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,
Departments of
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Pathology and
Surgery, and
University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ags bound by mannose receptor on dendritic cells are internalized and transported to a subcellular compartment in which Ag processing occurs (5, 6, 7). Peptide epitopes that are generated can bind to MHC proteins and subsequently be presented to Ag-specific T lymphocytes. It has been shown that addition of mannose residues to protein Ags such as BSA results in 1,000–10,000-fold increase in efficiency of presentation to Ag-specific CD4+ T cells, presumably due to much higher efficiency of internalization of the glycosylated Ag by mannose receptor (7, 8). The receptor is also responsible for uptake of the mycobacterial product lipoarabinomannan into dendritic cells, which binds to CD1b molecules and can be presented to CD4-, CD8- T cells (6). CD1b has been shown to bind several derivatives of mycophenolic acid, and exhibits structural homology to MHC class I molecules, which present peptide Ags to CD8+ T cells (9). Both CD1b and class II MHC proteins bind Ags in a specialized vesicular compartment within APC called the MHC class II compartment, which has also been shown to contain HLA-DM, lysosomal hydrolases, and in addition mannose receptor (6, 10). These data suggest that mannose receptor can deliver a variety of Ags to the MHC class II compartment for processing and presentation by class II MHC or CD1b molecules. It is therefore important to understand the structural basis for interaction of Ags with mannose receptor, and potentially to identify novel ligands for the receptor.
In the present study, we demonstrate that the agalactosyl form of IgG (G0 IgG)3 can be internalized by mannose receptor on macrophages and dendritic cells. IgG contains a biantennary oligosaccharide attached at asparagine 297 in the Fc region. Each arm of the glycan normally terminates in galactose and sialic acid residues, and normally glycosylated IgG has been called G2 IgG (11). G0 IgG lacks these terminal residues, thereby exposing N-acetylglucosamine residues (11, 12). This results in increased solvent accessibility and conformational flexibility of the G0 glycan, allowing binding to plant lectins specific for N-acetylglucosamine, and also to serum mannose-binding protein, which plays a role in triggering complement activation by microbes (13). This latter observation led to the hypothesis that G0 IgG could contribute to the pathogenesis of rheumatoid arthritis, and additional autoimmune disorders that are characterized by the presence of high circulating levels of G0 IgG, by contributing to inflammation via complement activation (13, 14). Mannose receptor has ligand-binding properties that are similar to mannose-binding protein, and we hypothesized that uptake of G0 IgG by potent APC such as macrophages and dendritic cells would result in the accumulation of IgG molecules in Ag-processing compartments within these cells. This could potentially result in the generation of abnormally large quantities of Ig-derived peptides that bind to class II molecules. Particularly in dendritic cells, which are highly potent APC, this could produce a very strong stimulus for activation of Ig-specific T cell clones, which might otherwise remain tolerized. Such T cells could potentially provide help to B cells specific for self IgG, resulting in production of anti-IgG Abs termed rheumatoid factors. Although rheumatoid factors are often IgM, other isotypes have been identified, and particularly for production of the moderate to high affinity rheumatoid factors showing evidence of affinity maturation, T cell help would likely be required (15, 16, 17, 18).
We examined whether G0 IgG could interact with mannose receptor by measuring internalization of fluorescently labeled IgG by dendritic cells and macrophages at 37°C. Uptake of G0 IgG, but not G2 IgG, was highly efficient and could be blocked either by specific sugars or Abs reactive with mannose receptor. These data demonstrate that interaction of G0 IgG with both of these cell types can be mediated by mannose receptor.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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FITC-celite was obtained from Sigma (St. Louis, MO). Conjugation of different proteins with FITC was done according to protocol supplied by the manufacturer. FITC-conjugated mannosylated BSA (mBSA), mannan derived from Saccharomyces cerevisiae, and the monosaccharides D-galactose, D-glucose, L-fucose, D-mannose, and N-acetylglucosamine were purchased from Sigma. IgG from normal human serum or derived from human plasmacytomas were purchased from either Sigma or Calbiochem (San Diego, CA).
Cell cultures
The B lymphoblastoid cell line WS was generated from a healthy donor by transformation with EBV using standard methods (19). Dendritic cells and macrophages were prepared from human adherent PBMC, as previously described (20). Briefly, PBMC were isolated from leukopheresed blood from healthy donors using density centrifugation on Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) gradients. After washing in HBSS (Life Technologies, Gaithersburg, MD), cells were resuspended in AIM-V serum-free medium (Life Technologies) at approximately 107/ml and incubated in T75 cell culture flasks for 1–2 h at 37°C. The plastic-adherent cells were further cultured (37°C, 5% CO2) in AIM-V medium supplemented with 1000 U/ml IL-4 and 1000 U/ml GM-CSF for dendritic cells (both cytokines were provided by Schering-Plough, Kenilworth, NJ), or 1000 U/ml GM-CSF alone (for macrophages). In most experiments, except where noted, cells were harvested for use in assays after 4 to 5 days in culture.
Antibodies
Hybridomas w6/32 (anti-HLA-A,B,C), IV.3 (anti-CD32), 32.2 (anti-CD64), and 3C10 (anti-CD14) were obtained from American Type Culture Collection (Manassas, VA). FITC-conjugated mAbs reactive with CD1a, CD32, CD40, CD44, CD64, CD83, CD86, and isotype-matched controls (IgG1 and IgG2a) were obtained from PharMingen (San Diego, CA). FITC-conjugated anti-HLA-DR, CD16, CD20 mAbs were purchased from Becton Dickinson (Mountain View, CA). FITC-conjugated anti-CD80 mAb was obtained from Ancell (Bayport, MN). mAbs reactive with human mannose receptor were obtained from the following sources: clone 19 (21) from Dr. Phil Stahl (St. Louis, MO) or purchased from PharMingen; Ab 15-2-2 (22) from Dr. D. C. Rijken (Leiden, Netherlands); and 2.1D10 from Dr. Sun-sang J. Sung (Charlottesville, VA). mAb MR15 reactive with the human macrophage marker CD68 was obtained as part of a typing panel for the Fifth International Workshop on Human Leukocyte Differentiation Antigens.
Generation of G0 IgG by digestion of normal human IgG
Normal human IgG was digested for 20 h at 37°C with neuraminidase (2 U/ml; Sigma) and ß-galactosidase (40 U/ml; Sigma) in sodium acetate buffer (50 mM, pH 5.5) containing 10 mM MnCl2 and 0.02% NaN3 (23). The digested IgG was purified through HiTrap protein G column (Pharmacia) following the manufacturer’s protocol to remove glycosidases. Ricinus communis agglutinin I (RCA-I) obtained from Sigma and Bandeiraea simplicifolia lectin (BS-II) from E-Y Lab (San Mateo, CA) were used in ELISA for detection of terminal galactose and glucosamine residues on IgG, respectively (24, 25, 26). To measure binding of IgG to RCA-I lectin, 0.5 µg recombinant Staphylococcus protein G in 0.1 ml carbonate buffer, pH 9.3, was added to individual wells of 96-well plates (Costar, Cambridge, MA) at 4°C overnight. Wells were then incubated with a 1% BSA solution, and 0.1 ml of IgG (10 µg/ml) was added, followed by biotinylated RCA-I (24) and streptavidin-peroxidase conjugate (Sigma). The chromogenic substrate o-phenylenediamine (Sigma) was then added, and absorbance was measured at 490 nm. For measuring interaction of IgG with BS-II lectin, 0.5 µg of BS-II was added directly to individual wells of 96-well plates. Following blocking with BSA, 1 µg IgG was added, followed by anti-human IgG conjugated to peroxidase (Sigma), and the substrate o-phenylenediamine. Detection of binding was the same as for RCA-I above.
Flow cytometry
Cells (2 x 105) were incubated with mAbs specific for cell surface markers for 45 min at 4°C. After two washes in PBS containing 1% BSA, FITC-conjugated goat anti-mouse IgG Ab (Sigma) was added for 30 min. Cells were then washed three times in PBS, then fixed with 4% paraformaldehyde in PBS. Samples were then analyzed by FACScan (Becton Dickinson), and mean fluorescence channels were recorded.
Quantitative analysis of ligand uptake by cells
Cells were harvested by gentle pipetting from flasks, and resuspended in RPMI 1640 medium (without serum) at 5 x 106/ml. Fluorescein-labeled ligands were diluted in RPMI medium, then added to 2.5 x 104 resuspended cells, followed by incubation at either 37°C or 4°C. For studies using inhibitors, mAbs or saccharides were added at the onset of incubation. Cells were washed three times in PBS, then fixed with 4% paraformaldehyde in PBS before examination by flow cytometry or fluorescence microscopy. Flow-cytometric data were collected as described above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To generate G0 IgG, we used the exoglycosidases ß-galactosidase
and neuraminidase to remove the terminal galactose and sialic acid
residues from normal G2 IgG, resulting in exposure of
N-acetylglucosamine residues. To test the efficiency of
digestion, control and digested IgG preparations were analyzed by ELISA
for binding to the plant lectins RCA-I and BS-II, which are selective
for galactose and N-acetylglucosamine, respectively. As
shown in a representative experiment, digested IgG showed decreased
RCA-I binding and increased BS-II binding compared with native IgG
(Fig. 1
). We estimated that greater than
75% of galactose residues on IgG were removed typically by
digestion.
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Dendritic cells and macrophages derived from PBMC used in these
studies were analyzed for expression of a number of cell surface
markers, as shown in Table I. Dendritic
cells cultured for 4 to 5 days expressed CD32, CD40, CD44, CD86, MHC
class I and class II, mannose receptor, and low levels of CD80 and
CD1a, and were negative for CD14, CD20, CD64, CD68, and CD83. This
surface marker profile is consistent with a relatively immature
phenotype shown previously to be characteristic of dendritic cells that
efficiently take up and process exogenous Ags. Macrophages cultured
from the same preparation of PBMC as described in Materials and
Methods expressed CD14, CD68, CD32, mannose receptor, and weakly
CD64. This distinct pattern of surface marker expression is consistent
with previous analyses of cultured macrophages. Cell cultures routinely
contained 85–95% dendritic cells or macrophages based on phenotyping
and morphology, with the remainder consisting mainly of T cells.
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Dendritic cells and macrophages were cultured for 5 days and then
analyzed for expression of cell surface markers or uptake of ligands,
together with a B lymphoblastoid cell line derived from the same donor.
Fig. 2 shows that all three lines express
CD32, but that only macrophages and dendritic cells express mannose
receptor. Cells were incubated with fluoresceinated ligands at either
37°C or 4°C, as shown in Fig. 3.
Incubation at 37°C, but not at 4°C, resulted in significant cell
fluorescence, suggesting that the ligand is internalized at 37°C. As
previously reported, mannosylated BSA (mBSA) is taken up much more
efficiently than unmodified BSA into both dendritic cells (Fig. 3a) and macrophages (Fig. 3b) after incubation
for 1 h at 37°C. In contrast, exposure of B cell lines to
fluoresceinated mBSA, which do not express mannose receptor, did not
result in significant labeling of cells (Fig. 3c).
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), and demonstrate that G0 IgG is taken
up selectively by dendritic cells and macrophages. Since the uptake of
both G0 IgG and mBSA depended upon their glycosylation status, these
results supported the hypothesis that a receptor with lectin-like
properties such as mannose receptor was required.
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When dendritic cells were incubated with 40 µg/ml of
fluoresceinated G0 IgG, internalization increased over the incubation
period without reaching a plateau (Fig. 4). This demonstrated that uptake was
highly efficient, and would be consistent with the previously reported
ability of mannose receptor to bind and internalize ligands, then
recycle to the plasma membrane (27, 28). Dendritic cells
incubated for 1 h with increasing concentrations of ligands were
analyzed (Fig. 5), and showed that
exposure to G0 IgG resulted in the cells becoming increasingly
fluorescent. Although this suggests that G0 IgG uptake is nonsaturable,
it may simply reflect on limitations in the concentration of ligand
that are achievable. Uptake of G0 IgG by dendritic cells was inhibited
with 100 µg/ml mannan, which has been used previously to block uptake
of other ligands by mannose receptor (5). In contrast,
mannan did not significantly inhibit uptake of aggregated G2 IgG (Table III), which is internalized by Fc
receptors on dendritic cells and macrophages (unpublished data). This
confirms that mannan is able to selectively block uptake of
carbohydrate ligands, as previously demonstrated (5).
Similar results were obtained using macrophage preparations
(unpublished data).
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To further characterize the interaction of G0 IgG with dendritic
cells, uptake of either G0 IgG or mBSA was measured in the presence of
100 mM monosaccharides (Fig. 6). Uptake
of both ligands was blocked by mannose and fucose, and somewhat less
well by N-acetylglucosamine. Galactose was unable to inhibit
uptake, either for G0 IgG (Fig. 6a) or for mBSA (unpublished
data). The pattern of inhibition is consistent with uptake being
mediated by mannose receptor, based on previously published studies. To
provide further evidence for a role for the receptor, we measured
uptake of labeled ligands by dendritic cells in the presence of Abs
reactive with mannose receptor (Fig. 6b). Ab 19 used at the
highest concentration tested (10 µg/ml) inhibited uptake of G0 IgG
and mBSA by 69% and 55%, respectively, while Abs 15-2-2 and 2.1D10
blocked only slightly at this concentration. At higher concentrations,
clone 19 blocked internalization of both ligands by 70–80%
(unpublished data). All three Abs bound to dendritic cells comparably,
as determined by flow cytometry (unpublished data). These results
strongly support the conclusion that uptake of G0 IgG and mBSA are both
mediated primarily by mannose receptor. Although the lack of complete
inhibition indicates that a secondary mechanism for uptake of these
ligands may exist, we believe it equally possible that the highly
efficient nature of mannose receptor uptake and recycling would be
difficult to block completely with Abs, perhaps due to internalization
of the specific Ab itself.
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To determine whether the subclass of IgG influenced its ability to
interact with mannose receptor following removal of galactose residues,
we subjected nine different preparations of IgG isolated from myelomas
grown in culture to enzymatic digestion to remove galactose residues.
Table IV shows the 
-chain subclass and
light chain isotype of each preparation, and compares the ratio of
BS-II to RCA lectin binding following digestion to uptake of
fluoresceinated ligand by dendritic cells. There were significant
differences in the ability of individual preparations to be digested,
with IgG in some samples appearing to be resistant to digestion, as
indicated by a low ratio of BS-II/RCA binding, even after redigestion.
However, in these samples, binding of both lectins was quite low
relative to total protein, suggesting that the preparations might
contain significant amounts of IgG with missing or aberrant glycan
structures that would not interact with lectins, including mannose
receptor (unpublished data). A clear correlation was seen between
efficient digestion to obtain G0 IgG and the level of uptake of each
IgG preparation. Furthermore, IgG1, IgG2, and IgG3 preparations bearing
either 
- or 
-light chains were each able to be efficiently
converted to the G0 form and subsequently internalized by dendritic
cells. We were unable to obtain additional IgG4 preparations, and thus
cannot further assess this subclass. These results suggest that Ig
subclass and light chain type do not influence uptake by mannose
receptor.
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As dendritic cells mature, either in vivo or in vitro, they
undergo phenotypic changes in cell surface protein expression and also
in their ability to internalize ligands. In the previous experiments,
we examined dendritic cells grown in vitro for 4 to 5 days that have an
immature phenotype, characterized by the ability to take up Ags very
efficiently via macropinocytosis, receptor-mediated endocytosis, or
phagocytosis (5, 29, 30). As previously reported, immature
dendritic cells treated with LPS, which induces a more mature
phenotype, undergo a rapid down-modulation in their ability to
internalize mBSA (5). A similar process of maturation
occurs spontaneously with increasing time in culture in the absence of
LPS or other stimulatory substances, although the phenotypic changes
appear more gradually. To examine whether mannose receptor expression
and function change over time, we isolated dendritic cells at different
times after initiation of culture, and measured the expression of
several surface markers, including mannose receptor, using specific Abs
and also the ability of cells to internalize labeled G0 IgG or mBSA
(Fig. 7). Between days 2 and 6, mannose
receptor expression was high, and both ligands were internalized. At
days 8 and 10, however, ligand uptake had stopped, although mannose
receptor expression was still quite high, as detected by specific two
Abs. This experiment demonstrates that mannose receptor is active in
immature dendritic cells, as measured by ligand uptake, but its
function decreases drastically following maturation of cells to a
phenotype that expresses higher levels of CD86 and cell surface class
II MHC (30, 31, 32). In addition, it is clear that the
presence of mannose receptor on the plasma membrane is not sufficient
for receptor function, since dendritic cells express the receptor
following 8 days in culture (Fig. 7), but do not take up ligand.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our results are potentially relevant to the last possibility, since dendritic cells, which are among the most potent APC in the body, and macrophages can take up large amounts of G0 IgG via mannose receptor. This may lead to processing and presentation of IgG-derived peptides in class II MHC, which could stimulate CD4+ T cell reponses. Although T cells are selected to be tolerant of self proteins, presumably including IgG, uptake of much larger amounts than normal by APC could potentially break tolerance by stimulating T cells cross-reacting with IgG peptides. It is also possible that uptake of IgG by mannose receptor results in its shunting to intracellular compartments, in which processing is altered such that normally unexposed or cryptic epitopes are revealed. Precedents for such altered processing have been reported recently, in which generation of particular epitopes depends upon the receptor used for Ag uptake (36).
In any case, to effect an immune response, IgG-derived peptides would
have to bind to class II MHC proteins and be transported to the surface
of the APC, before being able to stimulate T cell responses. To address
the question directly then, it will be necessary to determine whether
peptides from IgG can be generated and bind to class II MHC proteins.
This has been determined for the human class II molecule HLA-DR4
expressed on B cell lines, which binds peptides 145–159 and 188–203
from endogenously synthesized -light chains (37, 38).
These peptides bind to HLA-DR4 subtypes that are associated with
rheumatoid arthritis, suggesting a particular relevance to the disease.
However, it is not known if other APC, such as dendritic cells and
macrophages, process IgG into the same or different peptides, or if IgG
taken up exogenously by these cells would lead to differential
processing. Ultimately, it will be necessary to determine whether APC
generate IgG peptides that can bind to class II MHC proteins, and then
stimulate T cell immune responses in individuals with autoimmune
disorders.
The results presented in this study also have important implications for understanding how potential Ags other than IgG may be taken into macrophages and dendritic cells. It is likely that a variety of self proteins have glycan side chains that can interact with mannose receptor, including but not limited to those with fucose, mannose, and N-acetylglucosamine. Most of these ligands, even if taken up by APC in large quantities, would not be expected to provoke an immune response since T cells appropriately selected in the thymus should be tolerant of peptides derived from self proteins. However, if the amount of the protein taken into a potent APC changed drastically, as seen in tissue culture for G0 IgG uptake by macrophages and dendritic cells, it might be possible to break self-tolerance. It is not clear what alters the glycosylation state of IgG in the disease, or if other less abundant proteins have altered glycosylation in rheumatoid arthritis or other disease states. A further possibility is that immune complexes containing G0 IgG might allow internalization of Ags bound to specific IgG via the Ag-combining site. This could permit increased uptake of even nonglycosylated proteins indirectly via specific Ab and mannose receptor. In therapeutic situations in which it would be desirable to enhance Ag uptake for use in vaccines, it may be possible to exploit this effect by generating the G0 form of Ab against a desired Ag for targeting into APC.
Abs reactive with mannose receptor that we tested have differential
ability to interfere with uptake of ligands, with some blocking quite
efficiently, while others promoted uptake to a slight extent (Fig. 6
).
This suggests the interesting possibility that the latter set might be
used to stimulate uptake of Ags by mannose receptor-positive cells,
either by addition with the Ag, or by covalent coupling of Ag and
receptor-specific Ab. In mice, uptake of Ags via the receptor DEC-205,
which is structurally related to mannose receptor, was facilitated by
conjugation of a peptide Ag to an Ab against DEC-205 (39).
Nonblocking Abs against mannose receptor in humans may be useful for
promoting Ag uptake in a similar way.
In addition to identifying a novel ligand for mannose receptor, this
study demonstrates that dendritic cells alter their ability to
internalize ligands via mannose receptor during maturation. This is
consistent with previous reports suggesting that Ag uptake is stopped
once dendritic cells mature from a processing to a presenting
phenotype, either by exposure to inflammatory stimuli such as LPS or
TNF-
, or with increasing time in culture (5). In our
study, dendritic cells cultured for 8 or 10 days were shown to express
mannose receptor, but were unable to internalize ligands via the
receptor. This suggests that cells at this stage of maturation are
poised between the processing and presenting phenotypes. In addition,
this result implies that the presence of mannose receptor on the plasma
membrane is not sufficient for its function. It may be that
internalization via mannose receptor requires additional molecules that
are not present in our dendritic cell cultures at day 8, or that
broader changes are involved, such as rearrangement of cytoskeletal
elements during cell maturation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Russell D. Salter, Department of Pathology, University of Pittsburgh School of Medicine, W957 Biomedical Science Tower, 200 Lothrop Street, Pittsburgh, PA 15213. E-mail address:
3 Abbreviations used in this paper: G0 IgG, agalactosyl IgG; BS-II, Bandeiriea simplicia lectin; G2 IgG, galactosylated IgG; mBSA, mannosylated BSA; RCA, Ricinus communis agglutinin.
Received for publication April 14, 1999. Accepted for publication August 25, 1999.
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References |
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-galactosyl residues from human serum: comparison of their binding in bovine testicular tissue with that of the Griffonia simplicifolia lectin (GSI-B4) and impact of labeling on epitope localization. Eur. J. Cell Biol. 68:96.[Medline]
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), or G0 IgG in the presence of 100 µg/ml mannan (
).
Cells were washed, fixed, and analyzed as in Fig. 3
