manner (Figure 3A and data not shown). The 2D flow cytometric profiles indicated that the degree of {alpha}-GalNAc polymer bindings exactly coincided with the intensity of the binding of mAb LOM-14 specific for mMGL (Figure 1, 3A). This finding was confirmed by three-color flow cytometric analysis (Figure 3B). The binding of {alpha}-GalNAc polymers was inhibited by the presence of 0.1 M GalNAc, but not by 0.1 M Gal or GlcNAc (Figure 3C).



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Fig. 3. Binding of {alpha}-GalNAc polymers to BM-derived DCs. (A) Cell surface binding of {alpha}-GalNAc and ß-GlcNAc polymers was examined by flow cytometry. Cells were incubated first with biotinylated {alpha}-GalNAc or ß-GlcNAc polymers, then with FITC-streptavidin, and subsequently with PE-conjugated anti-MHC class II. PBS containing 10 mM EDTA, 0.1% BSA, and 0.1% sodium azide was used as the incubation medium without Ca2+. (B) Three-color flow cytometric analysis for {alpha}-GalNAc polymers, mMGL, and MHC class II. Cells (day 6) were incubated with biotinylated {alpha}-GalNAc polymers and mAb LOM-14, then tagged with streptavidin-RED670 and FITC-conjugated anti-rat IgG individually, and subsequently with PE-conjugated anti-MHC class II. (C) Inhibition of {alpha}-GalNAc polymer binding to day 6 DCs (MHC class II–positive cells) by monosaccharides. Cells were incubated with FITC-GalNAc polymers in the presence of indicated monosaccharides. Mean fluorescence intensity (MFI) of MHC class II positive cells is shown, and the results represent the mean ± SD of three independent experiments.

 
Internalization of GalNAc polymers by day 6 DCs
Internalization of bound fluorescein isothiocyanate (FITC)-conjugated {alpha}-GalNAc or ß-GlcNAc polymers by immature DCs was examined on a flowcytometer. Ethylenediamine tetra-acetic acid (EDTA) solution was applied at various time points to remove {alpha}-GalNAc polymers at the cell surfaces, and the polymers not removed by this treatment was considered to be internalized. Increase in the fluorescence intensity after incubation with polymers at 37°C indicated that polymers were internalized and accumulated (Figure 4). FITC-GlcNAc polymers were not internalized by day 6 DCs. By two-color flow cytometry with MHC class II, it was also confirmed that the cells that internalized {alpha}-GalNAc polymers were MHC class IIlow/moderate (data not shown). To confirm that the internalization of FITC-GalNAc polymers were mediated by mMGL, DCs were incubated with FITC-GalNAc polymers in the presence of anti-mMGL mAb LOM-14 or mAb LOM-8.7. mAb LOM-14 and mAb LOM-8.7 were previously reported as a nonblocking and blocking antibodies, respectively (Kimura et al., 1995Go). The binding pattern of mAb LOM-8.7 and mAb LOM-14 to DCs were almost the same in two-color flow cytometric analysis with MHC class II (data not shown). The internalization of FITC-GalNAc polymers was partially but not statistically significant, inhibited by both mAb LOM-14 and mAb LOM-8.7, although the binding of FITC-GalNAc polymers did not seem to be inhibited by either of these mAbs (Figure 5).



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Fig. 4. Flow cytometric profiles showing time-dependent uptake of {alpha}-GalNAc polymers by day 6 DCs. Uptake of {alpha}-GalNAc polymers was examined using flow cytometry. Day 6 DCs were incubated with FITC-GalNAc polymers as indicated in the figure. Dotted lines indicate the peak of the fluorescence intensity resulted from the binding at 4°C.

 


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Fig. 5. Inhibition of binding and internalization of {alpha}-GalNAc polymers by day 6 MHC class II–positive DCs by anti-mMGL mAbs. The cells were incubated with FITC-GalNAc polymers in the presence of indicated antibodies. MFI of MHC class II positive cells is shown, and the results represent the mean ± SD of four independent experiments. NRS: normal rat serum.

 
Day 6 DCs adherent to cover slips were pulsed with {alpha}-GalNAc polymers for 1 h, and the localization of {alpha}-GalNAc polymers was examined by confocal laser scanning microscopy. {alpha}-GalNAc polymers colocalized with mMGL detected by mAb LOM-14 even after the polymers were internalized (Figure 6A). Furthermore, internalized {alpha}-GalNAc polymers seemed to colocalize with intracellular populations of lysosome-associated membrane protein-1 (LAMP-1) and with MHC class II (Figure 6B, C). These results suggest that internalized {alpha}-GalNAc polymers are transported to MHC class II compartments (MIICs).



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Fig. 6. Intracellular localization of {alpha}-GalNAc polymers, mMGL, LAMP-1, and MHC class II. Day 6 DCs were seeded on coverslips, incubated with FITC-GalNAc polymers at 37°C for 1 h, fixed, and stained with anti-mMGL (A), anti-LAMP-1 (B), or anti–MHC class II (C) antibodies.

 

    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
In the present study, we detected significant levels of mMGL expression reactive with mAb LOM-14 on immature DCs. BM cells and the majority of mature DCs did not seem to express this molecule on their surfaces. The levels of mMGL mRNA determined by RT-PCR analysis were also high in immature DCs and low in mature DCs. In our preliminary experiment, mMGLlow-MHC class IIhigh cells could be generated after culturing mMGL-positive cells sorted from day 6 DCs by magnetic beads. We thus hypothesize that mMGL is only transiently expressed on DCs that are developing and maturing, during which stage they are capable of antigen uptake but do not yet present MHC class II–peptide complexes. Although we could not detect mMGL proteins in BM cells by cytostaining (data not shown), mRNA corresponding to mMGL is detectable in BM cells prior to the incubation with GM-CSF, which suggests that the cell surface expression may be due to changes in translation or intracellular transport of this molecule. Among other C-type lectins known to be expressed on DCs, DEC-205 was shown to be upregulated during the maturation (Figure 1), which was consistent with a previous report (Inaba et al., 1995Go). Expression of DC-SIGN and DCIR on both immature and mature human DCs was previously reported (Bates et al., 1999Go; Geijtenbeek et al., 2000Go). DCIR was also expressed on monocytes, a DC precursor, whereas the level on mature DCs obtained by LPS activation was slightly lower than that on immature DCs (Bates et al., 1999Go). Therefore, mMGL expression on immature DCs seems to be unique.

It has already been reported that many C-type lectins, such as MMR, DEC-205, DCIR, Langerin, DC-SIGN, Dectin-1, and Dectin-2, were expressed on DCs. None of them recognize Gal or GalNAc residues. Alterations in O-glycosylation such as an increased expression of T (Gal-GalNAc-Thr/Ser) and Tn (GalNAc-Thr/Ser) antigens are known to be associated with malignant cells. These epitopes are potential targets for antitumor immunity. It is suggested that terminal sialic acid residues of glycoproteins are lost and Gal or GalNAc residues are exposed on apoptotic cells (Savill, 1997Go). Structural alterations in O-glycans are known to occur during the differentiation, activation, and migration of T-lymphocytes (Baum et al., 1995Go; Gillespie et al., 1993Go; Lowe, 2001Go; Reisner et al., 1976Go). Therefore, cell surface receptors for truncated O-glycans should play important roles in the immune system in the protection from infection, antitumor immunity, and the clearance of apoptotic cells.

{alpha}-GalNAc polymers were used to test whether cell surface mMGL on DCs functions as a lectin, because mMGL and hMGL were shown to have high affinity with clusters of O-linked GalNAc residues (Iida et al., 1999Go;Yamamoto et al., 1994Go). {alpha}-GalNAc polymers, but not ß-GlcNAc polymers, bind to immature DCs in a Ca2+-dependent and GalNAc-specific manner, but the binding of {alpha}-GalNAc polymers was not inhibited by Gal. This was probably due to differences in the affinity of mMGL expressed on the surfaces of DCs toward these monosaccharides. The mMGL expression profile clearly correlated with the profile of {alpha}-GalNAc polymer binding in day 6 and day 8 DC populations. Furthermore, flow cytometric analyses and confocal microscopic studies indicated that cells strongly reactive with {alpha}-GalNAc polymers expressed high levels of mMGL. Therefore, the binding of {alpha}-GalNAc polymers is highly likely to be mediated by mMGL although the presence of another lectin expressed in parallel with mMGL remains to be possible. It has been suggested by several observations that mMGL can participate in carbohydrate binding at the cell surfaces. First, macrophages elicited by thioglycolate treatment are able to internalize glycoproteins through mMGL (Kawakami et al., 1994Go). Second, CTLL-2 T lymphoma cells transfected with mMGL cDNA acquire the ability to bind galactose or lactose-conjugated microspheres (Hosoi et al., 1998Go; Ichii et al., 1997Go). Third, K562 human erythroleukemia cells transfected with hMGL are able to internalize {alpha}-GalNAc polymers in an MGL-dependent manner (Fujita et al., unpublished data). Recently, we have also found that hMGL expressed on monocyte-derived immature DCs was involved in the uptake of {alpha}-GalNAc polymers (Higashi et al., 2002).

Immature DCs that expressed high levels of mMGL bound and internalized carbohydrate antigens. The binding of {alpha}-GalNAc polymers was not inhibited anti-mMGL mAb LOM-14 or LOM-8.7, although it has already been reported that mAb LOM-8.7, but not LOM-14, blocked the binding of galactosylated poly-L-lysine to recombinant mMGL (Kimura et al., 1995Go). On the other hand, the internalization of {alpha}-GalNAc polymers was partially inhibited by both mAb LOM-14 and mAb LOM-8.7. It has already been shown that mAb LOM-14 was internalized by immature DCs (data not shown). Furthermore, internalized {alpha}-GalNAc polymers colocalized with mAb LOM-14-reactive mMGL as observed by confocal microsopy. Therefore, it is strongly suggested that {alpha}-GalNAc polymers were internalized with mMGL. However, taking into account the fact that Gal and anti-mMGL blocking mAb LOM-8.7 could not inhibit the binding of {alpha}-GalNAc polymers, we cannot exclude a possibility that mMGL on DCs functions in accordance with another molecule having high affinity to GalNAc polymers.

The internalization of mMGL was expected from the fact that mMGL had tyrosine-based amino acid sequences (YENL) involved in endocytosis in the cytoplasmic domain (Sato et al., 1992Go). Macrophages elicited by thioglycolate were previously shown to internalize glycoproteins through mMGL (Kawakami et al., 1994Go). The antigens internalized by immature DCs were known to enter the endocytic pathway and transported to early endosomes, late endosomes, or lysosomes (Banchereau and Steinman, 1998Go). In immature DCs, newly synthesized MHC class II molecules accumulate in late endosomes or lysosomes (MIICs). During the DC maturation, antigen processing and peptide loading are known to occur in MIICs (Inaba et al., 2000Go; Pierre et al., 1997Go; Turley et al., 2000Go). The MMR and DEC-205 are involved in the antigen presentation. The MMR mainly releases its ligand in early endosomes and recycles to plasma membranes (Engering et al., 1997Go; Tan et al., 1997Go), although some of them are also located in MIICs (Prigozy et al., 1997Go). In contrast to the MMR, DEC-205 targets to MIICs and is more efficient than the MMR in antigen presentation (Mahnke et al., 2000Go). Thus, the delivery of antigens into MIICs is considered important event in the antigen presentation. To assess whether mMGL is involved in antigen presentation, the fate of {alpha}-GalNAc polymers bound to the surface of immature DCs was examined by confocal laser scanning microscopy. {alpha}-GalNAc polymers were internalized and colocalized with MGL, LAMP-1, and MHC class II. These results suggest that mMGL is also capable of targeting antigens to MIICs and presenting effectively. When DCs were cultured with {alpha}-GalNAc polymers for a day in our preliminary experiment, their maturation did not occur unless a maturation signal was provided by LPS, judging from the expression levels of cell surface markers, such as MHC class II, CD86, and DEC-205. Therefore, glycan binding to mMGL alone does not seem to provide a maturation signal.

In conclusion, we observed that mMGL, which is involved in the recognition and internalization of glycoproteins, is transiently expressed on BM-derived immature DCs. mMGL should be useful as a novel marker for immature DCs.


    Materials and methods
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Mice
C57BL/6 mice (5–12 weeks old) were obtained from Charles River Japan (Yokohama, Japan) and housed under SPF conditions.

Generation of BM-DCs
BM-DCs were generated as previously described with slight modifications (Inaba et al., 1992Go). Briefly, BM cells were collected and erythrocytes were lysed by ammonium chloride treatment (day 1). Cells (1.0 x 106 cells/ml) were plated in 24-well plates in 1 ml per well of RPMI1640 medium supplemented with 10% fetal calf serum, 10 mM HEPES, 50 µM 2-ME, 100 U/ml penicillin, 100 µg/ml streptomycin, and 1000 U/ml recombinant murine GM-CSF (Kirin Brewery, Takasaki, Japan). On day 4, the top two-thirds of the media were removed and replaced with fresh medium. On day 6, nonadherent cells were collected and replated into fresh 24-well plates, and the next day they were cultured with 1 µg/ml LPS (Bacto LPS B from Escherichia coli 0111:B4, Difco, Detroit, MI) for 24 h.

Two-color flow cytometric analysis
Nonadherent cells in the culture wells were gently suspended and used for flow cytometric analyses. Adherent cells were not included. Approximately 70% of the cells plated at day 6 were recovered at day 8 even in the presence of LPS. Cells (2–5 x 105 cells) were incubated with hybridoma culture supernatants or purified antibodies (0.5–5 µg/ml) for 30 min on ice. The following antibodies were used to analyze the DC surface phenotype. Hybridoma culture supernatants: mMGL (LOM-14, rat IgG2b, 1/10 dilution) (Kimura et al., 1995Go), DEC-205 (NLDC-145, rat IgG2a, 1/2 dilution, ATCC), CD11c (N418, hamster IgG, 1/10 dilution, ATCC), B220 (RA3-3A1/6.1, rat IgM, 1/2 dilution, ATCC), and MHC class I (M1/42, rat IgG2a, 1/2 dilution, ATCC). Purified antibodies: CD40 (3.23, rat IgG2a, 5 µg/ml, Immunotec Coulter, Marseilles, France), F4/80 (F4/80, rat IgG2b, 5 µg/ml, Dainippon Pharmaceutical), CD31 (MEC13.3, rat IgG2a, 2.5 µg/ml, BD Pharmingen, San Diego, CA), and Ly-6G (RB6-8C5, rat IgG2b, 2.5 µg/ml, Southern Biotechnology, Birmingham, AL). FITC-conjugated antibodies: CD11b (M1/70.15, rat IgG2b, 0.5 µg/ml, Caltag, Burlingame, CA) and CD14 (rmC5-2. rat IgG1, 2.5 µg/ml, BD Pharmingen). Biotinylated antibody: CD86 (RMMP1, rat IgG2a, 0.5 µg/ml, Caltag). For isotype controls, purified rat IgG (5 µg/ml, ICN, Costa Mesa, CA), purified rat IgM (5 µg/ml, Zymed, South San Francisco, CA), purified hamster IgG (1 µg/ml, ICN), and PE-conjugated rat IgG2b (0.2 µg/ml, Immunotech Coulter) were used. Primary rat and hamster antibodies were followed by FITC-conjugated rabbit anti-rat IgG (1/200 dilution, Zymed) and FITC-conjugated goat anti-hamster IgG (1/200 dilution, Southern Biotechnology), respectively. Incubation with biotinylated antibodies was followed by incubation with FITC-labeled streptavidin (1/200 dilution, Zymed). Following applications of the first and the second antibodies, the cells were then stained with PE-conjugated anti-mouse MHC class II (M5/114.15.2, 1/5000 dilution, rat IgG2b, BD Pharmingen). Antibodies and reagents were diluted in phosphate buffered saline (PBS) containing 0.1% bovine serum albumin (BSA), 0.1% sodium azide, and 2% normal goat serum. The cells were rinsed twice with PBS containing 0.1% BSA and 0.1% sodium azide at the end of each incubation. Samples were analyzed on an EPICS XL flowcytometer (Beckman Coulter, Fullerton, CA).

Isolation of immature and mature DCs and RT-PCR analysis
Cells were incubated with anti-mMGL mAb LOM-14 for 30 min on ice, with FITC-conjugated rabbit anti-rat IgG and then with PE-conjugated anti-mouse MHC class II. These antibodies were diluted in PBS containing 0.1% BSA and 2% normal goat serum. MHC class IIlow/moderate and mMGL-positive cells were sorted from day 6 DCs to obtain immature DCs and MHC class IIhigh and mMGL–/low cells were sorted from day 8 LPS-treated DCs to obtain mature DCs by an EPICS ELITE flowcytometer (Beckman Coulter). The >90% purity was confirmed by flow cytometric analysis on an EPICS XL. Total RNAs were extracted by using Ultraspec RNA zol (BIOTECX, Houston, TX), according to the manufacturer’s instructions. First-strand cDNA synthesis was carried out using oligo (dT)12–18 and Superscript II (Gibco BRL, Rockville, MD). The cDNA was used as the template in PCR reactions using Ampli Taq Gold polymerase (Perkin Elmer). PCR was performed with specific primers for mMGL (bp 465–486 and 1239–1216). The condition was 94°C for 30 s, 65°C for 30 s, and 72°C for 1 min 30 s for 40 and 45 cycles. Amplification of ß-actin (bp 42–63 and 1047–1024) was used as a control for cDNA quantity. The condition was 94°C for 30 s, 61°C for 30 s, and 72°C for 1 min for 25 and 30 cycles. The PCR products were then separated on 1.5% agarose gels, stained with ethidium bromide, and visualized with the Fluor-S image analyzer (Bio-Rad, Hercules, CA).

Carbohydrate binding and internalization assessed by flow cytometric analysis
Cell surface binding of biotinylated {alpha}-N-acetylgalactosaminide ({alpha}-GalNAc) or ß-N-acetylglucosaminide (ß-GlcNAc) conjugated with polyacrylamide carriers (GlycoTech, Rockville, MD) was examined using flow cytometry. Cells were incubated with {alpha}-GalNAc or ß-GlcNAc polymers (10 µg/ml) for 30 min on ice. Subsequently, the cells were incubated with FITC-streptavidin (10 µg/ml) and then PE-conjugated anti-mouse MHC class II. Reagents were diluted in Dulbecco’s modified PBS (DPBS, contains 0.91 mM CaCl2, 0.49 mM MgCl2) supplemented with 0.1% BSA and 0.1% sodium azide. The cells were rinsed twice with this buffer at the end of each incubation. PBS containing 10 mM EDTA, 0.1% BSA, and 0.1% sodium azide was used as the incubation medium devoid of Ca2+. To confirm the carbohydrate specific binding, cells were preincubated with 0.1 M GalNAc, GlcNAc, or Gal for 5 min on ice and then incubated with FITC-conjugated {alpha}-GalNAc polymers (GlycoTech) in the presence of monosaccharides. In the inhibition assay data was analyzed after gating on MHC class II expression. In three-color flow cytometric analysis, {alpha}-GalNAc polymers were monitored by streptavidin-RED670 (Gibco BRL).

For the internalization assays, FITC-conjugated {alpha}-GalNAc or ß-GlcNAc polymers (GlycoTech) were used as model compounds for glycosylated antigens. Day 6 DCs were incubated with FITC-{alpha}-GalNAc or ß-GlcNAc polymers (10 µg/ml) in RPMI 1640 medium supplemented with 10% fetal calf serum at 37 °C for various time periods and washed with PBS containing 10 mM EDTA, 0.1% BSA, and 0.1% sodium azide to remove the polymers attaching on the cell surfaces. In the inhibition assays DCs were incubated in the presence of anti-mMGL mMAb LOM-14 (partially purified, diluted to equivalent to 1/10 of hybridoma culture supernatants), anti-mMGL mAb LOM-8.7 (rat IgG2a, partially purified, diluted to equivalent to 1/2 of hybridoma culture supernatants) (Kimura et al., 1995Go), or normal rat serum (1/40 dilution). Data were analyzed after gating on MHC class II expression.

Confocal laser scanning microscopy
Day 6 DCs (5 x 105 cells) were plated on poly-L-lysine-coated coverslips (Iwaki, Tokyo) and allowed to adhere for 20 min at 37°C. The cells were incubated with 10 µg/ml FITC-GalNAc polymers for 1 h at 37°C, washed with DPBS, and then fixed with acetone for 30 s at room temperature. Nonspecific bindings were blocked with a blocking solution (2% normal goat serum and 3% BSA in DPBS) for 10 min, and DCs were incubated with anti-mMGL (mAb LOM-14, 1/10 dilution of hybridoma culture supernatant in the blocking solution), anti-LAMP-1 (mAb 1D4B, rat IgG2b, 1 µg/ml, BD Pharmingen), or anti-MHC class II (M5/114.15.2, 1/10 dilution of hybridoma culture supernatants, ATCC) for 40 min. After fixation in 4% paraformaldehyde dissoloved in 0.1 M sodium phosphate (pH 7.0) containing 0.91 mM CaCl2, DCs were incubated with biotinylated mouse anti-rat {kappa}/{lambda} mAb (1/100 dilution, Sigma, St. Louis, MO) for 30 min and with Texas Red–avidin D (1/100 dilution, Vector, Burlingame, CA) for 30 min. The coverslips were mounted in PermaFluor aqueous mounting medium (Shandon, Pittsburgh, PA) on glass slides and observed on a confocal microscope (MRC-1024; Bio-Rad).


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We thank Dr. Kayo Inaba for advice in generating BM-DCs, Ms. Chizu Hiraiwa for assistance in preparing this manuscript, and Kirin Brewery for supplying rGM-CSF. This work was supported by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (11557180, 11672162, and 12307054) the Research Association for Biotechnology; the Program for Promotion of Basic Research Activities for Innovative Biosciences; and the Cosmetology Research Foundation.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
{alpha}-GalNAc polymers, {alpha}-N-acetylgalactosaminides conjugated to soluble polyacrylamide; BM, bone marrow; BSA, bovine serum albumin; DCs, dendritic cells; DPBS, Dulbecco’s modified PBS; EDTA, ethylenediamine tetra-acetic acid; FITC, fluorescein isothiocyanate; GM-CSF, granulocyte-macrophage colony-stimulating factor; hMGL, human MGL; LAMP-1, lysosome-associated membrane protein-1; LPS, lipopolysaccharide; mAb, monoclonal antibody; MFI, mean fluorescence intensity; MGL, macrophage galactose-type C-type lectin; MHC, major histocompatibility complex; MIIC, MHC class II compartment; mMGL, murine MGL; MMR, macrophage mannose receptor; PBS, phosphate buffered saline; PE, phycoerythrin; RT-PCR, reverse transcriptase polymerase chain reaction.


    Footnotes
 
1 To whom correspondence should be addressed; E-mail:irimura{at}mol.f.u-tokyo.ac.jp Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Ariizumi, K., Shen, G.L., Shikano, S., Ritter, R. III, Zukas, P., Edelbaum, D., Morita, A., and Takashima, A. (2000a) Cloning of a second dendritic cell-associated C-type lectin (dectin-2) and its alternatively spliced isoforms. J. Biol. Chem., 275, 11957–11963.[Abstract/Free Full Text]

Ariizumi, K., Shen, G.L., Shikano, S., Xu, S., Ritter, I., Kumamoto, T., Edelbaum, D., Morita, A., Bergstresser, P.R., and Takashima, A. (2000b) Identification of a novel, dendritic cell-associated molecule, Dectin-1, by subtractive cDNA cloning. J. Biol. Chem., 275, 20157–20167.[Abstract/Free Full Text]

Banchereau, J. and Steinman, R.M. (1998) Dendritic cells and the control of immunity. Nature, 392, 245–252.[CrossRef][ISI][Medline]

Bates, E.E., Fournier, N., Garcia, E., Valladeau, J., Durand, I., Pin, J.J., Zurawski, S.M., Patel, S., Abrams, J.S., Lebecque, S., and others. (1999) APCs express DCIR, a novel C-type lectin surface receptor containing an immunoreceptor tyrosine-based inhibitory motif. J. Immunol., 163, 1973–1983.[Abstract/Free Full Text]

Baum, L.G., Pang, M., Perillo, N.L., Wu, T., Delegeane, A., Uittenbogaart, C.H., Fukuda, M., and Seilhamer, J.J. (1995) Human thymic epithelial cells express an endogenous lectin, galectin-1, which binds to core 2 O-glycans on thymocytes and T lymphoblastoid cells. J. Exp. Med., 181, 877–887.[Abstract]

Engering, A.J., Cella, M., Fluitsma, D., Brockhaus, M., Hoefsmit, E.C., Lanzavecchia, A., and Pieters, J. (1997) The mannose receptor functions as a high capacity and broad specificity antigen receptor in human dendritic cells. Eur. J. Immunol., 27, 2417–2425.[ISI][Medline]

Geijtenbeek, T.B., Torensma, R., van Vliet, S.J., van Duijnhoven, G.C., Adema, G.J., van Kooyk, Y., and Figdor, C.G. (2000) Identification of DC-SIGN, a novel dendritic cell-specific ICAM-3 receptor that supports primary immune responses. Cell, 100, 575–585.[ISI][Medline]

Gillespie, W., Paulson, J.C., Kelm, S., Pang, M., and Baum, L.G. (1993) Regulation of alpha 2, 3-sialyltransferase expression correlates with conversion of peanut agglutinin (PNA)+ to PNA- phenotype in developing thymocytes. J. Biol. Chem., 268, 3801–3804.[Abstract/Free Full Text]

Higashi, N., Fujioka, K., Denda-Nagai, K., Hashimoto, S., Nagai, S., Sato, T., Fujita, Y., Morikawa, A., Tsuiji, M., Miyata-Takeuchi, M., and others. (2002) The macrophage C-type lectin specific for galactose/N-acetylgalactosamine is an endocytic receptor expressed on monocyte-derived immature dendritic cells. J. Biol. Chem., forthcoming.

Hosoi, T., Imai, Y., and Irimura, T. (1998) Coordinated binding of sugar, calcium, and antibody to macrophage C-type lectin. Glycobiology, 8, 791–798.[Abstract/Free Full Text]

Ichii, S., Imai, Y., and Irimura, T. (1997) Tumor site-selective localization of an adoptively transferred T cell line expressing a macrophage lectin. J. Leukoc. Biol., 62, 761–770.[Abstract]

Iida, S., Yamamoto, K., and Irimura, T. (1999) Interaction of human macrophage C-type lectin with O-linked N-acetylgalactosamine residues on mucin glycopeptides. J. Biol. Chem., 274, 10697–10705.[Abstract/Free Full Text]

Imai, Y., Akimoto, Y., Mizuochi, S., Kimura, T., Hirano, H., and Irimura, T. (1995) Restricted expression of galactose/N-acetylgalactosamine-specific macrophage C-type lectin to connective tissue and to metastatic lesions in mouse lung. Immunology, 86, 591–598.[ISI][Medline]

Inaba, K., Inaba, M., Romani, N., Aya, H., Deguchi, M., Ikehara, S., Muramatsu, S., and Steinman, R.M. (1992) Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J. Exp. Med., 176, 1693–1702.[Abstract]

Inaba, K., Swiggard, W.J., Inaba, M., Meltzer, J., Mirza, A., Sasagawa, T., Nussenzweig, M.C., and Steinman, R.M. (1995) Tissue distribution of the DEC-205 protein that is detected by the monoclonal antibody NLDC-145. I. Expression on dendritic cells and other subsets of mouse leukocytes. Cell. Immunol., 163, 148–156.[CrossRef][ISI][Medline]

Inaba, K., Turley, S., Iyoda, T., Yamaide, F., Shimoyama, S., Reis e Sousa, C., Germain, R.N., Mellman, I., and Steinman, R.M. (2000) The formation of immunogenic major histocompatibility complex class II-peptide ligands in lysosomal compartments of dendritic cells is regulated by inflammatory stimuli. J. Exp. Med., 191, 927–936.[Abstract/Free Full Text]

Jiang, W., Swiggard, W.J., Heufler, C., Peng, M., Mirza, A., Steinman, R.M., and Nussenzweig, M.C. (1995) The receptor DEC-205 expressed by dendritic cells and thymic epithelial cells is involved in antigen processing. Nature, 375, 151–155.[CrossRef][ISI][Medline]

Kato, M., Neil, T.K., Clark, G.J., Morris, C.M., Sorg, R.V., and Hart, D.N. (1998) cDNA cloning of human DEC-205, a putative antigen-uptake receptor on dendritic cells. Immunogenetics, 47, 442–450.[CrossRef][ISI][Medline]

Kawakami, K., Yamamoto, K., Toyoshima, S., Osawa, T., and Irimura, T. (1994) Dual function of macrophage galactose/N-acetylgalactosamine-specific lectins: glycoprotein uptake and tumoricidal cellular recognition. Jpn. J. Cancer Res., 85, 744–749.[ISI][Medline]

Kimura, T., Imai, Y., and Irimura, T. (1995) Calcium-dependent conformation of a mouse macrophage calcium-type lectin. Carbohydrate binding activity is stabilized by an antibody specific for a calcium-dependent epitope. J. Biol. Chem., 270, 16056–16062.[Abstract/Free Full Text]

Lowe, J.B. (2001) Glycosylation, immunity, and autoimmunity. Cell, 104, 809–812.[ISI][Medline]

Mahnke, K., Guo, M., Lee, S., Sepulveda, H., Swain, S.L., Nussenzweig, M., and Steinman, R.M. (2000) The dendritic cell receptor for endocytosis, DEC-205, can recycle and enhance antigen presentation via major histocompatibility complex class II-positive lysosomal compartments. J. Cell Biol., 151, 673–684.[Abstract/Free Full Text]

Mitchell, D.A., Fadden, A.J., and Drickamer, K. (2001) A novel mechanism of carbohydrate recognition by the C-type lectins DC-SIGN and DC-SIGNR. Subunit organization and binding to multivalent ligands. J. Biol. Chem., 276, 28939–28945.[Abstract/Free Full Text]

Mizuochi, S., Akimoto, Y., Imai, Y., Hirano, H., and Irimura, T. (1997) Unique tissue distribution of a mouse macrophage C-type lectin. Glycobiology, 7, 137–146.[Abstract]

Pierre, P., Turley, S.J., Gatti, E., Hull, M., Meltzer, J., Mirza, A., Inaba, K., Steinman, R.M., and Mellman, I. (1997) Developmental regulation of MHC class II transport in mouse dendritic cells. Nature, 388, 787–792.[CrossRef][ISI][Medline]

Prigozy, T.I., Sieling, P.A., Clemens, D., Stewart, P.L., Behar, S.M., Porcelli, S.A., Brenner, M.B., Modlin, R.L., and Kronenberg, M. (1997) The mannose receptor delivers lipoglycan antigens to endosomes for presentation to T cells by CD1b molecules. Immunity, 6, 187–197.[ISI][Medline]

Reisner, Y., Linker-Israeli, M., and Sharon, N. (1976) Separation of mouse thymocytes into two subpopulations by the use of peanut agglutinin. Cell. Immunol., 25, 129–134.[ISI][Medline]

Sallusto, F., Cella, M., Danieli, C., and Lanzavecchia, A. (1995) Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial products. J. Exp. Med., 182, 389–400.[Abstract]

Sato, M., Kawakami, K., Osawa, T., and Toyoshima, S. (1992) Molecular cloning and expression of cDNA encoding a galactose/N-acetylgalactosamine-specific lectin on mouse tumoricidal macrophages. J. Biochem. (Tokyo), 111, 331–336.[Abstract]

Savill, J. (1997) Recognition and phagocytosis of cells undergoing apoptosis. Br. Med. Bull., 53, 491–508.[Abstract]

Soilleux, E.J., Barten, R., and Trowsdale, J. (2000) DC-SIGN; a related gene, DC-SIGNR; and CD23 form a cluster on 19p13. J. Immunol., 165, 2937–2942.[Abstract/Free Full Text]

Suzuki, N., Yamamoto, K., Toyoshima, S., Osawa, T., and Irimura, T. (1996) Molecular cloning and expression of cDNA encoding human macrophage C-type lectin. Its unique carbohydrate binding specificity for Tn antigen. J. Immunol., 156, 128–135.[Abstract]

Tan, M.C., Mommaas, A.M., Drijfhout, J.W., Jordens, R., Onderwater, J.J., Verwoerd, D., Mulder, A.A., van der Heiden, A.N., Scheidegger, D., Oomen, L.C., and others. (1997) Mannose receptor-mediated uptake of antigens strongly enhances HLA class II-restricted antigen presentation by cultured dendritic cells. Eur. J. Immunol., 27, 2426–2435.[ISI][Medline]

Tsuiji, M., Fujimori, M., Seldin, M.F., Taketo, M.M., and Irimura, T. (1999) Genomic structure and chromosomal location of the mouse macrophage C-type lectin gene. Immunogenetics, 50, 67–70.[CrossRef][ISI][Medline]

Turley, S.J., Inaba, K., Garrett, W.S., Ebersold, M., Unternaehrer, J., Steinman, R.M., and Mellman, I. (2000) Transport of peptide-MHC class II complexes in developing dendritic cells. Science, 288, 522–527.[Abstract/Free Full Text]

Valladeau, J., Ravel, O., Dezutter-Dambuyant, C., Moore, K., Kleijmeer, M., Liu, Y., Duvert-Frances, V., Vincent, C., Schmitt, D., Davoust, J., Caux, C., and others. (2000) Langerin, a novel C-type lectin specific to Langerhans cells, is an endocytic receptor that induces the formation of Birbeck granules. Immunity, 12, 71–81.[ISI][Medline]

Yamamoto, K., Ishida, C., Shinohara, Y., Hasegawa, Y., Konami, Y., Osawa, T., and Irimura, T. (1994) Interaction of immobilized recombinant mouse C-type macrophage lectin with glycopeptides and oligosaccharides. Biochemistry, 33, 8159–8166. [ISI][Medline]