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doi:10.1182/blood-2002-04-1044
Prepublished online June 14, 2002;
van Kooyk
Gangaram-Panday, Gerard C van Duijnhoven, Georg Kraal, Antoon J M van Oosterhout and Yvette
Teunis B H Geijtenbeek, Peter C Groot, Martijn A Nolte, Sandra J van Vliet, Shanti T
that captures blood-born antigens in vivo
Marginal zone macrophages express a murine homologue of DC-SIGN
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Copyright 2011 by The American Society of Hematology; all rights reserved.
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by the American Society of Hematology, 2021 L St, NW, Suite 900,
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly
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Marginal zone macrophages express a murine homologue of DC-
SIGN that captures blood-born antigens in vivo
Running title: A murine homologue of DC-SIGN captures antigens in vivo
Teunis B.H. Geijtenbeek†§, Peter C. Groot¶‡, Martijn A. Nolte †‡, Sandra J. van Vliet†, Shanti
T. Gangaram-Panday†, Gerard C.F. van Duijnhoven*, Georg Kraal†, Antoon J.M. van
Oosterhout ¶ and Yvette van Kooyk†
†Department of Molecular Cell Biology, Vrije Universiteit Medical Center Amsterdam, v.d.
Boechorststraat 7, 1081 BT Amsterdam, The Netherlands.
*Department of Tumor Immunology, University Medical Center St Radboud, Geert
Grooteplein Zuid 30, 6525 GA Nijmegen, The Netherlands.
¶ Department of Pharmacology & Pathophysiology, Faculty of Pharmacy, Utrecht University,
Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands
‡ P.C. Groot and M.A. Nolte contributed equally to this paper.
§To whom correspondence should be addressed:
T.B.H. Geijtenbeek, Department of Molecular Cell Biology, Vrije Universiteit Medical Center
Amsterdam, v.d. Boechorststraat 7, 1081 BT Amsterdam, The Netherlands. Tel: +(31)20-
4448080; Fax: +(31)20-4448081; e-mail: T.Geijtenbeek.Cell@med.vu.nl
Word count. abstract: 244 words, text: 4701 words
Scientific heading: Immunobiology
Copyright 2002 American Society of Hematology
Blood First Edition Paper, prepublished online June 14, 2002; DOI 10.1182/blood-2002-04-1044
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Abstract
Antigen presenting cells are localized in essentially every tissue, where they operate at the
interface of innate and acquired immunity by capturing pathogens and presenting pathogen-
derived peptides to T cells. C-type lectins are important pathogen recognition receptors and
the C-type lectin DC-SIGN is unique in that, in addition to pathogen capture, it regulates
adhesion processes such as DC trafficking and T cell synapse formation. We have isolated a
murine homologue of DC-SIGN that is identical to the previously reported murine homologue
mSIGNR1. mSIGNR1 is more closely related to the human DC-SIGN homologue L-SIGN
than to DC-SIGN self since mSIGNR1 is specifically expressed by liver sinusoidal endothelial
cells, similar as L-SIGN, and not by DC. Moreover, mSIGNR1 is also expressed by medullary
and subcapsular macrophages in lymph nodes, and by marginal zone macrophages (MZM) in
spleen. Strikingly, these MZM are in direct contact with the blood stream and efficiently
capture specific polysaccharide-antigens present on the surface of encapsulated bacteria. We
have investigated the in vivo function of mSIGNR1 on MZM in spleen. We demonstrate here
that mSIGNR1 functions in vivo as a pathogen recognition receptor on MZM that captures
blood-born antigens, which are rapidly internalized and targeted to lysosomes for processing.
Moreover, the antigen-capture is completely blocked in vivo by the blocking mSIGNR1-
specific antibodies. Thus, mSIGNR1, a murine homologue of DC-SIGN, is important in the
defense against pathogens and this study will facilitate further investigations into the in vivo
function of DC-SIGN and its homologues.
e-mail: T.Geijtenbeek.Cell@med.vu.nl
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Introduction
Dendritic cells (DC) are professional antigen presenting cells (APC) that sample foreign
pathogens in the periphery and migrate to the lymphoid tissues where they present processed
antigens to naïve T cells, initiating an immune response1. DC express a repertoire of
specialized receptors to perform their unique functions. We have identified the human DC-
specific adhesion receptor DC-SIGN (CD209), which mediates several key-functions
throughout the lifetime of a DC; DC-SIGN binding to its endothelial ligand ICAM-2 mediates
transendothelial migration of DC2, and DC-SIGN enables primary immune responses by
initiating transient DC-T cell interactions through binding of ICAM-3 on naïve T cells3.
Recently, we demonstrated that DC-SIGN also functions as an antigen receptor by rapidly
internalizing soluble ligands that are targeted to late endosomes/lysosomes; here the
internalized antigen is efficiently processed and subsequently presented to CD4+ T cells4.
Besides playing an important role in the initiation of immune responses, DC-SIGN also
functions as a novel HIV-1 trans-receptor important in the dissemination of HIV-1 from sites
of infection5. Resident mucosal DC capture HIV-1 through the interaction of DC-SIGN with
the HIV-1 envelope glycoprotein gp120, and subsequently migrate to the lymphoid tissues
where DC-SIGN facilitates the transmission of HIV-1 to T cells5. The DC-specific expression
of human DC-SIGN (hDC-SIGN) in situ and in vitro correlates well with both its
immunological functions3 and its role in HIV-1 dissemination5.
The DC-SIGN homologue L-SIGN6, which has also been called DC-SIGN-related (DC-
SIGNR)7, has a similar activity to DC-SIGN, but is not expressed by DC6. Human L-SIGN
(hL-SIGN) is specifically expressed by liver sinusoidal endothelial cells (LSEC), a liver-
resident APC of monocyte origin and in lymph nodes6;8. LSEC interact strongly with
leukocytes and appear to constitute a central mechanism of peripheral immune tolerance in the
liver9. The tissue location and ligand binding properties of hL-SIGN strongly implicate a
physiological role for this receptor in antigen clearance6 as has been reported for other C-type
lectins10;11, as well as in LSEC-leukocyte adhesion.
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Functional studies into the in vivo function of DC-SIGN have been hampered by the lack of
murine homologues. Therefore we set out to identify and isolate the murine homologue of
DC-SIGN. We have isolated a cDNA clone OtB7 which is homologous to hDC-SIGN and is
identical to murine SIGNR1 (mSIGNR1), a recently reported murine homologue of DC-
SIGN12. Here we describe the cell-specific expression and in vivo function of this murine
homologue of DC-SIGN. We generated antibodies against this homologue and demonstrated
that its cell-specific expression is similar to that of hL-SIGN. mSIGNR1 has a similar binding
activity in vitro to both hDC-SIGN and hL-SIGN. Its high affinity for murine ICAM-2, and its
localization at sites that are in close contact with blood, suggest a role for mSIGNR1 in
lymphocyte migration from the blood into tissues. Moreover, we demonstrate that marginal
zone macrophages (MZM) in spleen efficiently capture blood-born antigens in vivo through
mSIGNR1. mSIGNR1 rapidly internalizes blood-born antigens which are targeted to
lysosomes. These results demonstrate that a murine homologue of DC-SIGN is expressed by
highly phagocytic macrophages in spleen, and functions in vivo as an pathogen recognition
receptor.
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Materials and Methods
Antibodies. The following antibodies were used: N148 (CD11c), anti-ICAM-2 (BD
Pharmingen, NJ), NLDC-40 (anti-DEC-205), M1/70 (CD11b), MECA-32, MOMA-1, ER-
TR913. Polyclonal antisera against mDC-SIGN were raised in rabbits against two mSIGNR1-
specific peptides, KTPNTERQKEQEKILQ and SRFQKYWNRGEPNNI (Eurogentec,
Seraing, Belgium).
Mice. BALB/c mice were bred and maintained in the laboratory animal facilities at the Faculty
of Medicine of the Vrije Universiteit Medical Center Amsterdam under conventional
conditions. Mice were used at ages between 8-15 weeks.
Characterization of the cDNA encoding the murine homologues of hDC-SIGN. The cDNA
encoding mSIGNR1 was amplified by RT-PCR on total RNA derived from lung. The PCR
primers used for isolation of mSIGNR1 were as follows: forward primer,
ATG.AGT.GAC.TCC.ACA.GAA.GCC.AAG.ATG.CAG; reverse primer , AAG.AAG.AAT.
CCC.AGA.GCC.TTT.TTCACG.ATC.C. The PCR product was sequenced and subsequently
cloned into the eukaryotic expression vector pRc/CMV. The homologue was expressed either
by transient transfections of 293T cells, or by stable transfections in the erythroleukemic cell
line K562. We have submitted the full mSIGNR1 cDNA sequence to GenBank under
accession number AF422108.
Generation of murine ICAM-2 and mSIGNR1 fusion proteins. The extracellular domain of
mICAM-2 was isolated by RT-PCR on total liver RNA using primers based on the murine
ICAM-2 cDNA sequence14. The primers were as follows: forward primer
CTA.AGC.TTC.CCC.ACC.TGA.GAT.GTC.TTC.TTT.TGC; reverse primer
CGG.ATC.CGC.CAC.GGG.ACG.TGC.CCG.AAA.G. The identity of the cDNA was
confirmed by sequencing, and the cDNA encoding mICAM-2 was cloned into the Sig-pIgG1-
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Fc vector15. The resulting ICAM-2 Fc chimera contains the extracellular domain of ICAM-2
fused to the IgG1 Fc domain. ICAM-2-Fc was produced in COS cells by co-transfection of
ICAM-2-Sig-pIgG1-Fc and the pEE14 vector. ICAM-2-Fc concentrations in the supernatant
were determined by an anti-IgG1 ELISA.
mSIGNR1 Fc consists of the extracellular portion of mSIGNR1 (amino acid residues 75-225)
fused at the C-terminus to a human IgG1-Fc fragment into the Sig-pIgG1-Fc vector15.
mSIGNR1 Fc was produced in COS cells similarly as described for mICAM-2 Fc.
mSIGNR1-specific ELISA. mSIGNR1-specific antibody ER-TR9 (5 µg/ml) was coated in
ELISA plates for 18 hours at 4°C, followed by blocking with 1% BSA for 30 min. at 4°C.
Soluble mSIGNR1 Fc (1 µg) was added and incubated for 2 hours at 4°C. Unbound protein
was washed away and mSIGNR1 was detected by the mSIGNR1-specific antiserum 698
(1:200), 1 hour at 4°C, followed by peroxidase-conjugated goat-anti- rabbit IgG.
Fluorescent bead adhesion assay. Carboxylate-modified TransFluorSpheres (488/645 nm, 1.0
µm; Molecular Probes, Eugene, OR) were coated with ICAM-2-Fc, ICAM-3-Fc and HIV-1
gp120MN as described previously3;5. The fluorescent bead adhesion assay was performed as
described3. Briefly, 5x106 cells in adhesion buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl,
1mM CaCl2, 2 mM MgCl2, 0.5% BSA) were pre-incubated with 20µg/ml mAb for 10 minutes
at room temperature. Ligand-coated fluorescent beads (20 beads/cell) were added and the
suspension was incubated for 30 minutes at 37°C. Adhesion was determined using flow
cytometry (FACScan, Becton Dickinson, Oxnard, CA), by measuring the percentage of cells,
which had bound fluorescent beads.
Bone marrow-derived DC. DC were generated as described previously16, with modifications.
Briefly, femurs were dissected, placed in 70% alcohol for 1 min, and washed with PBS.
Marrow was flushed and passed through nylon mesh to remove debris. After washing,
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lymphocytes, granulocytes, and I-A-positive cells were removed by immunomagnetic
depletion against the CD45R, CD4, CD8, and I-A-antigens. Remaining cells were cultured
overnight, and the non-adherent cells were seeded at 2 x 105 cells/ml and 4 ml/well in the
presence of 20 ng/ml rmGM-CSF and 20 ng/ml rmIL-4 in 6-well plates (Costar,
Badhoevedorp, the Netherlands). On day 4, the cultures were refreshed by adding 1 ml of
culture medium supplemented with GM-CSF and IL-4 (both at 10 ng/ml). At day 7, non-
adherent and loosely adherent proliferating DC aggregates were collected and replated in fresh
medium with cytokines(1 x 106 cells/ml).
CD11c+ DC isolation. Spleens were digested with collagenase IV for 40 minutes at 37°C,
followed by erythrocyte lysis. Splenic CD11c+ DC were isolated using MACS CD11c
microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to manufacturer’s
protocol.
Macrophage depletion in vivo. Systemic depletion of macrophages was performed by
treatment with clodronate-filled liposomes, as described previously17. Mice received a single
intravenous injection of 0.2 ml multilamellar liposomes in PBS, containing 2 mg liposome-
encapsulated clodronate (dichloromethylene biphosphonate, a kind gift from Roche
Diagnostics GmbH, Mannheim, Germany). Depletion of macrophages from the spleen was
assessed two days after liposome injection by immunofluorescence analysis.
Antigen capture. For studying the interaction of antigens with MZM, 200 µl FITC-dextran (1
mg/ml, 500 kDa dextran; Molecular Probes, Eugene, OR) was injected intravenously into the
tail veins of naïve mice. Where appropriate mice were treated 30 minutes before dextran
administration with either 200 µl mannan (1 mg/ml) or 200 µl ER-TR9 by intravenous
injection into the tail vein. Mice were sacrificed 40 minutes after dextran injection by
immediate CO2 asphyxiation. The spleen was rapidly removed and directly frozen in Tissue-
Tek (Sakura Finetek, Torrance, CA).
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Immuno-fluorescence analysis. Tissues were collected and frozen in liquid nitrogen.
Cryosections (7 µm) of the tissues were fixed in 100% acetone (10 minutes), washed with
PBS and incubated with the first antibody (10 µg/ml) for 60 minutes at 37°C. The final
staining was performed with secondary antibodies labelled with either FITC or Texas Red for
30 minutes at room temperature.
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Results
Isolation of a murine homologue of DC-SIGN
A 415 nucleotide long cDNA fragment, called OtB7, was isolated by differential gene
expression from a mouse model of allergic asthma, using lung-draining lymph node mRNA
obtained from control and ovalbumin-challenged mice, which display phenotypic
characteristics resembling human asthma18. The cDNA fragment was homologous to hDC-
SIGN, and to two mouse genomic sequences present in the high-throughput division of
GenBank (accession numbers AC073804.1 and AC073706.2). Using a combination of
BLAST searches and multiple alignment analyses we were able to construct a contiguous
19619 bp long genomic sequence composed of overlapping sequences present in both
Genbank entries (Figure 1a). A gene with homology to hDC-SIGN was predicted, and the
1101 bp cDNA encoding the predicted mDC-SIGN homologue (Genbank entry AF422108)
was isolated by RT-PCR from lymph node mRNA. The OtB7 cDNA encodes a type II
transmembrane C-type lectin consisting of 325 amino acid residues. The C-type lectin domain
of the murine homologue has a 74% similarity with that of hDC-SIGN, and the amino acid
residues essential for ligand- as well as Ca2+-binding are identical to those of hDC-SIGN19
(Figure 1b), suggesting a similar ligand specificity. The predicted transmembrane-spanning
region is 85% identical to that of both hDC-SIGN and hL-SIGN. Comparison of the
cytoplasmic region of OtB7 with that of the human homologues demonstrates that OtB7 lacks
the LL sequence motif which is important for internalization of hDC-SIGN4;20. The C-type
lectin domain of hDC-SIGN is separated from the cell-membrane by a stalk of 8 repeats which
form an -helix21, whereas several splice variants of hL-SIGN exist, differing in the number
of repeats from 3 to 96. The stalk of the murine DC-SIGN homologue OtB7 is considerably
shorter and lacks these repeats, however the region contains regularly-spaced hydrophobic
sequences that have been observed in other -helices. The predicted amino acid sequence of
OtB7 is as homologous to hL-SIGN as it is to hDC-SIGN.
Sequence alignment demonstrates that the putative protein encoded by clone OtB7 is identical
to murine SIGNR1 (Genbank AF373409; results not shown), one of five recently identified
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murine homologues of hDC-SIGN12. We will hereafter refer to OtB7 as murine SIGNR1
(mSIGNR1).
mSIGNR1 binds both murine ICAM-2 and HIV-1 gp120
We raised polyclonal antibodies against two peptides present in the C-terminal region of
mSIGNR1 to investigate the expression of this putative DC-SIGN homologue in different
tissues. The human kidney 293T cell-line was transfected with mSIGNR1 cDNA and
mSIGNR1 was detected on the cell-surface by the mSIGNR1-specific polyclonal antibody 698
(Figure 2a). Thus, as predicted, mSIGNR1 is a type II transmembrane protein since the
antibodies, raised against the C-terminal region, stain intact 293T cells transfected with
mSIGNR1 (Figure 2a). The antibodies stained neither mock- nor hDC-SIGN-transfected cells
(results not shown). The monoclonal antibodies against hDC-SIGN (AZN-D1, -D2 and -D3)
and hL-SIGN (AZN-D2 and -D3) did not cross-react with the murine homologue mSIGNR1
(results not shown).
The C-type lectin domain of mSIGNR1 contains the amino acid residues which have been
shown to be essential in both ligand- and Ca2+-binding by hDC-SIGN19. Therefore we
investigated whether mSIGNR1 was functionally similar to both hDC-SIGN and hL-SIGN.
293T cells transfected with mSIGNR1 cDNA interacted with human ICAM-2, ICAM-3 and
HIV-1 gp120, but not with human ICAM-1, as is also observed for both hDC-SIGN- and hL-
SIGN-expressing cells (Figure 2b). Thus, as predicted by the high homology between C-type
lectin domains, mSIGNR1 has a similar ligand binding activity to both of these human
homologues.
Next we investigated whether mSIGNR1 is able to interact with murine ICAM-2, since mice
do not express a homologue of human ICAM-3. Indeed, mSIGNR1-expressing cells interacted
strongly with murine ICAM-2, in contrast to mock transfected cells (Figure 2c). The adhesion
was similar to that observed for both hDC-SIGN- and hL-SIGN-transfected cells (Figure 2c).
Moreover, the adhesion was inhibited by both the Ca2+-chelator EGTA and the
polycarbohydrate mannan, demonstrating that mSIGNR1 functions as a mannose-binding C-
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type lectin (Figure 2c). The binding site on ICAM-2 for DC-SIGN and its homologues is
conserved between species since both hL-SIGN and hDC-SIGN interact with murine ICAM-2
(Figure 2c), and mSIGNR1 binds to human ICAM-2 (Figure 2b). These data demonstrate that
mSIGNR1 functions as an adhesion molecule with similar specificity to hDC-SIGN and hL-
SIGN.
The expression pattern of mSIGNR1 is similar to that of hL-SIGN
Next, we used our polyclonal antibodies to investigate whether DC express mSIGNR1, since
mRNA encoding mSIGNR1 has been detected by RT-PCR from bone marrow-derived DC22
and splenic CD11c+ DC12. Strikingly, no mSIGNR1 was detected on the cell-surface of either
bone marrow-derived DC or splenic CD11c+ DC (Figure 3a), demonstrating that mSIGNR1,
in contrast to hDC-SIGN, is not expressed by either in vitro- or in vivo-derived murine DC.
Immuno-fluorescence analysis demonstrated that mSIGNR1 is expressed by cells that line the
sinusoids in the liver (Figure 3b).
The mSIGNR1 staining was similar to that observed for hL-SIGN6 and demonstrates that
mSIGRN1 is expressed by LSEC. This is further confirmed by the absence of mSIGNR1
expression from Kupffer cells, which were stained with the marker F4/80 (Figure 3c).
Both hDC-SIGN and hL-SIGN are expressed in lymph nodes, and staining of murine lymph
node tissue sections demonstrated that mSIGNR1 is also highly expressed in lymph nodes
(Figure 3d-g), but not by lymph node-resident DC since neither CD11c nor DEC205 co-
localize with mSIGNR1 expression (Figure 3d and e). The mSIGNR1+ cells co-express the
antigens for SER-4 (Figure 3f) and MOMA-1 (Figure 3g), markers for both medullary and
subcapsular macrophages in lymph nodes23. Thus, mSIGNR1 is specifically expressed by
medullary and subcapsular macrophages in lymph nodes. These data demonstrate that the cell-
specific expression of mSIGNR1 is similar to that of hL-SIGN and this indicates mSIGNR1 is
more closely related to hL-SIGN than to hDC-SIGN.
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mSIGNR1 is specifically expressed by marginal zone macrophages, and is recognized by the
MZM-specific antibody ER-TR9
We next investigated the expression of mSIGNR1 in the spleen, since mSIGNR1 mRNA has
been detected in spleen (results not shown and 12). We performed in situ immuno-fluorescence
analysis using the polyclonal antibodies 698 to identify those spleen cells expressing
mSIGNR1, since no mSIGNR1 was expressed on the cell-surface of splenic CD11c+ DC
(Figure 3a). mSIGNR1 is highly expressed by cells present in the marginal zone bordering the
white pulp (Figure 4a-d). These mSIGNR1+ cells are not DC, since they do not express the
DC markers CD11c and DEC-205 (Figure 4a and b). However, the mSIGNR1+ cells are
specifically stained with the marginal zone macrophage (MZM)-specific antibody ER-TR9
(Figure 4c). Thus, mSIGNR1 is specifically expressed by MZM in the spleen and these cells
are present in the marginal zone bordering the white pulp (Figure 4d). MZM are highly
phagocytotic cells whose dendrites that are in direct contact with the arterial blood stream
(reviewed in 24), and they can be effectively killed by a single intravenous injection of
liposomes containing dichloromethylene diphosphonate25. Indeed, all MZM were depleted
from the spleen after injection of the liposomes, since no staining of the MZM-marker ER-
TR9 is observed (Figure 4e). Moreover, no staining was observed with the polyclonal
antibodies against mSIGNR1, following MZM-depletion from the spleen, confirming that
mSIGNR1 is MZM-specific (Figure 4f).
The antigen of the MZM-specific antibody ER-TR9 has not been identified to date. The
similar expression patterns of the polyclonal antibodies 698 against mSIGNR1, and the MZM-
specific antibody ER-TR9 in spleen (Figure 4), liver and lymph node (results not shown), led
us to investigate whether mSIGNR1 is the antigen for this antibody. Indeed, 293T cells
transfected with mSIGNR1 were stained with the monoclonal antibody ER-TR9, whereas
mock transfected 293T cells are not (Figure 5a). Thus, the receptor recognized by ER-TR9 on
MZM is mSIGNR1. Moreover, this was further confirmed by the specific recognition of the
recombinant mSIGNR1 fusion protein by ER-TR9, in a mSIGNR1-specific sandwich ELISA
(Figure 5b). Comparison of the ER-TR9-specific expression pattern in spleen, lymph node and
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liver with that of the polyclonal antibodies 689 against mSIGNR1 demonstrates that ER-TR9
specifically recognizes mSIGNR1 but not the other murine homologues of DC-SIGN (results
not shown and Figure 4).
mSIGNR1 captures blood-born antigens in vivo
The spleen plays an important role in the defense against pathogens and MZM are important
in this defense (reviewed in 24). MZM are in direct contact with the blood stream and are
highly phagocytotic cells. These cells have been demonstrated to bind specific polysaccharide
antigens present at the surface of encapsulated bacteria26;27. We examined the molecular basis
for this specificity by determining the mechanisms by which these antigens preferentially
targeted to MZM. Polysaccharide antigens rapidly localize to the MZM as demonstrated by
intravenous injection of naïve wild-type mice with the model polysaccharide antigen
fluorescein isothiocyanate (FITC)-dextran (Figure 5c and d). The FITC-dextran staining is
localized in the marginal zone bordering the white pulp (Figure 5c) and co-localizes with the
anti- mSIGNR1 polyclonal antibodies 698 (Figure 5d). Because mSIGNR1 contains a C-type
lectin domain similar to hDC-SIGN which has been shown to bind polycarbohydrates3;28, we
next determined whether mSIGNR1 was integral to this dextran capture by MZM. Naïve mice
were injected with purified ER-TR9 and after 40 minutes FITC-dextran was injected
intravenously. In these mice, the FITC-dextran was not retained in the spleen by MZM (Figure
5e) since no FITC-dextran was detected in the marginal zone. This demonstrates that
mSIGNR1 is responsible for the efficient capture of dextran by MZM. These results were
further confirmed by injecting naïve mice with the yeast-derived polysaccharide mannan prior
to FITC-dextran injection. Strikingly, MZM were then no longer able to capture FITC-dextran
(Figure 5f) because mannan is efficiently bound by mSIGNR1 and blocks its function (Figure
2b and c). Thus, the well-known function of MZM to filter blood-born antigens from blood is
mediated by mSIGNR1, and this mSIGNR1 function is inhibited by ER-TR9, and other
polycarbohydrates such as mannan.
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MZM rapidly internalize antigens upon binding and we investigated whether mSIGNR1 also
mediates the internalization of ligands. K562 transfectants expressing mSIGNR1 have a high
affinity for FITC-dextran, whereas mock transfected cells do not (Figure 6a), confirming the
in vivo function of mSIGNR1. The dextran interaction with mSIGNR1 in vitro was
specifically blocked by the ER-TR9 antibody (Figure 6a). The fate of the bound dextran was
followed by immunofluorecence analysis. FITC-dextran was rapidly internalized and targeted
to the lysosomes (Figure 6b), demonstrating that mSIGNR1 is responsible for both capture
and internalization of dextran by MZM, and suggesting that mSIGNR1 captures antigens from
blood for degradation and antigen processing.
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Discussion
APC are present in essentially every tissue where they operate at the interface of innate and
acquired immunity by recognizing pathogens and presenting pathogen-derived peptides to T
cells. C-type lectins play an important role as pathogen recognition receptors that capture and
destroy pathogens for antigen processing to permit MHC class II-restricted presentation. It is
becoming clear that not all C-type lectins serve as antigen receptors recognizing pathogens
through carbohydrate structures. The DC-specific C-type lectin hDC-SIGN is unique in that it
regulates adhesion processes, such as DC trafficking through ICAM-2, and T cell synapse
formation through ICAM-3, as well as antigen capture3;4. Moreover, even though several C-
type lectins have been shown to bind HIV-1, hDC-SIGN not only captures HIV-1, but also
protects it in early endosomes allowing HIV-1 transport by peripheral DC to the lymphoid
tissues, where it enhances trans-infection of T cells5.
However, functional studies into the in vivo function of DC-SIGN have been hampered by the
lack of murine homologues. Here we report the identification and isolation of a cDNA called
OtB7 which encodes a murine protein that is homologous to both hDC-SIGN and hL-SIGN
(Figure 1b). During the preparation of this manuscript, five murine DC-SIGN homologues
were identified by RT-PCR from CD11c+ DC although only one, called murine DC-SIGN
(mDC- SIGN) was expressed at high mRNA levels in CD11c+ DC12. In contrast the other
cDNAs, designated mSIGNR1-4, were hardly detectable in DC, but were detected at various
mRNA levels in B and T cells by RT-PCR12. The OtB7 clone that we have isolated from
lymph node cDNA is identical to the DC-SIGN homologue mSIGNR1. We generated
polyclonal antibodies against mSIGNR1 to investigate the cell-specific expression of this
hDC-SIGN homologue, because of the complex mRNA expression patterns of the reported
DC-SIGN homologues. mSIGNR1 is expressed neither by in vitro-generated DC nor by in situ
CD11c+ DC in spleen and lymph nodes (Figure 3 and 4). However, mSIGNR1 is expressed by
LSEC in liver and by medullary and subcapsular macrophages in the lymph nodes (Figure 3).
This specific expression is very similar to that of hL-SIGN, which is expressed by LSEC6;8
and by a specific subpopulation of non-endothelial macrophage-like cells in lymph nodes (A.
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Engering, personal communication), indicating that, based on expression, mSIGNR1 is more
closely related to hL-SIGN than to hDC-SIGN.
Intermediate mRNA levels of mSIGNR1 were detected in murine spleen by RNA
hybridization (data not shown and 12). Here, we demonstrated using mSIGNR1-specific
antibodies that mSIGNR1 is specifically expressed by ER-TR9+ MZM in the spleen (Figure
4). These data were further confirmed by the intravenous injection of liposomes containing
dichloromethylene diphosphonate, which systemically depletes macrophages from the red
pulp and marginal zone25. Injection of the liposomes depleted the spleen of MZM as shown by
the absence of ER-TR9 staining (Figure 4e). No mSIGNR1 was present in MZM-depleted
spleens confirming the specific expression of mSIGNR1 by MZM (Figure 4f). Interestingly,
hDC-SIGN is expressed in spleen in contrast to hL-SIGN (T.G., unpublished results). Thus,
the expression pattern of mSIGNR1 is similar but not identical to that of hL-SIGN, and
mSIGNR1 has attributes of both hDC-SIGN and hL-SIGN suggesting that the distinction
between hL-SIGN and hDC-SIGN may be different and for less defined in the murine system.
The C-type lectin domain of mSIGNR1 has a 74% similarity with that of hDC-SIGN and the
amino acid residues important for both Ca2+- and ligand-binding19 are conserved in the murine
homologue. Indeed, mSIGNR1 has a similar binding activity for hICAM-2, hICAM-3 and
HIV-1 gp120 to both hDC-SIGN and hL-SIGN (Figure 2). Thus, mSIGNR1 can function as
an adhesion receptor. However, mice do not express an ICAM-3 homologue and therefore
binding of mSIGNR1 to ICAM-3 is not physiological. mSIGNR1 also interacts with murine
ICAM-2, which is widely expressed on murine lymphocytes29, and could therefore function as
the leukocyte ligand for mSIGNR1 mediating contact between mSIGNR1-postive cells and
leukocytes in mice. Strikingly, mSIGNR1 is abundantly expressed in liver and spleen by
LSEC and MZM respectively. Both cell-types are in direct contact with the blood and are
important in lymphocyte interactions. LSEC-leukocyte interactions appear to constitute a
central mechanism of peripheral immune surveillance in the liver30. The expression and
ligand-specificity of mSIGNR1 suggests a physiological role for this receptor in LSEC-
leukocyte adhesion through binding of ICAM-2 on lymphocytes, similar to hL-SIGN6. The
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strategic localization of MZM in the spleen resembles that of LSEC in the liver, and the
splenic marginal zone is important as a gateway for migrating lymphocytes (reviewed in 24).
MZM interact with incoming lymphocytes and antigens in the marginal sinus since part of the
arterial blood supply opens into the wider space of the marginal sinus resulting in a blood flow
with lower resistance. The only entry into the white pulp is via the marginal zone, and
lymphocytes entering the marginal zone are either directed into the white pulp or will leave
the marginal zone through the red pulp side. Little is known about the cellular interactions
important for this lymphocyte migration but carbohydrate interactions are known to be
important in this migration to the white pulp since polysaccharides such as mannan inhibit this
process26;31;32. Strikingly, here we demonstrate that the mSIGNR1-dependent binding of
ICAM-2 is inhibited by mannan suggesting that mSIGNR1 provides the interactions that
enable lymphocyte migration into the white pulp. This hypothesis is further supported by the
clustering of MZM with lymphocytes26 and the high expression of ICAM-2 on murine
lymphocytes29.
Besides being a gateway for migrating lymphocytes, the splenic marginal zone is also
important as a defense against pathogens. Because of their position adjacent to the marginal
sinuses, MZM are amongst the first cells that can interact with blood-born antigens and are
presumed to have a critical role in host defense against bacterial pathogen (reviewed in 24).
MZM are specifically stained by the monoclonal antibody ER-TR913 and although its antigen
had not been identified, results suggested that the ER-TR9-antigen is involved in antigen
capture. Strikingly, mSIGNR1 is specifically expressed by MZM and we demonstrate here
that mSIGNR1 is the antigen of ER-TR9 (Figure 5a and b). Thus, the C-type lectin mSIGNR1
may have a function as a pathogen recognition receptor. C-type lectins play an important role
as pathogen recognition receptors that capture and destroy pathogens for antigen processing to
permit MHC class II-restricted presentation. Recently, we have demonstrated that hDC-SIGN
functions as a pathogen recognition receptor in vitro that rapidly internalizes captured antigen
into lysosomal compartments for processing and antigen presentation4. Therefore, we
investigated whether mSIGNR1 is involved in pathogen recognition and capture in vivo.
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MZM are highly phagocytotic cells as demonstrated by their depletion after injection of
liposomes containing dichloromethylene diphosphonate (Figure 4) and their capacity to filter
pathogens from blood27. Indeed, bacterial antigens rapidly localize to the MZM as
demonstrated by intravenous injection of naïve wild-type mice with the model antigen, FITC-
dextran (Figure 5c). The FITC-dextran is specifically captured by the MZM in the marginal
zone and the FITC-staining co-localizes with antibodies against mSIGNR1 (Figure 5d).
Treatment of naïve mice with purified anti-SIGNR1 antibody ER-TR9 prior to the FITC-
dextran injection completely abrogates dextran binding by MZM (Figure 5e), demonstrating
that mSIGNR1 mediates the dextran capture by MZM. The anti-SIGNR1 antibody ER-TR9
also inhibits the in vitro binding of mSIGNR1 to dextran-FITC (Figure 6a) confirming that
ER-TR9 indeed inhibits the in vivo observed antigen uptake by mSIGNR1. Injection of the
yeast-derived polysaccharide mannan prior to FITC-dextran injection also prevents dextran-
binding by MZM, demonstrating that mSIGNR1 also binds mannan in vivo (Figure 5f) and
that mannan blocks the in vivo function of mSIGNR1. These data demonstrate that mSIGNR1
is involved in pathogen recognition by MZM in vivo. Moreover, antigens captured by
mSIGNR1 are rapidly internalized and targeted to lysosomes for degradation (Figure 6). Rapid
internalization of mSIGNR1 upon ligand binding (Figure 6b) suggests that ER-TR9 blocks
binding in vivo through downregulation of mSIGNR1 on the surface of MZM. The
cytoplasmic region of mSIGNR1 lacks the di-leucine motif present in hDC-SIGN, which
mediates the internalization of hDC-SIGN. However, the murine receptor contains a tri-acid
cluster (DDD) in its cytoplasmic region that functions as an internalization motif in DEC-205
where it is responsible for targeting to lysosomes for antigen processing33. The observed
lysosomal targeting of internalized ligand-mSIGNR1 complexes is likely be due to this tri-
acidic cluster. Thus, mSIGNR1 is important in the defense against pathogens by capturing
pathogens from the blood, which are then rapidly internalized into lysosomes and processed.
MZM do not express MHC class II molecules and it is thought that pathogen degradation
products are shed from the cell-surface and taken up by marginal zone B cells after
opsonization by complement. The marginal zone B cells migrate into the follicle and present
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the captured antigens to elicit an immune response34. Thus, the function of mSIGNR1 links
innate immunity with adaptive immunity. Furthermore, the affinity of mSIGNR1 for HIV-1
gp120 suggests that mSIGNR1 may be able to interact with both pathogens containing
dextran- and mannan-like structures, and viruses containing viral components with structures
similar to gp120.
In conclusion, we have isolated a murine homologue of hDC-SIGN, mSIGNR1, and
investigated its cell-specific protein expression using antibodies against mSIGNR1. We have
demonstrated that mSIGNR1 functions as a pathogen recognition receptor in vivo and thus is
important in the defense against pathogens. This in vivo model and the identification of
blocking anti-mSIGNR1 antibodies will facilitate studies into the in vivo function of DC-
SIGN.
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Acknowledgements
We are grateful to E. van Kesteren-Hendrix and N. van Rooijen for liposome-depleted tissues.
We thank J. Samsom, W. Jansen and R. Mebius for helpful discussions and providing
antibodies, and L. Colledge for suggestions and editing of the manuscript.
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21
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33. Mahnke K, Guo M, Lee S et al. The dendritic cell receptor for endocytosis, DEC-205,
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Legends
Figure 1. Clone OtB7 is identical to the murine DC-SIGN homologue SIGNR1
a. Schematic representation of the genomic structure of clone OtB7. Exons are numbered and
depicted as black bars. The genomic sequence was constructed by a combination of BLAST
searches and multiple alignment analyses, and is composed of overlapping sequences present
in the Genbank entries (AC073804.1 and AC073706.2).
b. Clone OtB7 is homologous to both human DC-SIGN and L-SIGN as demonstrated by the
amino acid sequence alignments of clone OtB7 (Genbank AF422108) with human DC-SIGN
(AAK20997) and L-SIGN (AAK20998). Amino acid residues involved in direct ligand
binding, as was shown for DC-SIGN 19, are denoted by filled arrows.
Figure 2. mSIGNR1 binds both mouse ICAM-2 and HIV-1 gp120, similar to both hDC-
SIGN and hL-SIGN.
a. Polyclonal antibodies against mSIGNR1 stain 293T cells transfected with mSIGNR1
cDNA. 293T cells were transfected with different cDNAs and subsequently stained with
antibodies against hDC-SIGN (AZN-D1; filled histogram) and mSIGNR1 (698; filled
histogram). Isotype controls are shown as open histograms. One representative experiment out
of three is shown.
b. mSIGNR1 binds human ICAM-2, ICAM-3 and HIV-1 gp120 but not human ICAM-1,
similar to hDC-SIGN and hL-SIGN. The adhesion of 293T transfected with mSIGNR1 to the
ligands was determined using the fluorescent bead adhesion assay. Specificity was determined
in the presence of blocking antibodies (AZN-D2, 20 µg/ml), mannan (5 µg/ml) or EGTA (5
mM). Cells were also pre-incubated with the polyclonal antibody against mSIGNR1 (1:10).
One representative experiment out of three is shown.
c. mSIGNR1 binds murine ICAM-2 with high affinity, similar to hDC-SIGN and hL-SIGN.
The adhesion assay was performed as described in Figure 2b. One representative experiment
out of three is shown.
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Figure 3. The expression pattern of mSIGNR1 is similar to that of hL-SIGN.
a. mSIGNR1 is not expressed by bone-marrow-derived DC or by splenic CD11c+ DC. Bone-
marrow derived DC and splenic CD11c+ DC were stained with the anti-mSIGNR1 antibody
698. Bone marrow DC were more than 90% pure as determined by CD11c staining (data not
shown). Dotted and open histogram represent isotype and mSIGNR1 staining, respectively.
Splenic DC were double stained with CD11c and the mSIGNR1-specific antibody 698.
b,c. Immuno-fluorescence staining of murine liver tissue sections with the polyclonal antibody
against mSIGNR1 698 (green, b and c) and double staining with F4/80 (red, c) (magnification
200x).
d-g. Immuno-fluorescence double staining of murine lymph node tissue sections with the
polyclonal antibody against mSIGNR1 698 (green) and in red CD11c (d), DEC-205 (e), SER-
4 (f), MOMA-1 (g). (magnification 200x).
Figure 4. mSIGNR1 is specifically expressed by splenic marginal zone macrophages.
a-d. Immuno-fluorescence double staining of murine spleen tissue sections with the polyclonal
antibody against mSIGNR1 698 (green) and in red CD11c (a), DEC-205 (b), ER-TR9 (c),
B220 (d) (magnification 200x).
e, f. Immuno-fluorescence of double staining of murine spleen tissue sections after depletion
with dichloromethylene diphosphonate liposomes. Double staining with (e) CD11c (red) and
ER-TR9 (green); and (f) CD11b (red) and polyclonal mSIGNR1 antibody 698 (green)
(magnification 200x).
Figure 5. mSIGNR1 efficiently captures polysaccharide antigens in vivo.
a. The MZM-specific antibody ER-TR9 stains 293T cells transfected with mSIGNR1 cDNA.
293T cells were transfected with mSIGNR1 cDNA and subsequently stained either with the
mSIGNR1-specific antibodies 698 or ER-TR9 (filled histograms). Isotype controls are
represented by open histograms.
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b. Recombinant mSIGNR1 is specifically detected by ER-TR9 using a sandwich ELISA. ER-
TR9-coated wells are incubated with recombinant mSIGNR1 and binding is detected by the
mSIGNR1-specific antibody 698. Standard deviation < 0.05.
c-d. MZM efficiently capture dextran-FITC in vivo. Naïve mice were injected intravenously
with FITC-dextran. Spleens were isolated and immuno-fluorescence analyses of murine
spleen tissue sections were performed. Double stainings with FITC-dextran (green) and B220
(red, c) or mSIGNR1-specific antibody 698 (red, d) are shown (magnification 200x).
e-f. Dextran capture by MZM in vivo is blocked by the mSIGNR1-specific antibody ER-TR9
and mannan. Naïve mice were treated with either purified ER-TR9 (e) or the yeast-derived
polysaccharide mannan (f) prior to intravenous injection with FITC-dextran. Double stainings
were performed with FITC-dextran (green) and the mSIGNR1-specific antibody 698 (red)
(magnification 200x).
Figure 6. mSIGNR1 mediates rapid internalization of captured dextran.
a. Dextran is efficiently captured by mSIGNR1. K562 cells expressing mSIGNR1 and mock
transfected K562 cells were incubated with FITC-dextran (1 µg/ml) at 37°C and unbound
dextran was washed away after 45 minutes. Specificity was determined in the presence of the
antibody ER-TR9. Binding was analyzed by flow cytometry. Standard deviation < 5%.
b. Dextran is rapidly internalized by SIGNR1. The fate of bound FITC-dextran was followed
by analyzing FITC-staining of K562 cells expressing mSIGNR1 after incubation with FITC-
dextran using immuno-fluorescence microscopy (magnification 200x).
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