The Mannose Receptor Mediates Uptake of Soluble but Not of
Cell-Associated Antigen for Cross-Presentation1
Sven Burgdorf,2Veronika Lukacs-Kornek,2and Christian Kurts3
The mannose receptor (MR) has been implicated in the recognition and clearance of microorganisms and serum glycoproteins. Its
endocytic function has been studied extensively using macrophages, although it is expressed by a variety of cell types, including
dendritic cells (DC). In this study, we investigated its role in Ag presentation by DC using MR?/?mice. Uptake of the model Ag,
soluble OVA, by bone marrow-derived DC and in vitro activation of OVA-specific CD8 T cells (OT-I cells) strictly depended on
the MR. In vivo, MR deficiency impaired endocytosis of soluble OVA by DC and concomitant OT-I cell activation. No alterations
in the DC subtype composition in MR?/?mice were accountable. Uptake of cell-associated OVA was unaffected by MR deficiency,
resulting in unchanged activation of OT-I cells. These findings demonstrate that DC use the MR for endocytosis of a particular
Ag type intended for cross-presentation. The Journal of Immunology, 2006, 176: 6770–6776.
organisms (1). Furthermore, the MR plays a homeostatic role in
the clearance of glycoproteins, such as ?-glucuronidase and pro-
collagen, which are up-regulated in the blood serum during in-
flammation (2). The MR consists of an N-terminal cysteine-rich
domain, a fibronectin type II repeat domain, eight carbohydrate
recognition domains (CRD), a transmembrane domain, and a short
intracellular region (3). The cysteine-rich region mediates binding
to sulfated sugar moieties, whereas the CRD bind glycoproteins
bearing (for instance) terminal mannose, fucose, and, with a lower
affinity, glucose residues (4). Most studies addressing the function
of the MR have used macrophages (5), which use it for uptake of
mannosylated structures such as dextrans (1). In addition to these
cells, the MR has also been detected in liver endothelial cells,
dermal microvascular endothelial cells, monocytes, Langerhans
cells, and dendritic cells (DC) (6).
DC play a central role in the induction of adaptive immune
responses (7). After capturing and internalizing Ag in peripheral
organs, they migrate toward the draining lymph nodes, where they
can activate naive T cells. For activation of CD8?T cells, captured
extracellular Ag are presented on MHC class I molecules (8)—a
process termed cross-presentation—which contributes to the in-
duction of cytotoxicity against many viruses and tumors (9). The
murine DC subpopulation expressing the CD8? homodimer has
he mannose receptor (MR)4is a 180-kDa transmembrane
C-type lectin that functions as an endocytic receptor. It
has been implicated in the recognition of various micro-
been shown to be particularly relevant for cross-presentation of
foreign Ag (10) and of self Ag under homeostatic conditions (11,
12). In the presence of inflammatory stimuli, also CD8?-deficient
DC were able to cross-present (13, 14).
Several receptors mediating Ag uptake in DC have been iden-
tified, such as Fc-receptors, DC-SIGN and DEC205. A role of the
MR in Ag uptake and presentation by DC has been proposed based
on the finding that mannosylated proteins are presented more ef-
ficiently than nonmannosylated ones (15, 16). It is unclear, how-
ever, whether this uptake was due to the MR, because DC express
other receptors, such as DC-SIGN, with affinity for mannosylated
proteins (6, 17). For the same reason, mannan, a polymer of man-
nose, which competitively blocks endocytosis of mannose-rich
structures, cannot be considered a specific inhibitor of the MR. To
overcome these limitations, we have used MR?/?mice to eluci-
date the role of the MR in the uptake and presentation of soluble
vs cell-associated OVA.
Materials and Methods
MR?/?mice on a C57BL/6 (B6) background were generated and provided
by Dr. M. C. Nussenzweig (Rockefeller University, New York, NY) (2).
B6 mice bearing the Kbmutant bm1 (bm1 mice) and OT-I Rag-1?/?mice
on a B6 background were provided by Dr. W. R. Heath (Walter and Eliza
Hall Institute of Medical Research, Melbourne, Australia) (18). For all
experiments, mice between 8 and 16 wk of age bred under specific patho-
gen-free conditions were used in accordance with local animal experimen-
Abs and reagents
All mAb used were purchased from BD Biosciences, except anti-MR Ab
(Serotec) and SF1, which were purified from hybridoma supernatant
(American Type Culture Collection) and used after conjugation with al-
exa488. All reagents, if not specified otherwise, were obtained from
Generation of bone marrow-derived dendritic cells (BMDC)
BMDC were generated using GM-CSF as described previously (19). At
day 7, CD11c?cells isolated by magnetic separation with the autoMACS
system (Miltenyi Biotec) were used for all in vitro experiments. Purity of
CD11c?cells was typically higher than 98%.
Preparation of fluorescent soluble and cell-associated OVA
Soluble OVA was conjugated to a fluorochrome using an alexa647labeling
kit (Invitrogen Life Technologies) according to the manufacturer’s guide-
lines. The labeling procedure involved gel filtration as a final step for
Institute of Molecular Medicine and Experimental Immunology (IMMEI), Friedrich-
Wilhelms-Universita ¨t, Bonn, Germany
Received for publication January 26, 2006. Accepted for publication March 15, 2006.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by was supported by a junior research group grant from
the German state of Nordrhein-Westfalen (to C.K.). S.B. was supported by BONFOR
Grant O-173.0009 of the University of Bonn Clinic, Bonn, Germany.
2S.B. and V.L-K. contributed equally to this work.
3Address correspondence and reprint requests to Dr. Christian Kurts, Institute of
Molecular Medicine and Experimental Immunology, Friedrich-Wilhelms-Universita ¨t,
53105 Bonn, Germany. E-mail address: firstname.lastname@example.org
4Abbreviations used in this paper: MR, mannose receptor; DC, dendritic cell; OT-I
cell, OVA-specific CD8 T cell; CRD, carbohydrate recognition domain; bm1 mice,
C57BL/6 mice bearing the Kbmutant bm1; BMDC, bone marrow-derived DC; cLN,
cutaneous lymph node; MFI, mean fluorescence intensity.
The Journal of Immunology
Copyright © 2006 by The American Association of Immunologists, Inc.0022-1767/06/$02.00
removal of low molecular mass molecules such as unbound fluorochrome.
For cell-associated OVA, splenocytes from bm1 mice (2 ? 108cells/ml)
were incubated with 10 mg/ml OVA-FITC for 10 min at 37°C, UV-irra-
diated with 15 mJ for 5 min, and washed extensively. For cell culture
experiments, 106splenocytes were coincubated with 4 ? 105BMDC for
OT-I cells were isolated from OT-I rag?/?mice as previously described
(18) and further purified by a nanobead-based CD8 T cell isolation kit
(Miltenyi Biotec). Purity was typically higher than 96% of viable cells;
contaminating CD11c?cells were typically rarer than 0.2%, NK1.1?cells
rarer than 0.03%, and CD4?cells below 1%. For presentation of soluble
OVA, 4 ? 105BMDC were stimulated with 10 ?g/ml LPS for 2 h and
incubated with 500 ?g/ml OVA or 20 nM SIINFEKL (OVA peptide).
After another 3 h, cells were washed, fixed with 0.008% glutaraldehyde for
3 min, and coincubated with 2 ? 105OT-I cells. IL-2 concentrations were
determined after 18 h by ELISA. For the analysis of cell-associated Ag,
bm1 splenocytes were coated with OVA as described above. Splenocytes
(106) were coincubated with 4 ? 105BMDC and 2 ? 105OT-I cells. IL-2
concentrations in the supernatant were determined by ELISA after 40 h.
Isolation of DC from experimental animals
Cells were isolated from spleen and cutaneous lymph node (cLN) as de-
scribed before (12). For the preparations from bone marrow, cells were
collected by flushing femurs and tibias with PBS. CD11c?cells from all
organs were enriched by magnetic separation using MS25 columns (Milte-
nyi Biotec). Purity was typically higher than 85%.
Flow cytometry, data analysis, and statistics
Flow cytometry was performed on an LSR (BD Biosciences). Dead cells
were excluded by Hoechst-33342 dye. Data were analyzed using Flow-Jo
software (Tristar), including calculation of division indices, which indicate
the average number of cell divisions. Statistical analysis was done using
Excel (Microsoft). All experiments reported here have been reproduced at
The MR is essential for uptake of soluble but not of
cell-associated OVA by BMDC
When we studied uptake of OVA in BMDC, we noticed that only
some of the CD11c?cells took up this model Ag in vitro (Fig. 1,
A and B), as others have noted recently (20). We reasoned that this
might be explained by selective expression of an endocytic recep-
tor that mediated uptake of OVA. In support of this hypothesis, we
observed a close correlation between the uptake of OVA and the
extent of MR expression (Fig. 1A). To investigate whether this
association was due to a functional role of the MR in Ag uptake,
we preincubated the DC with mannan, which competitively inhib-
its MR-mediated endocytosis (21). This agent blocked the uptake
of OVA completely (Fig. 1B). To exclude an influence of other
receptors with affinity to mannan, we performed this experiment
also with BMDC from MR?/?mice (2). These cells did not show
any uptake (Fig. 1, B and C), indicating that the MR was indis-
pensable for endocytosis of soluble OVA by BMDC in vitro. Re-
markably, under these experimental conditions, no other receptor
appeared to compensate even partially for the absence of the MR.
To exclude the possibility that BMDC generated from MR?/?
mice differed from those from wild-type mice in terms of subtype
composition or maturation status, we phenotypically characterized
these DC. Consistent with reports from others (22, 23), BMDC
expressed CD11b but not CD8? (Fig. 1D). CD11c and CD11b
expression was identical in both BMDC populations (Fig. 1D), as
was constitutive and LPS-induced expression of costimulatory
molecules (Fig. 1E), suggesting equivalent states of maturation.
Next, we investigated whether the MR was important also for
the uptake of cell-associated OVA, using splenocytes loaded with
fluorochrome-labeled OVA (Fig. 2A). To demonstrate intracellular
uptake, loading was performed at 4°C and at 37°C, because intra-
cellular uptake is energy-dependent, as opposed to extracellular
coating. Indeed, uptake at 37°C was significantly higher than at
4°C, suggesting that some of the Ag was transported into the
spleen cells. We then induced apoptosis by UV irradiation and
cocultured these cells with BMDC from MR?/?mice or controls.
After 18 h, the uptake of cell-associated OVA by wild-type and
MR?/?cells was indistinguishable (Fig. 2B), indicating that re-
ceptors other than the MR had mediated endocytosis of
The MR is required for cross-presentation of soluble but not of
cell-associated OVA in vitro
The requirement of the MR for OVA uptake suggested a role in Ag
presentation. To address this hypothesis, we studied cross-presen-
tation of OVA in coculture experiments with BMDC prepared
from B6 or MR?/?donor mice and OVA-specific CD8 T cells
isolated from transgenic OT-I mice. Their activation was moni-
tored by measuring IL-2 release into the culture supernatant. This
release correlated with the Ag amount in a dose-dependent fashion
(Fig. 3A). To ensure that OT-I cell activation was in fact due to
cross-presentation, and not to coating of the BMDC with peptide
fragments present in the OVA solution, we performed incubation
with soluble OVA in the presence of the proteasome inhibitor
MG132, which inhibits intracellular generation of peptides for
loading onto MHC class I molecules (24). This inhibitor prevented
IL-2 release nearly completely (Fig. 3A), indicating that OT-I cells
were indeed activated by OVA peptides generated intracellularly.
MG132 did not affect OT-I cell activation by BMDC coated with
the OVA peptide SIINFEKL, which is recognized by OT-I cells,
demonstrating that in this experimental setting, the ability of OT-I
cells to produce IL-2 was not compromised (Fig. 3A).
When BMDC from MR?/?mice were used, they induced se-
verely reduced IL-2 production by OT-I cells as compared with
wild-type cells (Fig. 3B), demonstrating that MR-mediated uptake
of soluble OVA could provide Ag for cross-presentation. MR-
deficient DC externally loaded with SIINFEKL activated OT-I
cells equally well as DC from wild-type mice (Fig. 3B), indicating
that BMDC from MR?/?mice were not compromised in their
general ability to activate T cells. Thus, the MR mediated not only
uptake of OVA, but also the resulting activation of OT-I cells.
Because the uptake of cell-associated OVA was not reduced in
MR-deficient DC, we tested whether OT-I cell activation remained
operative as well. To this end, we loaded splenocytes from bm1
mice with OVA. These mice bear a mutant H2-Kbprotein that
cannot present OVA to OT-I cells (18). After coating with OVA,
apoptosis was induced by UV irradiation. These cells were then
cocultured with wild-type and MR-deficient BMDC and OT-I
cells. No differences in T cell activation between the two DC types
could be observed in vitro (Fig. 3C), indicating that the MR not
only was dispensable for the uptake but also for intracellular pro-
cessing of cell-associated OVA for cross-presentation.
The MR contributes to in vivo uptake of soluble but not of
cell-associated OVA by DC
Next, we decided to investigate the in vivo role of the MR in Ag
uptake. To this end, we injected fluorochrome-labeled soluble
OVA into B6 mice. Consistent with our in vitro findings, a close
correlation between the uptake of soluble OVA and the expression
of the MR in DC was found (Fig. 4, A and B). DC from MR?/?
mice showed significant but incomplete reduction of uptake of
soluble OVA (Fig. 4, C and D). This was most evident in the
spleen and in the bone marrow, which have been described as
locations in which cross-presentation takes place (8, 25). In the
6771The Journal of Immunology
conjugated OVA, stained for expression of the MR, and analyzed by flow cytometry. B, CD11c?BMDC from C57/BL6 mice were treated for 30 min with
3 mg/ml mannan and then cocultured for 10 min at 37°C with 10 ?g/ml alexa647-OVA. Histograms show the uptake profiles of fluorescent Ag by
mannan-treated and untreated DC, and by BMDC from MR?/?mice. Numbers indicate the MFI ? SD; the proportions of the CD11c?cells that had taken
up Ag are given above the histogram area indicators. C, Immunofluorescence images visualizing uptake of alexa647-OVA by BMDC. D, BMDC were
prepared from MR?/?and wild-type mice and stained for expression of the subtype markers CD11c, CD11b, and CD8? (solid lines) or control (gray areas).
E, BMDC from MR?/?and wild-type mice were stained for expression of the maturation markers CD80, CD86, CD40, and MHC class II following
stimulation for 24 h with 10 ?g/ml LPS (solid lines) or no stimulation (dashed lines).
The MR is essential for uptake of soluble OVA by BMDC. A, CD11c?BMDC generated from B6 mice were incubated with alexa647-
6772 MANNOSE RECEPTOR-MEDIATED AG UPTAKE FOR CROSS-PRESENTATION
cLN, only moderate uptake was observed, which was further re-
duced in MR?/?mice, albeit not significantly (Fig. 4, C and D).
This reduction was not due to changes of the DC subpopulations
present in MR?/?mice, because these were indistinguishable from
those in wild-type mice (Fig. 4E). These findings demonstrated
that the MR was involved also in the in vivo uptake of soluble
OVA, but it was not essential, as opposed to its role in vitro.
Indeed, in wild-type mice we found some DC that had taken up
OVA but did not express the MR (Fig. 4B). The mechanisms that
partially compensated for the absence of the MR in vivo remain to
Next, we examined the effect of the MR on the in vivo uptake
of cell-associated OVA by injection of UV-irradiated bm1 spleno-
cytes loaded with FITC-labeled OVA into MR?/?mice and con-
trols. DC from the spleen and from the bone marrow of C57/BL6
mice showed significant uptake (Fig. 5, A and B). The cLN showed
only marginal uptake of cell-associated Ag, consistent with reports
by others (26). In MR?/?mice, uptake was unaltered (Fig. 5, A
and B), implying that cell-associated Ag had been internalized by
mechanisms other than MR-mediated endocytosis.
The MR mediates cross-presentation of soluble but not of
cell-associated OVA in vivo
To assess the in vivo role of the MR for CD8 T cell activation by
cross-presentation, we injected CFSE-labeled OT-I transgenic T
cells into wild-type or MR-deficient recipient mice. After priming
with soluble OVA, we analyzed the proliferation of OT-I cells in
spleen, bone marrow, and cLN. In MR?/?mice, proliferation of
these T cells was substantially reduced (Fig. 6A). Also, CD69 ex-
pression on OT-I cells was substantially diminished on day 1 in all
organs tested in MR?/?mice (data not shown), demonstrating that
the diminished proliferation of OT-I cells was due to decreased
activation at the priming site, rather than to reduced recirculation
of OT-I cells activated elsewhere.
Finally, we examined the in vivo role of the MR in the presen-
tation of cell-associated OVA. Wild-type and MR?/?mice were
injected with OT-I cells and primed with OVA-coated, UV-irra-
diated bm1 splenocytes. Proliferation of OT-I cells in the spleen
and in the bone marrow was not reduced in MR?/?mice (Fig. 6B).
Moreover, no alterations in up-regulation of CD69 in the activated
T cells were observed (data not shown). Consistent with the mar-
ginal Ag uptake in the cLN, no proliferation of OT-I cells was
observed in this location (Fig. 6B). Thus, mechanisms other than
MR-mediated Ag uptake were responsible for CD8 T cell activa-
tion by cross-presentation of cell-associated Ag.
The molecular mechanisms that mediate Ag uptake for cross-pre-
sentation are unresolved. The present study is the first to identify
a receptor involved in this process, by demonstrating that Ag en-
docytosed via the MR gained access to the cross-presentation path-
way. We showed that the MR was important for the uptake of
by BMDC. A, UV-irradiated splenocytes from bm1 mice were incubated
for 10 min at 4°C or 37°C with FITC-labeled OVA, and then analyzed for
uptake of fluorescent soluble Ag. B, UV-irradiated bm1 splenocytes loaded
with FITC-OVA at 37°C were cocultured with BMDC from wild-type or
MR?/?mice. After 18 h, DC were analyzed for uptake of fluorescent
cell-associated Ag. Apoptotic bm1 splenocytes were excluded from anal-
ysis by forward scatter/side scatter gating or gating for Kb?cells revealed
by staining with SF1 Ab. Numbers indicate the MFI ? SD. The propor-
tions of the CD11c?cells that had taken up Ag are given above the his-
togram area indicator.
The MR is dispensable for uptake of cell-associated OVA
not of cell-associated OVA by BMDC to CD8 T cells. A, To establish an
in vitro system for CD8 T cell activation by cross-presentation, various
concentrations of soluble OVA or of the OVA peptide SIINFEKL were
coincubated for 2 h with BMDC from B6 mice in the absence (f) or
presence (?) of the proteasome inhibitor MG132. DC were then washed,
fixed, and cocultured with OT-I cells. After 18 h, IL-2 concentrations in the
culture supernatants were determined by ELISA. B, BMDC from wild-type
(f) or MR?/?(?) B6 mice were incubated with 1 mg/ml OVA or 200 nM
SIINFEKL. After an additional 3 h, cells were fixed and naive OT-I cells
were added. IL-2 concentrations in the culture supernatants were deter-
mined at 18 h after addition of OT-I cells. C, Splenocytes from bm1 mice
were incubated with OVA, UV irradiated, and then cocultured with BMDC
from wild-type or MR?/?mice and OT-I cells. After 40 h, IL-2 concen-
trations were determined.
The MR is essential for cross-presentation of soluble but
6773 The Journal of Immunology
soluble OVA by DC in vivo, and that it was even essential for
BMDC in vitro. Concomitantly, the activation of naive CD8 T
cells by soluble OVA was diminished and in fact abrogated when
BMDC were used. This was most likely due to reduced cross-
presentation of OVA, because the absence of the MR did not affect
expression of costimulatory molecules. Furthermore, MR-deficient
DC loaded with OVA peptide induced a T cell response equal to
that induced by wild-type DC, implying that the costimulatory
signals provided must have been similar. Also, differences in the
DC subtype composition of MR-deficient mice were unchanged, in
particular the content of CD8??DC, which have been reported to
mediate in vivo cross-presentation of soluble and cell associated
OVA (10, 14).
As opposed to the clear-cut in vitro situation, uptake and cross-
presentation of soluble OVA in vivo was not completely abolished
in MR?/?mice, yet it was significantly reduced. This may indicate
that BMDC do not mimic all mechanisms involved in Ag uptake
by DC in vivo. These mechanisms may involve the generation of
OVA peptides by endogenous proteases, or Ag uptake by addi-
tional cellular receptors, such as the lectins DC-SIGN and
DEC205, whose expression might differ between BMDC and DC
in living animals (17, 27). This may be the case for CD8??DC
that were shown to mediate cross-presentation in vivo (10, 14),
because these are known to be absent from BMDC generated by
standard methodology (22).
Uptake of cell-associated OVA and the resulting activation of
injected i.v. into wild-type and MR-deficient B6 mice. After 12 h, DC were isolated from spleen, bone marrow, and cLN, and the proportion of alexa647
cells in the viable CD11c?cells was determined (n ? 3 or 4 mice). B, MFI ? SD of the cells from A.
The MR is dispensable for in vivo uptake of cell-associated OVA. A, FITC-OVA-loaded bm1 cells (20 ? 106) were UV-irradiated and
mice were i.v. injected with alexa647-conjugated OVA (5 ?g/g body weight). After 45 min, single-cell suspensions from the spleen were stained for CD11c
and MR expression, and analyzed using the gate shown in A. C, Wild-type and MR-deficient C57/BL6 mice were i.v. injected with 5 ?g/g body weight
alexa647-conjugated OVA. After 45 min, DC were isolated from spleen, bone marrow, and cLN, and analyzed by flow cytometry. Numbers give the
proportions of alexa647
cells were stained for expression of the DC subtype markers CD11b and CD8?, and analyzed by flow cytometry.
The MR predominantly mediates in vivo uptake of soluble OVA. A, Gating scheme used for analysis of Ag uptake by splenic DC. B, B6
?cells ? SD within the viable CD11c?cells (n ? 3 or 4 mice). D, MFI ? SD of the cells from C. E, Alexa647
6774 MANNOSE RECEPTOR-MEDIATED AG UPTAKE FOR CROSS-PRESENTATION
OT-I cells were not impaired by the absence of the MR, either in
vivo or in vitro. The cell-associated Ag used in the present study
was located intracellularly, at least in part. Such Ag may be avail-
able for endocytosis by one or several distinct receptors. Reports
implicating CD36 in uptake of Ag borne by apoptotic cells are
controversial (28). Thus, the mechanisms mediating Ag uptake for
classical cross-priming, as described by Bevan (8), remain to be
resolved. Nevertheless, our findings demonstrated that additional
pathways exist for cross-presentation of Ag carried by microor-
ganisms, or of viral Ag within infected host cells.
The MR is mostly known for its role as a scavenger receptor of
macrophages. A role in Ag presentation has been proposed by
others based on its expression by DC and on its role in the uptake
of mannosylated structures such as dextrans or horseradish perox-
idase in a mannan-blockable fashion into cellular compartments
that contained MHC class II molecules (21). Such uptake could
result in stimulation of CD4 T cell clones in vitro (16), suggesting
a role for the MR in adaptive immunity. The findings reported here
support such a role by demonstrating MR-mediated activation of
naive CD8 T cells, and by providing evidence for in vivo relevance
of such activation. Furthermore, the use of MR?/?mice in the
present study precluded the specificity concerns that apply to man-
nan blockade of the MR (6).
Our findings suggest that targeting of the MR may be useful for
introducing extracellular Ag into the MHC class I-restricted Ag
presentation pathway. Targeting of a related lectin, DEC205, has
been shown to result in tolerogenic CD4 and CD8 T cell activation
(27). It will be interesting to investigate whether Ag uptake by the
MR would result in immunogenic or tolerogenic CTL priming, or
both, depending on the absence or presence of inflammatory stim-
uli. Finally, the indispensability of the MR for uptake of soluble
OVA by BMDC may allow further in vitro dissection of intracel-
lular mechanisms governing cross-presentation.
We thank Andreas Kautz and Petra Peters for excellent technical assistance
and Dr. Michel C. Nussenzweig for providing MR?/?mice. We acknowl-
edge technical support by the flow cytometry core facility of the Institute
of Molecular Medicine and Experimental Immunology and by the central
animal facilities of the Medical Faculty of the University of Bonn (House
for Experimental Therapy).
The authors have no financial conflict of interest.
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6776 MANNOSE RECEPTOR-MEDIATED AG UPTAKE FOR CROSS-PRESENTATION