Glycobiology vol. 12 no. 7 pp. 443–450, 2002
Macrophage C-type lectin on bone marrow–derived immature dendritic cells is involved
in the internalization of glycosylated antigens
Kaori Denda-Nagai, Nobuyoshi Kubota, Makoto Tsuiji,
Mika Kamata, and Tatsuro Irimura1
Laboratory of Cancer Biology and Molecular Immunology, Graduate School
of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113-0033, Japan
Received on February 4, 2002; revised on April 15, 2002; accepted on April
15, 2002
Bone marrow–derived dendritic cells (DCs) were examined
for the expression of the murine macrophage C-type lectin
specific for galactose and N-acetylgalactosamine (mMGL).
Flow cytometric analysis after double staining for MHC
class II and mMGL with specific monoclonal antibodies
indicated that mMGL was expressed on immature DCs
with low to moderate levels of MHC class II and downregulated during maturation. Immature DCs bound and
internalized α-N-acetylgalactosaminides conjugated to soluble
polyacrylamide (α-GalNAc polymers), whereas mature DCs
and bone marrow cells did not. The two-color flow cytometric
profiles indicated that the degree of α-GalNAc polymer
bindings exactly coincided with the intensity of the binding
of a mMGL-specific monoclonal antibody LOM-14. The
internalized α-GalNAc polymers seemed to be transported
to MHC class II compartments. Thus, mMGL is transiently
expressed on bone marrow–derived DCs during their
development and maturation and suggested to be involved
in the uptake of glycosylated antigens for presentation.
Key words: antigen uptake/C-type lectin/dendritic cells/
glycosylated antigen
Introduction
Dendritic cells (DCs) are known as antigen-presenting cells
that play a key role in the immune system. They capture
foreign antigens in peripheral tissues, process them into
peptides that bind to class II major histocompatibility complex
(MHC) antigens and migrate to lymphoid organs, where they
activate naive T cells by presenting their MHC–peptide
complexes on their cell surfaces (Banchereau and Steinman,
1998). During this process, DCs differentiate from an immature
state to a mature state as judged by their morphology, biological
functions, and cell surface phenotypes.
DCs are known to incorporate antigens by phagocytosis,
macropinocytosis, and receptor-mediated endocytosis through
a variety of putative antigen receptors having C-type lectin
1To whom correspondence should be addressed;
E-mail: [email protected]
© 2002 Oxford University Press
domains. At least two such receptors, DEC-205 and the
macrophage mannose receptor (MMR), type I Ca2+-dependent
C-type multilectins, appear to be involved in antigen uptake
and processing (Jiang et al., 1995; Mahnke et al., 2000;
Sallusto et al., 1995). Recently, many novel C-type lectins,
such as DCIR (Bates et al., 1999), Langerin (Valladeau et al.,
2000), DC-SIGN (Geijtenbeek et al., 2000), Dectin-1 (Ariizumi
et al., 2000b), and Dectin-2 (Ariizumi et al., 2000a), all type II
transmembrane proteins, have also been reported to be
expressed on DCs and Langerhans cells. Their contributions to
antigen recognition and processing are not clear, although
Langerin was shown to be an endocytic receptor (Valladeau
et al., 2000). Among them, MMR, DC-SIGN (Mitchell et al.,
2001), and Langerin were previously shown to be specific for
mannose. Other C-type lectins expressed on DCs do not seem
to recognize carbohydrates.
Mannose-specific recognition should be effective to incorporate
and to process glycoproteins with high-mannose type carbohydrate
chains expressed on pathogens and truncated or unprocessed
N-glycans expressed by defective and apoptotic cells.
However, none of these C-type lectins recognize galactose or
N-acetylgalactosamine, which also seem to be important
pathogenic substances. The macrophage galactose-type C-type
lectin (MGL) has a binding capacity to galactose and N-acetylgalactosamine as monosaccharides and is a type II transmembrane glycoprotein that contains a single carbohydrate
recognition domain. Murine MGL (mMGL) was originally
detected on tumoricidal peritoneal macrophages (Sato et al.,
1992) and human MGL (hMGL) was cloned from IL-2-treated
peripheral blood monocytes (Suzuki et al., 1996). The Mgl gene
consists of ten exons and has been mapped to mouse chromosome 11 (Tsuiji et al., 1999), and the MGL gene has been mapped
to human chromosome 17p13.2. The chromosome localization
of MGL is distinct from that of many C-type lectins, MMR,
DEC-205, DCIR, langerin, DC-SIGN, and Dectin-1, previously
claimed to be specific for DCs (Bates et al., 1999; Kato et al.,
1998; Soilleux et al., 2000; Valladeau et al., 2000). mMGL is
expressed on histiocytic macrophages but not on Langerhans
cells (Imai et al., 1995; Mizuochi et al., 1997). However, it has
not previously been examined whether or not DCs express MGL.
We have examined the expression of mMGL on bone marrow
(BM)–derived DCs by flow cytometry using mMGL-specific
monoclonal antibody (mAb) LOM-14. We found that only
immature DCs expressed high levels of mMGL and that such
immature cells were also able to bind and uptake soluble polyacrylamides with clusters of GalNAc residues. We hypothesize
that mMGL expressed on BM-derived immature DCs can
function as a recognition and internalization molecule with
distinct carbohydrate specificity from other DC lectins and
potentially participates in the presentation of antigens bearing
clusters of GalNAc.
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K. Denda-Nagai et al.
Results
Expression of mMGL on DCs
Surface phenotypes of BM-DCs
The expression of mMGL on the DC surface was examined by
flow cytometry using the specific mAb LOM-14 (Figure 1).
mMGL was not expressed on BM cells before incubation with
granulocyte-macrophage colony-stimulating factor (GM-CSF)
(data not shown). mAb LOM-14 binding was very low on the
majority of MHC class IIhigh cells (Figure 1). In contrast,
mMGL was detected by mAb LOM-14 on almost all MHC
class IIlow/moderate cells at significant levels (Figure 1). To assess
whether the surface expression of mMGL was regulated at the
level of transcription, the expression of mMGL transcripts was
examined by reverse transcriptase polymerase chain reaction
(RT-PCR) (Fig. 2). In contrast to the absence of mMGL on BM
cell surfaces, these cells expressed mMGL mRNA. The cell
surface expressions and mRNA levels of mMGL were elevated
at day 6. Immature DCs sorted for cell surface mMGL and
MHC class II from day 6 DCs showed high levels of mMGL
mRNA. Day 8 mature DCs sorted from day 8 LPS-treated DCs
for high levels of MHC class II exhibited low mMGL mRNA.
Surface phenotypes of BM-derived cells were examined by
two-color flow cytometry. BM-derived nonadherent cells
could be divided into three subpopulations according to their
expression levels of MHC class II, namely, MHC class II–, MHC
class IIlow/moderate, and MHC class IIhigh. The proportions of these
subpopulations in a given cell preparation were indicated by
staining with phycoerythrin (PE)-conjugated anti-MHC class II
antibody alone (Figure 1), and these proportions differed with
the duration of BM cell culture. On day 6, most cells (42.1%)
were MHC class II–, whereas 33.2% was MHC class IIlow/
moderate, and 10.5% was MHC class IIhigh (Figure 1A). On day 8
in the absence of lipopolysaccharide (LPS), there was a higher
percentage (28.2%) of MHC class IIhigh cells than on day 6
(Figure 1B), while if the cells were treated with LPS, 70.8% of
the cells was MHC class IIhigh on day 8 (Figure 1C). In all cases,
MHC class IIhigh cells coexpressed CD11c, DEC-205, CD40,
and CD86 and were thus considered to be mature DCs. MHC
class IIlow/moderate cells also expressed CD11c but not DEC-205,
CD40, or CD86. Judging from the time-dependent shift and the
effect of LPS, it is reasonable to speculate that the MHC class
IIlow/moderate population consists of immature DCs.
Binding of GalNAc polymers to DCs
To assess whether mMGL on DCs acts as a lectin, we
performed carbohydrate binding assays that were measured by
flow cytometry. α-GalNAc polymers, but not β-GlcNAc
Fig. 1. Surface phenotypes of BM-derived DCs at various stages of development and maturation. Nonadherent cells cultured with GM-CSF were collected and
analyzed by flow cytometry on day 6 (A), day 8 without LPS treatment (B), and day 8 after 24 h LPS treatment (C). Two-color flow cytometric analysis was
performed using antibodies tagged with FITC indicated below each pattern together with PE-labeled anti-mouse MHC class II (see Materials and methods). The
control was the staining pattern of PE-labeled anti-mouse MHC class II alone. When control rat or hamster antibodies were used, patterns were almost the same
(data not shown). Cells could be divided into three subpopulations according to MHC class II expression levels, and the percentage of each subpopulation is
indicated in the panels showing control staining patterns.
52
MGL on dendritic cells
Fig. 2. The expression of mMGL mRNA analyzed by RT-PCR. Lane 1, BM
cells; 2, day 6 DCs; 3, day 8 DCs without LPS treatment; 4, day 8 DCs after
LPS treatment for 24 h; 5, immature DCs sorted from day 6 DCs; 6, mature
DCs sorted from day 8 DCs after LPS treatment for 24 h.
polymers, bound to immature DCs but not to mature DCs and
BM cells in a Ca2+-dependent manner (Figure 3A and data not
shown). The 2D flow cytometric profiles indicated that the
degree of α-GalNAc polymer bindings exactly coincided with
the intensity of the binding of mAb LOM-14 specific for mMGL
(Figure 1, 3A). This finding was confirmed by three-color flow
cytometric analysis (Figure 3B). The binding of α-GalNAc
polymers was inhibited by the presence of 0.1 M GalNAc, but
not by 0.1 M Gal or GlcNAc (Figure 3C).
Internalization of GalNAc polymers by day 6 DCs
Internalization of bound fluorescein isothiocyanate (FITC)conjugated α-GalNAc or β-GlcNAc polymers by immature
DCs was examined on a flowcytometer. Ethylenediamine
tetra-acetic acid (EDTA) solution was applied at various time
points to remove α-GalNAc polymers at the cell surfaces, and
the polymers not removed by this treatment was considered to
be internalized. Increase in the fluorescence intensity after
incubation with polymers at 37°C indicated that polymers were
internalized and accumulated (Figure 4). FITC-GlcNAc polymers
were not internalized by day 6 DCs. By two-color flow cytometry
with MHC class II, it was also confirmed that the cells that
internalized α-GalNAc polymers were MHC class IIlow/moderate
(data not shown). To confirm that the internalization of
FITC-GalNAc polymers were mediated by mMGL, DCs were
incubated with FITC-GalNAc polymers in the presence of
anti-mMGL mAb LOM-14 or mAb LOM-8.7. mAb LOM-14
and mAb LOM-8.7 were previously reported as a nonblocking
and blocking antibodies, respectively (Kimura et al., 1995).
The binding pattern of mAb LOM-8.7 and mAb LOM-14 to DCs
were almost the same in two-color flow cytometric analysis with
MHC class II (data not shown). The internalization of FITCGalNAc polymers was partially but not statistically significant,
inhibited by both mAb LOM-14 and mAb LOM-8.7, although the
binding of FITC-GalNAc polymers did not seem to be inhibited
by either of these mAbs (Figure 5).
Day 6 DCs adherent to cover slips were pulsed with α-GalNAc
polymers for 1 h, and the localization of α-GalNAc polymers
was examined by confocal laser scanning microscopy. α-GalNAc
polymers colocalized with mMGL detected by mAb LOM-14
even after the polymers were internalized (Figure 6A). Furthermore, internalized α-GalNAc polymers seemed to colocalize
with intracellular populations of lysosome-associated membrane
protein-1 (LAMP-1) and with MHC class II (Figure 6B, C).
These results suggest that internalized α-GalNAc polymers are
transported to MHC class II compartments (MIICs).
Fig. 3. Binding of α-GalNAc polymers to BM-derived DCs. (A) Cell surface
binding of α-GalNAc and β-GlcNAc polymers was examined by flow
cytometry. Cells were incubated first with biotinylated α-GalNAc or
β-GlcNAc polymers, then with FITC-streptavidin, and subsequently with
PE-conjugated anti-MHC class II. PBS containing 10 mM EDTA, 0.1% BSA,
and 0.1% sodium azide was used as the incubation medium without Ca2+.
(B) Three-color flow cytometric analysis for α-GalNAc polymers, mMGL,
and MHC class II. Cells (day 6) were incubated with biotinylated α -GalNAc
polymers and mAb LOM-14, then tagged with streptavidin-RED670 and
FITC-conjugated anti-rat IgG individually, and subsequently with
PE-conjugated anti-MHC class II. (C) Inhibition of α-GalNAc polymer
binding to day 6 DCs (MHC class II–positive cells) by monosaccharides. Cells
were incubated with FITC-GalNAc polymers in the presence of indicated
monosaccharides. Mean fluorescence intensity (MFI) of MHC class II positive
cells is shown, and the results represent the mean ± SD of three independent
experiments.
Discussion
In the present study, we detected significant levels of mMGL
expression reactive with mAb LOM-14 on immature DCs. BM
cells and the majority of mature DCs did not seem to express
this molecule on their surfaces. The levels of mMGL mRNA
53
K. Denda-Nagai et al.
Fig. 5. Inhibition of binding and internalization of α-GalNAc polymers by day
6 MHC class II–positive DCs by anti-mMGL mAbs. The cells were incubated
with FITC-GalNAc polymers in the presence of indicated antibodies. MFI of
MHC class II positive cells is shown, and the results represent the mean ± SD
of four independent experiments. NRS: normal rat serum.
Fig. 4. Flow cytometric profiles showing time-dependent uptake of α-GalNAc
polymers by day 6 DCs. Uptake of α-GalNAc polymers was examined using
flow cytometry. Day 6 DCs were incubated with FITC-GalNAc polymers as
indicated in the figure. Dotted lines indicate the peak of the fluorescence
intensity resulted from the binding at 4°C.
determined by RT-PCR analysis were also high in immature
DCs and low in mature DCs. In our preliminary experiment,
mMGLlow-MHC class IIhigh cells could be generated after
culturing mMGL-positive cells sorted from day 6 DCs by
magnetic beads. We thus hypothesize that mMGL is only
transiently expressed on DCs that are developing and maturing,
during which stage they are capable of antigen uptake but do not
yet present MHC class II–peptide complexes. Although we could
not detect mMGL proteins in BM cells by cytostaining (data not
shown), mRNA corresponding to mMGL is detectable in BM
cells prior to the incubation with GM-CSF, which suggests that
the cell surface expression may be due to changes in translation
or intracellular transport of this molecule. Among other C-type
lectins known to be expressed on DCs, DEC-205 was shown to
be upregulated during the maturation (Figure 1), which was
consistent with a previous report (Inaba et al., 1995). Expression
of DC-SIGN and DCIR on both immature and mature human
DCs was previously reported (Bates et al., 1999; Geijtenbeek
et al., 2000). DCIR was also expressed on monocytes, a DC
precursor, whereas the level on mature DCs obtained by LPS
activation was slightly lower than that on immature DCs
(Bates et al., 1999). Therefore, mMGL expression on immature
DCs seems to be unique.
It has already been reported that many C-type lectins, such as
MMR, DEC-205, DCIR, Langerin, DC-SIGN, Dectin-1, and
54
Fig. 6. Intracellular localization of α-GalNAc polymers, mMGL, LAMP-1,
and MHC class II. Day 6 DCs were seeded on coverslips, incubated with
FITC-GalNAc polymers at 37°C for 1 h, fixed, and stained with anti-mMGL
(A), anti-LAMP-1 (B), or anti–MHC class II (C) antibodies.
MGL on dendritic cells
Dectin-2, were expressed on DCs. None of them recognize Gal
or GalNAc residues. Alterations in O-glycosylation such as an
increased expression of T (Gal-GalNAc-Thr/Ser) and Tn
(GalNAc-Thr/Ser) antigens are known to be associated with
malignant cells. These epitopes are potential targets for antitumor
immunity. It is suggested that terminal sialic acid residues of
glycoproteins are lost and Gal or GalNAc residues are
exposed on apoptotic cells (Savill, 1997). Structural alterations
in O-glycans are known to occur during the differentiation,
activation, and migration of T-lymphocytes (Baum et al.,
1995; Gillespie et al., 1993; Lowe, 2001; Reisner et al., 1976).
Therefore, cell surface receptors for truncated O-glycans
should play important roles in the immune system in the
protection from infection, antitumor immunity, and the clearance
of apoptotic cells.
α-GalNAc polymers were used to test whether cell surface
mMGL on DCs functions as a lectin, because mMGL and hMGL
were shown to have high affinity with clusters of O-linked GalNAc
residues (Iida et al., 1999;Yamamoto et al., 1994). α-GalNAc
polymers, but not β-GlcNAc polymers, bind to immature DCs
in a Ca2+-dependent and GalNAc-specific manner, but the
binding of α-GalNAc polymers was not inhibited by Gal. This
was probably due to differences in the affinity of mMGL
expressed on the surfaces of DCs toward these monosaccharides.
The mMGL expression profile clearly correlated with the
profile of α-GalNAc polymer binding in day 6 and day 8 DC
populations. Furthermore, flow cytometric analyses and
confocal microscopic studies indicated that cells strongly
reactive with α-GalNAc polymers expressed high levels of
mMGL. Therefore, the binding of α-GalNAc polymers is
highly likely to be mediated by mMGL although the presence
of another lectin expressed in parallel with mMGL remains to
be possible. It has been suggested by several observations that
mMGL can participate in carbohydrate binding at the cell
surfaces. First, macrophages elicited by thioglycolate treatment
are able to internalize glycoproteins through mMGL
(Kawakami et al., 1994). Second, CTLL-2 T lymphoma cells
transfected with mMGL cDNA acquire the ability to bind
galactose or lactose-conjugated microspheres (Hosoi et al.,
1998; Ichii et al., 1997). Third, K562 human erythroleukemia
cells transfected with hMGL are able to internalize α-GalNAc
polymers in an MGL-dependent manner (Fujita et al., unpublished
data). Recently, we have also found that hMGL expressed on
monocyte-derived immature DCs was involved in the uptake
of α-GalNAc polymers (Higashi et al., 2002).
Immature DCs that expressed high levels of mMGL bound
and internalized carbohydrate antigens. The binding of α-GalNAc
polymers was not inhibited anti-mMGL mAb LOM-14 or
LOM-8.7, although it has already been reported that mAb
LOM-8.7, but not LOM-14, blocked the binding of galactosylated poly-L-lysine to recombinant mMGL (Kimura et al.,
1995). On the other hand, the internalization of α-GalNAc
polymers was partially inhibited by both mAb LOM-14 and
mAb LOM-8.7. It has already been shown that mAb LOM-14
was internalized by immature DCs (data not shown). Furthermore, internalized α-GalNAc polymers colocalized with mAb
LOM-14-reactive mMGL as observed by confocal microsopy.
Therefore, it is strongly suggested that α-GalNAc polymers
were internalized with mMGL. However, taking into account
the fact that Gal and anti-mMGL blocking mAb LOM-8.7
could not inhibit the binding of α-GalNAc polymers, we
cannot exclude a possibility that mMGL on DCs functions in
accordance with another molecule having high affinity to
GalNAc polymers.
The internalization of mMGL was expected from the fact
that mMGL had tyrosine-based amino acid sequences (YENL)
involved in endocytosis in the cytoplasmic domain (Sato et al.,
1992). Macrophages elicited by thioglycolate were previously
shown to internalize glycoproteins through mMGL (Kawakami
et al., 1994). The antigens internalized by immature DCs were
known to enter the endocytic pathway and transported to early
endosomes, late endosomes, or lysosomes (Banchereau and
Steinman, 1998). In immature DCs, newly synthesized MHC
class II molecules accumulate in late endosomes or lysosomes
(MIICs). During the DC maturation, antigen processing and
peptide loading are known to occur in MIICs (Inaba et al.,
2000; Pierre et al., 1997; Turley et al., 2000). The MMR and
DEC-205 are involved in the antigen presentation. The MMR
mainly releases its ligand in early endosomes and recycles to
plasma membranes (Engering et al., 1997; Tan et al., 1997),
although some of them are also located in MIICs (Prigozy et al.,
1997). In contrast to the MMR, DEC-205 targets to MIICs and
is more efficient than the MMR in antigen presentation
(Mahnke et al., 2000). Thus, the delivery of antigens into
MIICs is considered important event in the antigen presentation.
To assess whether mMGL is involved in antigen presentation,
the fate of α-GalNAc polymers bound to the surface of immature
DCs was examined by confocal laser scanning microscopy.
α-GalNAc polymers were internalized and colocalized with
MGL, LAMP-1, and MHC class II. These results suggest that
mMGL is also capable of targeting antigens to MIICs and
presenting effectively. When DCs were cultured with α-GalNAc
polymers for a day in our preliminary experiment, their
maturation did not occur unless a maturation signal was
provided by LPS, judging from the expression levels of cell
surface markers, such as MHC class II, CD86, and DEC-205.
Therefore, glycan binding to mMGL alone does not seem to
provide a maturation signal.
In conclusion, we observed that mMGL, which is involved
in the recognition and internalization of glycoproteins, is
transiently expressed on BM-derived immature DCs. mMGL
should be useful as a novel marker for immature DCs.
Materials and methods
Mice
C57BL/6 mice (5–12 weeks old) were obtained from Charles River
Japan (Yokohama, Japan) and housed under SPF conditions.
Generation of BM-DCs
BM-DCs were generated as previously described with slight
modifications (Inaba et al., 1992). Briefly, BM cells were
collected and erythrocytes were lysed by ammonium chloride
treatment (day 1). Cells (1.0 × 106 cells/ml) were plated in 24-well
plates in 1 ml per well of RPMI1640 medium supplemented
with 10% fetal calf serum, 10 mM HEPES, 50 µM 2-ME,
100 U/ml penicillin, 100 µg/ml streptomycin, and 1000 U/ml
recombinant murine GM-CSF (Kirin Brewery, Takasaki, Japan).
On day 4, the top two-thirds of the media were removed and
replaced with fresh medium. On day 6, nonadherent cells were
collected and replated into fresh 24-well plates, and the next
55
K. Denda-Nagai et al.
day they were cultured with 1 µg/ml LPS (Bacto LPS B from
Escherichia coli 0111:B4, Difco, Detroit, MI) for 24 h.
Two-color flow cytometric analysis
Nonadherent cells in the culture wells were gently suspended
and used for flow cytometric analyses. Adherent cells were not
included. Approximately 70% of the cells plated at day 6 were
recovered at day 8 even in the presence of LPS. Cells (2–5 ×
105 cells) were incubated with hybridoma culture supernatants
or purified antibodies (0.5–5 µg/ml) for 30 min on ice. The
following antibodies were used to analyze the DC surface
phenotype. Hybridoma culture supernatants: mMGL (LOM-14,
rat IgG2b, 1/10 dilution) (Kimura et al., 1995), DEC-205
(NLDC-145, rat IgG2a, 1/2 dilution, ATCC), CD11c (N418,
hamster IgG, 1/10 dilution, ATCC), B220 (RA3-3A1/6.1, rat
IgM, 1/2 dilution, ATCC), and MHC class I (M1/42, rat IgG2a,
1/2 dilution, ATCC). Purified antibodies: CD40 (3.23, rat
IgG2a, 5 µg/ml, Immunotec Coulter, Marseilles, France), F4/80
(F4/80, rat IgG2b, 5 µg/ml, Dainippon Pharmaceutical), CD31
(MEC13.3, rat IgG2a, 2.5 µg/ml, BD Pharmingen, San Diego,
CA), and Ly-6G (RB6-8C5, rat IgG2b, 2.5 µg/ml, Southern
Biotechnology, Birmingham, AL). FITC-conjugated antibodies:
CD11b (M1/70.15, rat IgG2b, 0.5 µg/ml, Caltag, Burlingame,
CA) and CD14 (rmC5-2. rat IgG1, 2.5 µg/ml, BD Pharmingen).
Biotinylated antibody: CD86 (RMMP1, rat IgG2a, 0.5 µg/ml,
Caltag). For isotype controls, purified rat IgG (5 µg/ml, ICN,
Costa Mesa, CA), purified rat IgM (5 µg/ml, Zymed, South
San Francisco, CA), purified hamster IgG (1 µg/ml, ICN), and
PE-conjugated rat IgG2b (0.2 µg/ml, Immunotech Coulter)
were used. Primary rat and hamster antibodies were followed
by FITC-conjugated rabbit anti-rat IgG (1/200 dilution, Zymed)
and FITC-conjugated goat anti-hamster IgG (1/200 dilution,
Southern Biotechnology), respectively. Incubation with
biotinylated antibodies was followed by incubation with
FITC-labeled streptavidin (1/200 dilution, Zymed). Following
applications of the first and the second antibodies, the cells
were then stained with PE-conjugated anti-mouse MHC class
II (M5/114.15.2, 1/5000 dilution, rat IgG2b, BD Pharmingen).
Antibodies and reagents were diluted in phosphate buffered
saline (PBS) containing 0.1% bovine serum albumin (BSA),
0.1% sodium azide, and 2% normal goat serum. The cells were
rinsed twice with PBS containing 0.1% BSA and 0.1% sodium
azide at the end of each incubation. Samples were analyzed on
an EPICS XL flowcytometer (Beckman Coulter, Fullerton, CA).
Isolation of immature and mature DCs and RT-PCR analysis
Cells were incubated with anti-mMGL mAb LOM-14 for 30 min
on ice, with FITC-conjugated rabbit anti-rat IgG and then with
PE-conjugated anti-mouse MHC class II. These antibodies
were diluted in PBS containing 0.1% BSA and 2% normal goat
serum. MHC class IIlow/moderate and mMGL-positive cells were
sorted from day 6 DCs to obtain immature DCs and MHC class
IIhigh and mMGL–/low cells were sorted from day 8 LPS-treated
DCs to obtain mature DCs by an EPICS ELITE flowcytometer
(Beckman Coulter). The >90% purity was confirmed by flow
cytometric analysis on an EPICS XL. Total RNAs were
extracted by using Ultraspec RNA zol (BIOTECX, Houston,
TX), according to the manufacturer’s instructions. First-strand
cDNA synthesis was carried out using oligo (dT)12–18 and
Superscript II (Gibco BRL, Rockville, MD). The cDNA was
used as the template in PCR reactions using Ampli Taq Gold
56
polymerase (Perkin Elmer). PCR was performed with specific
primers for mMGL (bp 465–486 and 1239–1216). The condition
was 94°C for 30 s, 65°C for 30 s, and 72°C for 1 min 30 s for
40 and 45 cycles. Amplification of β-actin (bp 42–63 and
1047–1024) was used as a control for cDNA quantity. The
condition was 94°C for 30 s, 61°C for 30 s, and 72°C for 1 min
for 25 and 30 cycles. The PCR products were then separated on
1.5% agarose gels, stained with ethidium bromide, and visualized
with the Fluor-S image analyzer (Bio-Rad, Hercules, CA).
Carbohydrate binding and internalization assessed by flow
cytometric analysis
Cell surface binding of biotinylated α-N-acetylgalactosaminide
(α-GalNAc) or β-N-acetylglucosaminide (β-GlcNAc) conjugated
with polyacrylamide carriers (GlycoTech, Rockville, MD) was
examined using flow cytometry. Cells were incubated with
α-GalNAc or β-GlcNAc polymers (10 µg/ml) for 30 min on
ice. Subsequently, the cells were incubated with FITC-streptavidin
(10 µg/ml) and then PE-conjugated anti-mouse MHC class II.
Reagents were diluted in Dulbecco’s modified PBS (DPBS,
contains 0.91 mM CaCl2, 0.49 mM MgCl2) supplemented with
0.1% BSA and 0.1% sodium azide. The cells were rinsed twice
with this buffer at the end of each incubation. PBS containing
10 mM EDTA, 0.1% BSA, and 0.1% sodium azide was used as the
incubation medium devoid of Ca2+. To confirm the carbohydrate
specific binding, cells were preincubated with 0.1 M GalNAc,
GlcNAc, or Gal for 5 min on ice and then incubated with
FITC-conjugated α-GalNAc polymers (GlycoTech) in the
presence of monosaccharides. In the inhibition assay data was
analyzed after gating on MHC class II expression. In three-color
flow cytometric analysis, α-GalNAc polymers were monitored
by streptavidin-RED670 (Gibco BRL).
For the internalization assays, FITC-conjugated α-GalNAc
or β-GlcNAc polymers (GlycoTech) were used as model
compounds for glycosylated antigens. Day 6 DCs were incubated
with FITC-α-GalNAc or β-GlcNAc polymers (10 µg/ml) in
RPMI 1640 medium supplemented with 10% fetal calf serum
at 37 °C for various time periods and washed with PBS
containing 10 mM EDTA, 0.1% BSA, and 0.1% sodium azide
to remove the polymers attaching on the cell surfaces. In the
inhibition assays DCs were incubated in the presence of
anti-mMGL mMAb LOM-14 (partially purified, diluted to
equivalent to 1/10 of hybridoma culture supernatants), antimMGL mAb LOM-8.7 (rat IgG2a, partially purified, diluted to
equivalent to 1/2 of hybridoma culture supernatants) (Kimura
et al., 1995), or normal rat serum (1/40 dilution). Data were
analyzed after gating on MHC class II expression.
Confocal laser scanning microscopy
Day 6 DCs (5 × 105 cells) were plated on poly-L-lysine-coated
coverslips (Iwaki, Tokyo) and allowed to adhere for 20 min at
37°C. The cells were incubated with 10 µg/ml FITC-GalNAc
polymers for 1 h at 37°C, washed with DPBS, and then fixed
with acetone for 30 s at room temperature. Nonspecific bindings
were blocked with a blocking solution (2% normal goat serum
and 3% BSA in DPBS) for 10 min, and DCs were incubated
with anti-mMGL (mAb LOM-14, 1/10 dilution of hybridoma
culture supernatant in the blocking solution), anti-LAMP-1
(mAb 1D4B, rat IgG2b, 1 µg/ml, BD Pharmingen), or antiMHC class II (M5/114.15.2, 1/10 dilution of hybridoma
culture supernatants, ATCC) for 40 min. After fixation in
MGL on dendritic cells
4% paraformaldehyde dissoloved in 0.1 M sodium phosphate
(pH 7.0) containing 0.91 mM CaCl2, DCs were incubated with
biotinylated mouse anti-rat κ/λ mAb (1/100 dilution, Sigma,
St. Louis, MO) for 30 min and with Texas Red–avidin D (1/100
dilution, Vector, Burlingame, CA) for 30 min. The coverslips
were mounted in PermaFluor aqueous mounting medium
(Shandon, Pittsburgh, PA) on glass slides and observed on a
confocal microscope (MRC-1024; Bio-Rad).
Acknowledgments
We thank Dr. Kayo Inaba for advice in generating BM-DCs,
Ms. Chizu Hiraiwa for assistance in preparing this manuscript,
and Kirin Brewery for supplying rGM-CSF. This work was
supported by grants-in-aid from the Ministry of Education,
Culture, Sports, Science and Technology of Japan (11557180,
11672162, and 12307054) the Research Association for
Biotechnology; the Program for Promotion of Basic Research
Activities for Innovative Biosciences; and the Cosmetology
Research Foundation.
Abbreviations
α-GalNAc polymers, α-N-acetylgalactosaminides conjugated
to soluble polyacrylamide; BM, bone marrow; BSA, bovine
serum albumin; DCs, dendritic cells; DPBS, Dulbecco’s modified
PBS; EDTA, ethylenediamine tetra-acetic acid; FITC, fluorescein
isothiocyanate; GM-CSF, granulocyte-macrophage colonystimulating factor; hMGL, human MGL; LAMP-1, lysosomeassociated membrane protein-1; LPS, lipopolysaccharide;
mAb, monoclonal antibody; MFI, mean fluorescence intensity;
MGL, macrophage galactose-type C-type lectin; MHC, major
histocompatibility complex; MIIC, MHC class II compartment; mMGL, murine MGL; MMR, macrophage mannose
receptor; PBS, phosphate buffered saline; PE, phycoerythrin;
RT-PCR, reverse transcriptase polymerase chain reaction.
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