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Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan;
Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Osaka, Japan;
Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston MA 02115; and
§
Mammalian Genetics Laboratory, Advanced BioScience Laboratories-Basic Research Program, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD 21702
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-6, and IFN-
in wild-type
macrophages. In contrast, NF-IL6-deficient macrophages displayed a much
lower level of Mincle mRNA induction following treatment with these
inflammatory reagents. The mouse Mincle proximal promoter region
contains an indispensable NF-IL6 binding element, demonstrating that
Mincle is a direct target of NF-IL6. The Mincle gene
locus was mapped at 0.6 centiMorgans proximal to CD4 on
mouse chromosome 6. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Here, we report a novel C-type lectin, macrophage-inducible C-type
lectin (Mincle), which is a transcriptional target of NF-IL6 in
peritoneal macrophages (PM
). NF-IL6 was initially identified as a
nuclear factor that binds to the IL-1 responsive element in the IL-6
gene (6). Cloned NF-IL6 exhibits homology with
CCAAT/enhancer binding protein (C/EBP), a member of the basic leucine
zipper family of transcription factors (7). NF-IL6 has
been reported by other groups under the names AGP/EBP, LAP, IL-6DBP,
rNFIL-6, C/EBPß, and CRP2 (8, 9, 10, 11, 12, 13). NF-IL6 exhibits a low
transcriptional activity unless activated by inflammatory stimuli,
which induce phosphorylation of NF-IL6 and augment its transcriptional
activity (14, 15, 16). NF-IL6 appears to play an important
role in activated macrophages (17). NF-IL6-deficient
(-/-) mice are extremely susceptible to infections with
microorganisms such as Listeria monocytogenes and
Candida albicans (18, 19). PM from NF-IL6
(-/-) mice were defective in intracellular killing of L.
monocytogenes and displayed impaired tumoricidal and tumoristatic
activity. The macrophages used for these studies were able to produce
normal amounts of NO, which is thought to play an important role in the
elimination of intracellular bacteria and parasites, thus suggesting
that a NO-independent, NF-IL6-dependent pathway may be involved in
Listeria killing and tumoricidal activity
(18).
Overexpression of NF-IL6 protein have established that IL-6, macrophage
chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1
(MIP-1), MIP-1ß, osteopontin, CD14, and lysozyme are downstream
genes of NF-IL6 in several hemopoietic cell lines
(20, 21, 22). However, expressions of these genes by NF-IL6
(-/-) macrophages are unexpectedly comparable to those observed by
wild-type (WT) macrophages probably because other C/EBP families, such
as C/EBP
, could in part compensate for the lack of NF-IL6 in vivo
(23). So far, the transcriptional targets of NF-IL6 have
been unsuccessfully defined except G-CSF in macrophages
(18). Identification of other targets is essential to
clarify the NF-IL6 function in more detail.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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LPS (Escherichia coli O55: B5) and thioglycolate
broth (Brewer’s formula) were purchased by Difco (Detroit MI).
IFN-, GM-CSF, IL-6, and TNF- were obtained from Genzyme
(Cambridge, MA). Restriction and DNA modification enzymes were products
of Toyobo (Otsu, Japan). [-32P]dCTP
(3000Ci/mmol) and [-32P]ATP (3000Ci/mmol)
were obtained from Amersham Pharmacia Biotech (Little Chalfont,
U.K.).
Mice
The NF-IL6 (-/-) mice generated by homologous recombination have been described previously (18). NF-IL6 (-/-) and WT mice were littermates derived from intercrossing hemizygous females and males.
Preparation of PM
PM were collected by peritoneal lavage with PBS at 4 days
after i.p. injecting 2 ml of sterile thioglycolate into 8- to 12 wk-old
mice. PM were plated on 10-cm plastic dishes at 2.5 x
106 cells/dish in macrophage culture medium
(MCM). MCM consists of DMEM (Nissui, Tokyo, Japan) supplemented with
10% FBS, 2 mM L-glutamine, 50 U/ml of GM-CSF, 100 µg/ml
streptomycin, and 10 U/ml penicillin G. After 2 h incubation to
allow for adherence of macrophages, the dishes were washed vigorously
to remove nonadherent cells. Fresh MCM was added on day 2, and fresh
MCM without GM-CSF was added on day 4 of culture. PM were treated
with appropriate reagents on day 5 for harvesting RNA.
Cell lines
Murine B cell leukemia (BCL-1), myeloma cells (MOPC 315), thymoma cells (EL-4), and human monocytic leukemia cells (THP-1) were cultured in RPMI 1640 medium (Life Technologies, Gaithersburg, MD) containing 10% FBS, 100 µg/ml streptomycin, and 10 U/ml penicillin G. Murine NK cells (5E3) were cultured in RPMI 1640 supplemented with 10% FBS and 500 U/ml of IL-2 (Genzyme). NF-IL6 M1 cells were cultured in MEM (Life Technologies) supplemented with twice the normal concentration of amino acids and vitamins, 10% FBS, 400 µg/ml of geneticin (Life Technologies), and 50 µg/ml of hygromycin B (Boehringer Mannheim, Mannheim, Germany) (21). Murine macrophage cells (RAW264.7), embryonic fibroblast cells (NIH3T3), and human embryonic kidney cells (293T) were cultured in DMEM supplemented with 10% FBS, 100 µg/ml streptomycin, and 10 U/ml penicillin G.
Construction of subtracted cDNA library and differential screening
WT and NF-IL6 (-/-) PM at about 80% confluence were
treated with 100 ng/ml LPS and 100 U/ml IFN- for 16 h. Total
RNA was prepared by RNAeasy kit (Qiagen, Hilden, Germany) following
poly(A)+ RNA selection using Oligotex-dT30 Latex
beads (TaKaRa, Otsu, Japan). Then all procedures were performed
according to PCR-select cDNA subtraction kit (Clontech, Palo Alto,
CA).
Rapid amplification of cDNA ends (RACE)
RACE (5' and 3') were performed using a Marathon cDNA
amplification kit (Clontech). Using 1 µg of
poly(A)+ RNA from WT PM stimulated with 100
ng/ml LPS and 100 U/ml IFN-, a library of adaptor-ligated
double-stranded cDNA was constructed as described by the
manufacturer’s instruction. To obtain a full-length cDNA of mouse
Mincle (mMincle), an antisense primer, 270F
(5'-GAGAAAATGGGGCTCCAGGAAGAGTG-3') and a sense primer, 92R
(5'-CCCTAAAGGAACCTTCAGCAGCAGTC-3') were designed from the sequence
of the cDNA fragment obtained by subtraction cloning for 5'-RACE and
3'-RACE, respectively.
To obtain human Mincle (hMincle), cDNA synthesized from THP-1 cells mRNA extracted after LPS stimulation (5 µg/ml) was subjected to PCR using degenerate primers. The 5' primer (5'-GTGAGGCATCAGGTbTCAG-3') and 3' primer (5'-DATRTTGTTGGGYTCNCC-3') were designed to cover a portion of mMincle CRD. PCR conditions were 94°C for 30 s, followed by 35 cycles of 94°C for 5 s and 50°C for 30 s. The resulting 311-bp PCR product was subcloned into pT7Blue T vector (Novagen, Madison, WI) and sequenced. An antisense primer (5'-CCCAGTTCAATGGACAATTCTTG-3') and a sense primer (5'-ACGGCACACCTTTGACAAAGTCTCTG-3') were designed from the sequence and 5'- and 3'- RACE were performed using an adaptor-ligated double-stranded cDNA prepared from LPS-stimulated THP-1 cells.
Northern blot analysis
Total RNA (5 µg/lane) was separated on 1.0% agarose gels
containing 6.0% formaldehyde. After transfer to a
HybondN+ membrane (Amersham Pharmacia Biotech),
hybridization and wash were performed as described previously
(21). RsaI-RsaI cDNA fragment of
mMincle (nucleotide 1188–1404), SphI-PstI cDNA
fragment of mouse NF-IL6 (X62600; nucleotide 135–897), cDNA fragment
of mouse MIP-2 (X53798; nucleotide 149–840), and cDNA fragment of
mouse G3PDH (M32599; nucleotide 566-1017) were used as probes. cDNA
fragments were radiolabeled with [-32P]dCTP
(3000 Ci/mmol) by use of the Megaprime DNA labeling system (Amersham
Pharmacia Biotech).
Interspecific mouse back-cross mapping
Interspecific back-cross progeny were generated by mating
(C57BL/6J x Mus spretus)F1
females and C57BL/6J males as described (24). A total of
205 N2 mice were used to map the
Mincle locus. DNA isolation, restriction enzyme digestion,
agarose gel electrophoresis, Southern blot transfer, and hybridization
were performed essentially as described (25). All blots
were prepared with HybondN+ membrane. The mMincle
cDNA fragment (nucleotides 1549–2517) was labeled with
[32P]dCTP using a nick translation labeling
kit (Boehringer Mannheim); washing was done to a final stringency
of 1.0x SSCP, 0.1% SDS, 65°C. A fragment of 3.7 kb was
detected in BamHI-digested C57BL/6J DNA and a fragment of
6.5 kb was detected in BamHI-digested M.
spretus DNA. The presence or absence of the 6.5-kb
BamHI M. spretus-specific fragment was followed
in back-cross mice.
A description of the probes and RFLPs for the loci linked to Mincle including Atp6e, Slc2a3, and Cd4 has been reported previously (26). Recombination distances were calculated using Map Manager, version 2.6.5 (http://mcbio.med.buffalo.edu/mapmgr.html). Gene order was determined by minimizing the number of recombination events required to explain the allele distribution patterns.
Flow cytometric analysis
The mMincle expression vector was constructed in pcDNA3.1(+) (Invitrogen, Carlsbad, CA). To construct pcDNA3.1(+)-mMincleFlag, forward and reverse primers were devised to introduce an optimal Kozak consensus sequence and Flag epitope (DYKDDDDK), respectively. The forward primer sequence was 5'-GGTCGACCACCATGAATTCAACCAAATCG-3' and the reverse primer sequence was 5'-CTCACTTGTCATCGTCGTCCTTGTAGTCCAGAGGACTTAT-3'. PCR was performed using double-stranded cDNA generated in the course of RACE as template. Amplified product was verified by sequencing after subcloned into pT7Blue T vector and excised by Sal I digestion. Then mMincleFlag cDNA fragment was ligated to the XhoI site of pcDNA3.1(+) with correct orientation. The day before transfection, 293T cells were seeded on a 6-well plate at 2.0 x 105 cells/well. Four micrograms of pcDNA3.1(+)-mMincleFlag or pcDNA3.1 empty vector was transiently transfected by calcium-phosphate precipitation method. Cells were freed from culture plates using 0.02% EDTA in PBS at 48 h following transfection and washed in flow cytometry buffer (PBS with 2% FBS and 0.1% NaN3). Cells were incubated for 20 min on ice with 15 µg/ml biotin-conjugated anti-Flag M2 mAb (BioM2; Sigma-Aldrich, St. Louis, MO), washed in flow cytometry buffer, and labeled with 5 µg/ml FITC-streptavidin (PharMingen, San Diego, CA). Control consisted of cells treated with FITC-streptavidin alone. After a final wash in flow cytometry buffer, mMincle-Flag expression was analyzed using FACScalibur using CellQuest software (Becton Dickinson, Lincoln Park, NJ).
Western blot analysis
The day before transfection, 293T cells were seeded on a 100-mm plate at 2.0 x 106 cells. Twenty micrograms of pcDNA3.1(+)-mMincleFlag or pcDNA3.1 empty vector was transiently transfected by calcium-phosphate precipitation method. Cells were lysed with lysis buffer containing 0.5% Nonidet P-40, 150 mM NaCl, 1 mM EDTA, and 10 mM Tris-HCl, pH 7.4. Cell lysates were immunoprecipitated with protein-G Sepharose (Amersham Pharmacia Biotech) together with 10 µg/ml anti-Flag M2 mAb (Sigma-Aldrich). Immunoprecipitates were washed four times with lysis buffer and suspended with Laemmli sample buffer. After boiling for 5 min, samples were separated on a gradient (10–20%) SDS-polyacrylamide gel and electrically transferred to a nitrocellulose membrane. The membrane was incubated with BioM2 mAb, subsequently treated with HRP-conjugated streptavidin (Genzyme), and then analyzed for immunoreactivity by the enhanced chemiluminescence detection system (DuPont, Boston, MA).
Isolation of mMincle 5'-flanking region and reporter gene assays
A 129/Sv mouse liver genomic DNA library in the 
Fix II vector
was purchased from Stratagene (La Jolla, CA). Approximately 1 x
106 plaques were screened with the
32P-labeled EcoRI- EcoRI
cDNA fragment of mMincle (nucleotides 126–496). Positive plaques from
the genomic library were enriched after two further rounds of
screening. DNA from two independent clones was purified using the
Wizard prep kit (Promega, Madison, WI). Digestion with several
restriction enzymes revealed that these two clones contain the
identical sequence. Following digestion with BamHI and
SalI, five resulting fragments (5.4 kb, 5.1 kb, 3.2 kb, 0.8
kb, and 0.6 kb) were subcloned into pBluescript KS (+) and sequenced.
The 5.1kb BamHI-BamHI fragment containing exon I
and the 5'-flanking region was designated pBS/Bam1-8 and employed for
promoter-luciferase construction. Appropriate 5' primers and a common
3' primer were synthesized to amplify the promoter regions
of mMincle. The following primers were used to
generate pGL3-1783/+69, pGL3-1190/+69, pGL3-240/+69, and pGL3-61/+69;
-1783 (5'-CGACGCGTGGTTTGCAGCCCCATAGGAG-3'), -1190
(5'-CGACGCGTATGATGGCACACCATGATAG-3'), -240
(5'-CGACGCGTAAATCGGGACCAAGTTAGAC-3'), -61
(5'-CGACGCGTCAAGAGAGGAAATTCTGAC-3'), and +69
(5'-GAAGATCTCCCCTGGAAAGTGAGTCTTG-3'). Amplified products
were subcloned into pT7Blue T vector and sequenced for
confirmation. The inserted fragments were cut out with Mlu I
and BglII digestion and ligated to pGL3 basic vector
(Promega) at the same restriction sites. To create pGL3-166/+69,
pGL3-240/+69 was digested with Mlu I and Aat I,
blunt-ended, and re-ligated. NF-IL6 binding site mutation was generated
by Quick Change site-directed mutagenesis kit (Stratagene). The
mutagenic primer (with altered nucleotides underlined) was
5'-CCTTGTCCTTGTGCCCCAGAGGAAATTCTG-3'. The mutated
construct was confirmed by sequencing. Transient transfection into
NF-IL6 M1 cells and luciferease assay were done as described previously
(21).
Primer extension analysis
Primer extension was performed as described (27).
Briefly, an oligonucleotide primer complementary to nucleotides 81–112
of Mincle cDNA was synthesized and end-labeled with
[-32P]ATP and T4 polynucleotide kinase. Ten
micrograms of total RNA from LPS-stimulated PM was hybridized to
104 cpm of the labeled oligonucleotides in 10 mM
PIPES, pH 6.4, 1 mM EDTA, pH 8.0, and 0.4 M NaCl at 30°C for 16
h. Following ethanol precipitation, the samples were dissolved in 20
µl reaction buffer containing 50 mM Tris/HCl, pH 8.3, 10 mM
MgCl2, 1 mM DTT, 75 mM KCl, 1 mM dNTPs, and 20 U
RNase inhibitor. Reverse transcription was performed at 42°C for 30
min by adding 200 U Superscript II (Life Technologies). The extension
products were ethanol precipitated and analyzed on 6% polyacrylamide 7
M urea sequencing gels. A sequence reaction was set up separately with
the same nonradiolabeled primer, using the template of genomic clone
pBS/Bam1-8, which covered the mMincle 5'-flanking region, and was run
in parallel with the extension products on the same sequencing
gel.
EMSA
The following single-stranded oligonucleotides were
synthesized: P1 (position -76 to -45 of the mMincle promoter),
5'-CCTTGTCCTTGTGCAAGAGAGGAAATTCTG-3' and
5'-GTCAGAATTTCCTCTCTTGCACAAGGACAAGG-3'; mP1,
5'-CCTTGTCCTTGTGCCCCAGAGGAAATTCTG-3' and
5'-GTCAGAATTTCCTCTGGGGCACAAGGACAAGG-3'; IL-6 (position
-165 to -138 of the human IL-6 promoter),
5'-GGACGTCACATTGCACAATCTTAATAAT-3' and
5'-ATTATTAAGATTGTGCAATGTGACGTCC-3'. Underlined nucleotides
represent the mutant sequences. Complementary DNA oligonucleotides
were annealed by heating in a buffer containing 20 mM Tris-HCl, pH 7.5,
10 mM MgCl2, 50 mM NaCl at 75°C for
5 min and cooling at room temperature. Probes were then labeled by
filling in with [-32P]dCTP using a Klenow
fragment. NF-IL6 M1 nuclear extracts were prepared as described
previously (21). pGEX-NF-IL6 plasmid inserting human
NF-IL6 (amino acids 24–345; Ref. 7) downstream of the GST
gene in pGEX-4T-2 (Amersham Pharmacia Biotech) is a generous gift from
S. Hashimoto. GST protein and GST-NF-IL6 fusion protein were expressed
in E. coli BL21 and purified by incubation with
glutathione-coupled Sepharose beads (Amersham Pharmacia Biotech).
Protein concentration was determined by BCA protein assay reagent
(Pierce, Rockford, IL). Nuclear extracts (6 µg) or GST fusion
proteins (100 ng) were incubated for 20 min at room temperature with
1 x 104 cpm of the labeled DNA probe in 25
µl of binding buffer containing 10 mM HEPES-KOH, pH 7.8, 50 mM KCl, 1
mM EDTA, pH 8.0, 5 mM MgCl2, 10% glycerol, and 3
µg of poly(dI-dC) (Amersham Pharmacia Biotech). Competition assays
were conducted in the same manner, except that the above reaction
mixture was preincubated with a 100-fold molar excess of unlabeled
competitor oligonucleotides for 30 min at 4°C before the addition of
the labeled probe. Supershift assays were performed using 200 ng of
anti-C/EBPß Ab (C-19; Santa Cruz Biotechnology, Santa Cruz, CA)
and preincubated with the above reaction mixture at 4°C for 30 min
before the addition of the labeled probe. Samples were loaded on native
5% polyacrylamide gels, and electrophoresis was conducted at 30 mA in
25 mM Tris, pH 8.5, 190 mM glycine, and 1 mM EDTA. Gels were
subsequently dried for autoradiography.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We used the subtraction cloning method to compare the differences
of mRNA expression profiles between WT and NF-IL6 (-/-) PM. WT and
NF-IL6 (-/-) PM were stimulated for 16 h with 100 ng/ml LPS
and 100 U/ml IFN-. Tester and driver cDNAs were synthesized using
poly(A)+ RNA extracted from WT and NF-IL6 (-/-)
PM, respectively. Subtractive hybridization was performed to
concentrate cDNA species preferentially existing in tester cDNA. Two
independent subtractive libraries were constructed to reduce false
positives owing to individual differences. Differential hybridization
was performed against 1000 colonies for each library, and positive
clones were sequenced. Among these positive clones, commonly isolated
ones from both libraries were subjected to further study. Comparing
with WT PM, we identified four cDNAs whose expressions were
dramatically reduced in NF-IL6 (-/-) PM (M.M. and S.A.,
unpublished observations). One of these cDNAs was novel and designated
macrophage-inducible C-type lectin (Mincle) based on its expression
characteristics and primary structure of the encoded protein as
described below.
Mincle expression was assessed by Northern blotting in PM following
stimulation with LPS. Mincle mRNA expression was hardly detected in
untreated PM, but appeared within 2 h following LPS treatment
(Fig. 1
A). Three mRNA species
hybridized with this probe. The shortest mRNA (1.7 kb) is found to be
most abundant. Mincle mRNA expression sustained for at least 16 h
after LPS stimulation in WT PM. The levels of NF-IL6 mRNA were
elevated within 30 min following LPS treatment before the induction of
Mincle mRNA in WT PM (Fig. 1A). Although NF-IL6
(-/-) PM induced expression of Mincle mRNA by 2 h following
LPS stimulation, the expression levels were much lower than WT ones.
Comparable levels of MIP-2 mRNA in both cell types indicate that NF-IL6
(-/-) cells respond efficiently to LPS stimulation. Next, we examined
Mincle mRNA expression in WT and NF-IL6 (-/-) PM after various
inflammatory stimuli (Fig. 1B). Following 4 h
stimulation with IFN-, IL-6, TNF-, and LPS, total RNAs were
extracted and Northern blotting was performed using Mincle-specific
probe. A strong Mincle mRNA induction was observed in WT PM
following treatment with IFN-, IL-6, TNF-, or LPS (Fig. 1B). In contrast, much lower levels of expression were
detected in NF-IL6 (-/-) PM in response to these stimuli. IL-1ß,
phorbol myristate acetate, or ionomycin treatment could not induce
Mincle mRNA effectively both in WT and NF-IL6 (-/-) PM
(data not shown). Although we used thioglycolate-elicited PM that
were subsequently expanded with GM-CSF in this experiment, the similar
results were obtained from resident PM and thioglycolate-elicited
PM cultured without GM-CSF (data not shown). These results indicate
that several inflammatory stimuli strongly induce Mincle mRNA
expression in an NF-IL6-dependent manner. Because LPS stimulation
induced the strongest Mincle expression in PM, we tested Mincle
expression in various cell lines after LPS treatment (Fig. 1C). Mincle expression was observed in macrophage RAW264.7
cells and dramatically augmented after LPS stimulation. A low level of
expression was also detected in M1 myeloblastic leukemia cells after
stimulation with LPS. However, other cell lines BCL-1 (mature B cell),
MOPC (myeloma), 5E3 (NK cell), EL4 (thymoma), and NIH3T3 cells
(embryonic fibroblast) could not express any detectable levels of
Mincle mRNA. Furthermore, Mincle mRNA was undetectable in brain, heart,
lung, spleen, kidney, skeletal muscle, and testis (data not shown),
suggesting that it may display macrophage-restricted expression.
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24.4 kDa. This polypeptide was devoid of
NH2-terminal hydrophobic signal peptide but
contained a transmembrane domain of 24 amino acids predicted by
hydrophobicity analysis (data not shown) (28). These
features on the primary structure of Mincle indicated that it is a type
II integral membrane protein and is therefore composed of a
NH2-terminal cytoplasmic domain of 21 residues
and extracellular COOH-terminal domain of 169 aa (Fig. 2). We also identified a human homologue
by a combination of degenerative PCR and RACE techniques using
LPS-stimulated THP-1 cell cDNA and aligned it with mMincle (Fig. 2). We
have cloned 2.2- and 1.0-kb hMincle cDNAs (data not shown), both of
which include a single open reading frame of 657 bp encoding a
polypeptide of 219 aa. Sequence comparison of hMincle with mMincle
revealed overall identity of 67% and similarity of 85% (Fig. 2).
mMincle has a putative N-linked glycosylation site at
Asp107, while hMincle has two sites at
Asp62 and Asp107. Two
potential phosphorylation sites for protein kinase C
(Ser3 and Thr12) were found
within the cytoplasmic tail of both mouse and human Mincle (Fig. 2).
These sites are compatible with protein kinase C phosphorylation motif,
Ser/Thr-X-Arg/Lys.
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). The sequence of rat mannose binding
lectin A (rMBL-A) was included because it has been analyzed in detail
(30, 31, 32, 33). The alignment showed that the putative sequence
is 44, 40, 38, 36, and 31% identical with the CRDs of mMCL, mCD23,
mASGR-1, mM-ASGP-BP, and rMBL-A, respectively. In addition, it
indicated that apart from Asp153 and
Gln189, mMincle protein shares 12 of the 14
invariant and 18 of the 18 highly conserved amino acids used to define
C-type lectins (Fig. 3) (34). mMincle protein also
conserved all amino acids required for calcium cation association
within the rMBL-A CRD. Taken together, these observations suggested
that Mincle is indeed a C-type lectin.
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Chromosomal localization of the mMincle gene
The mouse chromosomal location of Mincle was determined
by interspecific back-cross analysis using progeny derived from matings
of [(C57BL/6J x M. spretus)F1
x C57BL/6J] mice. This interspecific back-cross mapping panel has
been typed for over 2700 loci that are well distributed among all the
autosomes as well as the X chromosome (24). C57BL/6J and
M. spretus DNAs were digested with several enzymes and
analyzed by Southern blot hybridization for informative RFLPs using a
mouse Mincle cDNA probe. The 6.5-kb BamHI
M. spretus RFLP (see Materials and Methods) was
used to follow the segregation of the Mincle locus in
back-cross mice. The mapping results indicated that Mincle
is located in the distal region of mouse chromosome 6 linked to
Atp6e, Slc2a3, and Cd4. Although 95 mice were
analyzed for every marker and are shown in the segregation analysis
(Fig. 4), up to 167 mice were typed for
some pairs of markers. Each locus was analyzed in pairwise combinations
for recombination frequencies using the additional data. The ratios of
the total number of mice exhibiting recombinant chromosomes to the
total number of mice analyzed for each pair of loci and the most likely
gene order are: centromere Atp6e (2/110), Slc2a3
(1/136), Mincle (1/167), Cd4. The recombination
frequencies (expressed as genetic distances in centiMorgans (cM)
± SE) are Atp6e (1.8 ± 1.3 cM), Slc2a3
(0.7 ± 0.7 cM), Mincle (0.6 ± 0.6 cM),
Cd4.
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The distal region of mouse chromosome 6 shares regions of homology with
human chromosomes 22 and 12 (summarized in Fig. 4
), suggesting that the
human homologue of Mincle will map to one of these two
chromosomes as well.
Surface expression of mMincle protein
The amino acid sequence suggests that Mincle is a type II integral
membrane protein as described above. To confirm the surface expression
of Mincle protein, a carboxyl-terminal Flag-tagged mMincle cDNA was
transiently transfected into 293T cells and mMincleFlag protein was
detected by flow cytometric analysis using anti-Flag M2 mAb without
permeabilizing cells. Staining of mMincleFlag-transfected cells with
the M2 mAb revealed strong surface expression of mMincleFlag protein
(Fig. 5A, left).
Mock-transfected cells showed no actual staining with the M2 mAb (Fig. 5A, right). Simultaneously, mMincleFlag protein expression
was detected by Western blot analysis (Fig. 5B).
mMincleFlag-transfected or mock-transfected 293T cells were
immunoprecipitated and blotted with anti-Flag M2 mAb. The observed
mMincleFlag protein corresponded to a molecular mass of 35 kDa,
although the calculated molecular mass of mMincleFlag is 25.4 kDa. This
result suggests that mMincle protein extensively acquired
posttranslational modifications.
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To determine the transcription initiation site of the mMincle
gene, we performed a primer extension analysis using a primer
corresponding to nucleotides 81–112 of the cDNA (Fig. 6). After hybridization with total RNA
from LPS-stimulated PM, the primer was extended with reverse
transcriptase. Identical reactions were conducted side by side with
yeast tRNA as negative control. The extension products are displayed
alongside the mMincle antisense sequence generated with the same
oligonucleotide primer (Fig. 6). We identified a single predominant
transcription initiation site, corresponding to 125 bases upstream of
the translation start codon. The nucleotide at this side is C, which we
tentatively assigned +1 as the transcriptional initiation
site.
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A mouse genomic DNA library was screened with the
32P-labeled EcoRI- EcoRI
cDNA fragment of mMincle (nucleotides 126–496). Sequencing of an
isolated clone revealed that it included segments of the 5'-flanking
region and the first five exons. The 1.8-kb promoter region of the
mMincle gene was completely sequenced from both strands (Fig. 7). Computer-assisted search using the
TFSEARCH program (36) revealed a number of potential
binding sites for various transcription factors. The location of a
select few of these thought to be potentially relevant to the
regulation of this gene by LPS are shown. These include motifs that may
bind NF-IL6, NF-
B, AP-1, and c-Ets. We found putative binding motifs
for NF-IL6 at positions -1222 to -1210, -1108 to -1095, -929 to
-917, -632 to -619, and -69 to -56. A canonical TATA box is
located 29–24 bp upstream of the transcription initiation
site.
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To examine whether NF-IL6 could trans-activate the
mMincle promoter, we constructed a series of the mMincle
promoter-luciferase plasmids with various 5' deletions. A 1852-bp
fragment, including 1783 bp of the 5'-flanking region and 69 bp of the
5'-noncoding region, was fused to a promoterless luciferase reporter
vector pGL3 and luciferase expression constructs with various 5'
deletions were prepared (Fig. 8).
Luciferase reporter constructs were transiently transfected into NF-IL6
M1 cells by an electroporation method. The cells were split into two
equal parts posttransfection and cultured in the presence (1 mM) or
absence of isopropyl-ß-D-thiogalactoside
(IPTG). NF-IL6 M1 cells inducibly expressed human NF-IL6 protein within
4 h after IPTG addition as described previously (data not shown)
(21). After 18 h, the cultures were harvested and
cellular extracts were prepared. The luciferase activity levels were
normalized to the cotransfected Renilla luciferase activities and
presented as values relative to that of the promoterless pGL3 basic
vector. Transfection of the pGL3-1783/+69, -1190/+69, -240/+69, and
-166/+69 constructs resulted in about 3- to 5-fold induction by NF-IL6
expression. Deletion of the sequence from position -166 to -61
impaired the responsiveness to IPTG exposure completely (Fig. 8). This
region contains one NF-IL6 binding motif (TKNNGNAAK) at position -66
to -58. Mutations were then introduced in the NF-IL6 consensus
sequence within pGL3-240/+69 vector to form pGL3-240m. Two adenine and
one guanine residues that were thought to be essential for NF-IL6
binding were all changed to cytosine residues. pGL3–240m activity was
assayed and found not to respond to NF-IL6 expression (Fig. 8). This
result indicates that the binding of NF-IL6 to position -66 to -58 is
required to activate the mMincle promoter.
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We next tested whether NF-IL6 could actually interact with
position -66 to -58 of the mMincle promoter. A double-stranded
oligonucleotide P1 spanning position -76 to -45 was radiolabeled and
used in EMSA. As shown in Fig. 9A, when P1 probe was
incubated with nuclear extracts prepared from IPTG-treated
NF-IL6 M1 cells, broad binding activities appeared (Fig. 9A, lane 2). Competition assays were done with an
authentic NF-IL6 binding site from the human IL-6 promoter (IL-6), P1,
and mutated P1 (mP1) harboring the same mutation as the reporter gene
assay. A 100-fold molar excess of unlabeled IL-6 and P1
oligonucleotides competed away the nuclear protein-DNA complexes (Fig. 9A, lanes 3 and 4), but mP1 could not
abolish them at all (Fig. 9A, lane 5). Polyclonal
Ab to NF-IL6 inhibited the formation of these complexes and resulted in
supershifted complexes (Fig. 9A, lane 6). To
confirm thoroughly that NF-IL6 protein binds to P1 oligonucleotide, a
purified GST-NF-IL6 fusion protein was examined instead of NF-IL6 M1
cells nuclear extracts and found to form a specific complex with the
DNA probe (Fig. 9B, lane 2). The DNA-protein
complex could be abolished by competition with unlabeled IL-6 and P1
oligonucleotides but not by mP1 oligonucleotide (Fig. 9B,
lane 3–5). These results clearly demonstrate that NF-IL6
binds to position -66 to -58 of the mMincle promoter, which accords
with the NF-IL6 binding consensus sequence.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-6, and TNF-, in WT PM. In the present
study, the Mincle mRNA expressions were observed only in PM,
macrophage cell line RAW264.7, and myeloblastic leukemia cell line M1
following inflammatory stimuli. Mincle gene expression may be
restricted to myelomonocytic lineage cells stimulated with inflammatory
mediators.
Macrophages infiltrate into various inflammatory regions such as
rheumatoid arthritis joints (37), many types of tumors
(38), and wounds (39), where concentrations
of proinflammatory cytokines are elevated. Mincle may be up-regulated
in those conditions and play some role on infiltrating macrophages.
NF-IL6 (-/-) macrophages lack activities of bacteria killing and
tumor cytotoxity even though they are fully activated by treatment with
LPS and IFN- (18). Low levels of Mincle induction in
NF-IL6 (-/-) PM could raise a possibility that Mincle may play a
role in bacteria killing and tumor cytotoxity. An expected function of
Mincle is the recognition of the surface carbohydrates of
microorganisms or tumor cells, followed by macrophage activation.
Activated macrophages could kill ingested bacteria or lyse tumor cells
as NK cells kill tumors and virally infected cells following
recognizing target cells through NKR-P1 (40, 41). Mincle
may be involved in such immune surveillance processes by activated
macrophages under transcriptional control of NF-IL6. However, extensive
studies are required to discuss the biological function of Mincle
exactly.
Although Mincle is a target gene of NF-IL6 in addition to
G-CSF, the promoter sequence of Mincle is quite different
from that of G-CSF. G-CSF promoter contains a segment between -165 and
-196 bp, G-CSF gene promoter element 1 (GPE1), which plays an
important role in the LPS-inducible expression of the G-CSF gene in
macrophages (42). Subsequent studies on the GPE1 revealed
that both an NF-B binding element and an adjacent NF-IL6
binding element within GPE1 are critical for induction of the G-CSF
promoter by TNF- and IL-1ß (43). It has also been
demonstrated that both NF-B p65 and NF-IL6 can bind to the GPE1 and
form a ternary complex with the DNA. Although the Mincle gene has three
putative NF-B binding motifs in the 1.7-kb promoter region, these
sites exist apart from the indispensable NF-IL6 binding
element. It is possible that NF-B is involved in the inflammatory
signal-induced Mincle gene transcription, but NF-B may not directly
interact with NF-IL6 on the Mincle promoter. The Mincle gene promoter
harbors potential binding elements for other inflammation-activated
transcription factors, such as AP-1 and c-Ets. These transcription
factors may cooperatively act with NF-IL6 for the Mincle gene induction
in macrophages.
Recent reports showed that the activities of C/EBP, -ß, -, and
-
are redundant in regard to the expression of IL-6 and MCP-1
(22, 23). The ectopic expression of C/EBP, -ß, -,
or - is sufficient to confer the LPS-inducible expression of IL-6
and MCP-1 to P388 lymphoblasts, which normally lack C/EBP factors and
do not display LPS induction of proinflammatory cytokines. In fact,
C/EBP exhibits an expression pattern similar to that of NF-IL6 and
has been shown to be involved in the regulation of several genes
induced during inflammation (44). It is likely that the
lack of NF-IL6 is partly compensated for by the induction of C/EBP
upon LPS treatment in vivo. The precise mechanism that other NF-IL6
family proteins could compensate for IL-6 and MCP-1 expression but not
G-CSF and Mincle expression in LPS-stimulated macrophages remains to be
elucidated.
Mincle protein sequence exhibits the highest similarity to the members of group II C-type lectins, most of which mediate glycoprotein endocytosis. The prototype for this group is the asialoglycoprotein receptor, a hepatic cell-surface protein that binds terminal galactose residues exposed upon partial desialylation of circulating glycoproteins (45). This receptor directs turnover of serum glycoproteins, leading to their internalization and delivery to lysosomes via an endocytic pathway. Human asialoglycoprotein receptor H1 subunit contains a single cytoplasmic tyrosine at position 5 that is located within a critical internalization motif for the receptor (46). Rapid internalization of cell-surface receptors generally requires a short stretch of amino acids containing a tyrosine residue (47). Other members of group II C-type lectins, such as M-ASGP-BP (4), CD23 (48), gp120-binding C-type lectin (49), and Kupffer cell fucose receptor (50) carry tyrosine residues in the cytoplasmic region. However, the cytoplasmic region of Mincle protein contains no tyrosine residues, indicating that Mincle protein could not mediate efficient endocytosis. Another C-type lectin that possesses no intracytoplasmic tyrosine residue is MCL, whose protein sequence exhibits the highest homology to Mincle.
Mincle and MCL present several common features; the protein sequences display the highest similarity to the members of group II C-type lectins, transcripts are abundantly expressed in macrophages, cytoplasmic regions contain no tyrosine residue, and the murine genes are mapped on chromosome 6. Thus, Mincle and MCL could be classified together into a derivative of group II C-type lectins.
In conclusion, we have isolated a novel C-type lectin, Mincle, whose expression is strongly induced in response to inflammatory stimuli under the regulation of NF-IL6 in macrophages. Identification of Mincle ligand and targeted disruption of Mincle gene should help elucidate its physiological role in vivo.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Shizuo Akira, Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail address:
3 The abbreviations used in this paper: C-type CRD, Ca2+-dependent carbohydrate-recognition domain; M-ASGP-BP, macrophage asialoglycoprotein binding protein; PM, peritoneal macrophages; C/EBP, CCAAT/enhancer binding protein; MCP-1, macrophage chemoattractant protein-1; MIP, macrophage inflammatory protein; WT, wild type; MCM, macrophage culture medium; RACE, rapid amplification of cDNA ends; Mincle, macrophage inducible C-type lectin; mMincle, mouse Mincle; hMincle; human Mincle; MCL, macrophage C-type lectin; ASGR, asialoglycoprotein receptor; MBL-A, mannose binding lectin A; cM, centimorgans; IPTG, isopropyl-ß-D-thiogalactoside; GPE1, G-CSF gene promoter element 1; BCL, B cell leukemia.
Received for publication June 7, 1999. Accepted for publication August 13, 1999.
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References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, osteopontin and CD14 in a monocytic leukemia cell line. Int. Immunol. 10:1825., -ß, and - in supporting the lipopolysaccharide-induced transcription of IL-6 and monocyte chemoattractant protein-1. J. Immunol. 160:2334.This article has been cited by other articles:
|
|
 |
|
  N. Shibata, T. Saitoh, Y. Tadokoro, and Y. Okawa The cell wall galactomannan antigen from Malassezia furfur and Malassezia pachydermatis contains {beta}-1,6-linked linear galactofuranosyl residues and its detection has diagnostic potential Microbiology, October 1, 2009; 155(10): 3420 - 3429. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Yamasaki, M. Matsumoto, O. Takeuchi, T. Matsuzawa, E. Ishikawa, M. Sakuma, H. Tateno, J. Uno, J. Hirabayashi, Y. Mikami, et al. C-type lectin Mincle is an activating receptor for pathogenic fungus, Malassezia PNAS, February 10, 2009; 106(6): 1897 - 1902. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Bugarcic, K. Hitchens, A. G Beckhouse, C. A Wells, R. B Ashman, and H. Blanchard Human and mouse macrophage-inducible C-type lectin (Mincle) bind Candida albicans Glycobiology, September 1, 2008; 18(9): 679 - 685. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. A. Wells, J. A. Salvage-Jones, X. Li, K. Hitchens, S. Butcher, R. Z. Murray, A. G. Beckhouse, Y.-L.-S. Lo, S. Manzanero, C. Cobbold, et al. The Macrophage-Inducible C-Type Lectin, Mincle, Is an Essential Component of the Innate Immune Response to Candida albicans J. Immunol., June 1, 2008; 180(11): 7404 - 7413. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Uematsu, T. Kaisho, T. Tanaka, M. Matsumoto, M. Yamakami, H. Omori, M. Yamamoto, T. Yoshimori, and S. Akira The C/EBPbeta Isoform 34-kDa LAP Is Responsible for NF-IL-6-Mediated Gene Induction in Activated Macrophages, but Is Not Essential for Intracellular Bacteria Killing J. Immunol., October 15, 2007; 179(8): 5378 - 5386. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Schwegmann, R. Guler, A. J. Cutler, B. Arendse, W. G. C. Horsnell, A. Flemming, A. H. Kottmann, G. Ryan, W. Hide, M. Leitges, et al. Protein kinase C {delta} is essential for optimal macrophage-mediated phagosomal containment of Listeria monocytogenes PNAS, October 9, 2007; 104(41): 16251 - 16256. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. M. Comelli, S. R. Head, T. Gilmartin, T. Whisenant, S. M. Haslam, S. J. North, N.-K. Wong, T. Kudo, H. Narimatsu, J. D. Esko, et al. A focused microarray approach to functional glycomics: transcriptional regulation of the glycome Glycobiology, February 1, 2006; 16(2): 117 - 131. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. A. Begum, K. Ishii, M. Kurita-Taniguchi, M. Tanabe, M. Kobayashi, Y. Moriwaki, M. Matsumoto, Y. Fukumori, I. Azuma, K. Toyoshima, et al. Mycobacterium bovis BCG Cell Wall-Specific Differentially Expressed Genes Identified by Differential Display and cDNA Subtraction in Human Macrophages Infect. Immun., February 1, 2004; 72(2): 937 - 948. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. A. Moore, A. Turler, M. A. Pezzone, K. Dyer, J. Grandis, and A. J. Bauer Tyrphostin AG 126 inhibits development of postoperative ileus induced by surgical manipulation of murine colon Am J Physiol Gastrointest Liver Physiol, February 1, 2004; 286(2): G214 - G224. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. kanazawa, K. Tashiro, K. Inaba, and Y. Miyachi Dendritic Cell Immunoactivating Receptor, a Novel C-type Lectin Immunoreceptor, Acts as an Activating Receptor through Association with Fc Receptor {gamma} Chain J. Biol. Chem., August 29, 2003; 278(35): 32645 - 32652. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Higashi, A. Morikawa, K. Fujioka, Y. Fujita, Y. Sano, M. Miyata-Takeuchi, N. Suzuki, and T. Irimura Human macrophage lectin specific for galactose/N-acetylgalactosamine is a marker for cells at an intermediate stage in their differentiation from monocytes into macrophages Int. Immunol., June 1, 2002; 14(6): 545 - 554. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Uematsu, M. Matsumoto, K. Takeda, and S. Akira Lipopolysaccharide-Dependent Prostaglandin E2 Production Is Regulated by the Glutathione-Dependent Prostaglandin E2 Synthase Gene Induced by the Toll-Like Receptor 4/MyD88/NF-IL6 Pathway J. Immunol., June 1, 2002; 168(11): 5811 - 5816. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Gorgoni, D. Maritano, P. Marthyn, M. Righi, and V. Poli C/EBP{beta} Gene Inactivation Causes Both Impaired and Enhanced Gene Expression and Inverse Regulation of IL-12 p40 and p35 mRNAs in Macrophages J. Immunol., April 15, 2002; 168(8): 4055 - 4062. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Rosati, A. Valentin, D. J. Patenaude, and G. N. Pavlakis CCAAT-Enhancer-Binding Protein {beta} (C/EBP{beta}) Activates CCR5 Promoter: Increased C/EBP{beta} and CCR5 in T Lymphocytes from HIV-1-Infected Individuals J. Immunol., August 1, 2001; 167(3): 1654 - 1662. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. S. Llera, F. Viedma, F. Sanchez-Madrid, and J. Tormo Crystal Structure of the C-type Lectin-like Domain from the Human Hematopoietic Cell Receptor CD69 J. Biol. Chem., March 2, 2001; 276(10): 7312 - 7319. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Xiao, L. Wang, X. Yang, T. Chen, D. Hodge, P. F. Johnson, and W. Farrar CCAAT/Enhancer-binding Protein beta Mediates Interferon-gamma -induced p48 (ISGF3-gamma ) Gene Transcription in Human Monocytic Cells J. Biol. Chem., June 22, 2001; 276(26): 23275 - 23281. [Abstract] [Full Text] [PDF]
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, aromatic; 
,
aliphatic; 
, either aromatic or aliphatic; O, oxygen containing.
Secondary structure (2°) and calcium binding are shown as determined
by Weis for rMBL-A (
