Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
Received on February 28, 2004; revised on June 3, 2004; accepted on June 4, 2004
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Abstract |
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Key words: antigen presentation / endocytosis / mannose receptor / MHC class II
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Introduction |
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One receptor proposed to be involved in uptake of glycoprotein antigens is the mannose receptor, which was initially characterized on macrophages and liver endothelial cells but has more recently been identified on dendritic cells (Avrameas et al., 1996). The mannose receptor binds and mediates endocytosis of glycoconjugates with terminal mannose, fucose, or N-acetylglucosamine residues. Calcium-dependent recognition of these sugars is mediated by several C-type carbohydrate-recognition domains in the extracellular region of the receptor (Taylor, 2001
). The mannose receptor is thought to act as a molecular scavenger, binding and internalizing potentially harmful glycoconjugates. Endogenous ligands recognized by the receptor include lysosomal hydrolases, tissue plasminogen activator, and collagen propeptides, and many different pathogenic microorganisms have also been shown to bind to the receptor.
The ability of the mannose receptor to mediate internalization of glycoproteins suggests that peptides derived from glycoconjugates could associate with MHC class II molecules resulting in enhanced presentation to T cells. The presence of the mannose receptor on dendritic cells is consistent with this proposal, but no direct evidence for enhancement of uptake and presentation of glycoprotein antigens by the mannose receptor has been obtained. Studies showing that mannosylated proteins or peptides are presented more efficiently by dendritic cells than nonmannosylated proteins or peptides (Engering et al., 1997; Tan et al., 1997
) do not provide conclusive evidence that the mannose receptor is involved in this process because dendritic cells are now known to express other mannose-specific receptors, such as DC-SIGN, which could be responsible for uptake of the glycoprotein antigens (Figdor, 2002
). Thus a more direct assessment of the possible role of the mannose receptor in enhancing presentation of glycoprotein antigens is required.
In this study, the ability of the mannose receptor to enhance presentation of a glycoprotein antigen has been assessed directly. The results suggest that the receptor does not have a general role in processing and presentation of glycoprotein antigens.
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Results |
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Processing and presentation of RNases A and B were first compared in cells not expressing the mannose receptor. Mouse fibroblasts (L cells) transfected with MHC class II IAk (Germain et al., 1985) were incubated with increasing concentrations of RNase A or B. RNase-specific T cells were used to recognize complexes of MHC class II and RNase peptide 4356 presented at the surface of the L cells (Lorenz et al., 1988
). IL-2 release in response to antigen presentation was quantified using an enzyme-linked immunosorbent assay (ELISA). For both RNase A and B, concentration-dependent release of IL-2 is seen up to an antigen concentration of about 10 µg/ml, above which no further increase in IL-2 production occurs (Figure 2). Identical responses are produced with RNases A and B, indicating that fibroblasts process these two proteins at similar rates. Thus RNases A and B are appropriate test antigens for studying whether the mannose receptor can enhance antigen uptake and presentation.
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The best rat-6 cell line expressing both mannose receptor and MHC class II IAk processes and presents RNase less efficiently than the L cell line (Figure 3). However, no IL-2 release is detected when the cells are fixed with paraformaldehyde before incubation with antigens (Figure 3b) and cells not expressing MHC do not stimulate any release of IL-2 (Figure 3d), indicating that the MHC class II presentation pathway is functioning in the doubly transfected cells. The levels of antigen processing and presentation were sufficient for comparison between RNases A and B to be made. When IL-2 release was measured 16 h, 24 h, and 48 h after incubation of the doubly transfected rat-6 cell line with antigens and T cells (Figure 3), no difference was observed between the responses to RNases A and B at any of the three incubation times. Thus RNase B is not presented any more efficiently than RNase A.
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Discussion |
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Two previous studies linking the mannose receptor to enhanced antigen presentation involved experiments with monocyte-derived dendritic cells and mannosylated BSA or mannosylated peptide antigens (Engering et al., 1997; Tan et al., 1997
). Both studies showed that mannosylated antigens are presented more efficiently than nonmannosylated forms. However, although the presence of the mannose receptor on the dendritic cells was documented, no direct link between the mannose receptor and the enhanced presentation of the mannosylated antigens was demonstrated. Involvement of the mannose receptor was assumed because mannan, which inhibits binding to the mannose receptor, blocks the enhanced presentation. However, it is now known that DC-SIGN, which has the same monosaccharide specificity as the mannose receptor and is inhibited by mannan, is expressed at higher levels than the mannose receptor on monocyte-derived dendritic cells (Mitchell et al., 2001
; Turville et al., 2002
). DC-SIGN mediates endocytosis of mannosylated and fucosylated ligands, including mannose-BSA (Frison et al., 2003
; Guo et al., 2004
). In addition, studies showing that peptides derived from an internalized anti-DC-SIGN antibody are efficiently presented by MHC class II molecules provide evidence for a role of DC-SIGN in antigen processing and presentation (Engering et al., 2002
). Thus DC-SIGN could have been responsible for the increased presentation of mannosylated antigens seen in the studies with monocyte-derived dendritic cells.
For the mannose receptor to have a role in processing and presentation of glycoprotein antigens, ligands internalized by the receptor would have to be efficiently routed to compartments containing MHC class II molecules. The mannose receptor releases its bound ligand in early endosomes and recycles to the cell surface (Wileman et al., 1984). Several studies have shown that the mannose receptor does not enter late endosomal or lysosomal compartments containing MHC class II molecules (Mahnke et al., 2000
; Tan et al., 1997
). Thus although the mannose receptor could certainly enhance uptake of glycosylated antigens, it is unlikely to route them efficiently to MHC class II compartments. In fact, lack of response to one glycoprotein tumor antigen, MUC1, has been attributed to uptake by the mannose receptor. Inhibition experiments with mannan and a mannose receptor-specific antibody suggest that MUC1 is internalized into dendritic cells by the mannose receptor, but it is retained in early endosomes and not transported to compartments containing MHC class II molecules (Hiltbold et al., 2000
).
In contrast to the early endosomal targeting of the mannose receptor, complexes of dendritic cell receptors DC-SIGN or DEC-205 with specific antibodies are targeted to late endosomal/lysosomal compartments containing abundant MHC class II molecules (Engering et al., 2002; Mahnke et al., 2000
). A three-amino-acid motif, EDE, found in the cytoplasmic tail of DEC-205 but not the mannose receptor, mediates this deeper routing. The cytoplasmic domain of DC-SIGN also contains a three-acidic-residue motif, EEE, but it is not yet known whether this motif is required for targeting of DC-SIGN to late endosomes. This deeper internalization into the cell is likely to explain why DC-SIGN and DEC-205 are effective in enhancing antigen presentation by MHC class II molecules. It should be pointed out, however, that although DEC-205 is related in structure to the mannose receptor, it does not bind mannose or other sugars; thus, unlike DC-SIGN, DEC-205 could not be responsible for processes attributed to the mannose receptor (Taylor, 1997
).
Other evidence for involvement of the mannose receptor in antigen processing represents a more specialized case. Internalization of lipoarabinomannan by the mannose receptor on monocyte-derived dendritic cells leads to delivery of this glycolipid antigen to compartments containing CD1b molecules that bind and present the antigen at the cell surface (Prigozy et al., 1997). Colocalization of the mannose receptor and CD1b in intracellular vesicles is observed. Mannan inhibits presentation of lipoarabinomannan, but in this case, an antiserum against the mannose receptor also inhibits the process. Thus involvement of the mannose receptor in this specialized case of uptake and presentation of a glycolipid antigen seems more plausible than a general role for the receptor in processing and presentation of glycoprotein antigens.
Analysis of mannose receptor knockout mice indicates that the major function of the receptor is in clearance of endogenous proteins bearing high-mannose oligosaccharides, such as lysosomal enzymes that are released as part of the inflammatory response (Lee et al., 2002). These mice exhibit defects in clearance of mannosylated glycoproteins by the liver and accumulate several lysosomal enzymes and other inflammatory glycoproteins in the serum. In addition, the knockout mice are no more susceptible than wild-type mice to infection by either Pneumocystis carinii or Candida albicans, suggesting that although these pathogens can bind to the mannose receptor in vitro, the receptor does not play an important role in the immune response against them (Swain et al., 2003
; Lee et al., 2003
). Thus the results presented here showing that the mannose receptor is unlikely to play a general role in processing and presentation of glycoprotein antigens are consistent with evidence that the primary physiological role of the receptor is in clearance.
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Materials and methods |
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Production of rat-6 fibroblast cell lines stably transfected with the human mannose receptor has been described (Taylor et al., 1990). cDNA clones for murine MHC class II IAK
and IAKß were provided by Dr. C. Benoist, INSERM. DNA coding for each MHC class II IAK chain was cloned into the expression vector pIREhyg2 (Invitrogen, Carlsbad, CA). A 1:1 of mixture of IAK
and IAKß expression vectors (5 µg each) was transfected into rat-6 cells expressing the mannose receptor using the calcium phosphate method (Wigler et al., 1979
). Following incubation with the calcium phosphateDNA coprecipitate for 4 h at 37°C, cells were grown overnight in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, before selection was initiated by inclusion of 400 µg/ml hygromycin in the medium.
After 2 weeks, colonies were isolated by trypsinization using cloning cylinders. Cells expressing functional MHC class II molecules were identified by assaying presentation of RNase A. Coexpression of MHC class II and mannose receptor was confirmed by immunofluorescence. Paraformaldehyde-fixed cells were incubated with a 1:1 mixture of monoclonal anti-mannose receptor antibody (BD Biosciences) labeled with Zenon Alexa Fluor 594 (Molecular Probes, Eugene, OR) and monoclonal anti-MHC I-Ak
-chain antibody (BD Biosciences) labeled with Zenon Alexa Fluor 488.
RNase B and mannose30-BSA (E-Y Labs, San Mateo, CA) were iodinated with Na125I (Amersham Pharmacia, Little Chalfont, UK) using the chloramine T method (Greenwood et al., 1963). Analysis of uptake and degradation of iodinated ligands was performed as described previously (Mellow et al., 1988
).
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Acknowledgements |
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Footnotes |
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Abbreviations |
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References |
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