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*
Laboratory for Immunological Research, Schering-Plough, Dardilly, France; and
DNAX, Research Institute, Palo Alto, CA 94304
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. Thus, DCIR is
differentially expressed on DC depending on their origin and stage of
maturation/activation. DCIR represents a novel surface molecule
expressed by Ag presenting cells, and of potential importance in
regulation of DC function. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The functions of DC are mediated in part by the expression of specialized surface receptors (3). In the case of Ag uptake and presentation, DC express the macrophage-mannose receptor (MMR) (4, 5, 6) and DEC-205 (7) that bind and internalize carbohydrate-bearing Ags by receptor-mediated endocytosis. Both MMR and DEC-205 are type I membrane proteins that belong to the Ca2+-dependent (C-type) lectin family (8). Like the pancreatic phospholipase A2 receptor (9, 10), they possess multiple Ca2+-dependent extracellular carbohydrate recognition domains (CRDs) on their NH2 terminus. This group of type I surface lectins also includes members of the selectin family that are implicated in adhesion events on endothelial cells, which mediate the tethering and rolling of leukocytes via oligosaccharide groups (11).
A second family of C-type lectin molecules are type II proteins with a single CRD at the COOH terminal end. This group includes cell surface receptors such as the hepatic asialoglycoprotein receptors (ASGPRs) 1 and 2 (12, 13) and the macrophage lectin (14), which bind oligosaccharide groups and are involved in ligand internalization and may thus be important in the uptake of Ag by macrophages.
The receptors coded for by genes localized on human chromosome
12p12-p13 in a region designated as the NK gene complex (15, 16) represent a closely related group of type II proteins,
although their extracellular C-terminal domains are relatively
divergent from the CRDs in the C-type lectins (17). This
group of molecules includes products of the NKG2 gene complex
(18), CD94 (19), NKR-P1A/CD161
(20), and CD69 (21, 22, 23). Some of these
receptors are involved in recognition of MHC class I molecules and in
the regulation of NK cell activity. In particular, inhibition of
cellular functions by NKG2A/B-CD94 heterodimers is linked to the
presence of immunoreceptor tyrosine-based inhibitory motifs (ITIM) in
the NKG2A/B intracellular domains (24). The ITIM, first
identified in Fc
RIIB (25, 26, 27), is also present in the
cytoplasmic tail of NK inhibitory receptors of the Ig-like superfamily
(28, 29, 30, 31).
To identify novel receptors of DC that may have important functions in Ag-presenting cells, we searched nucleotide databases with a sequence (8 aa) derived from the CRD of hepatic ASGPRs and the macrophage lectin. In the present report, we describe the identification of a cDNA encoding DCIR (for DC immunoreceptor), a novel type II molecule of the C-type lectin family containing an intracellular ITIM consensus sequence.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All cultures were performed in RPMI 1640 medium supplemented
with 10% heat-inactivated FCS, 2 mM L-glutamine (all from
Life Technologies, Gaithersburg, MD), and 160 µg/ml gentamicin
(Schering-Plough, Levallois-Perret, France). All hematopoietic factors
and cytokines are recombinant human proteins. GM-CSF (sp. act.: 2
x 106 U/mg; Schering-Plough Research Institute,
Kenilworth, NJ) was used at a saturating concentration of 100 ng/ml
(200 U/ml). TNF- (sp. act.: 2 x 107
U/mg; Genzyme, Boston, MA) was used at an optimal concentration of 2.5
ng/ml (50 U/ml). Stem cell factor (SCF; sp. act. 4 x
105 U/mg; R&D Systems, Abington, U.K.) and M-CSF
(sp. ac.: 2 x 106 U/mg; R&D Systems) were
used at an optimal concentration of 25 ng/ml. G-CSF
(ED50, 0.01–0.03 ng/ml; R&D Systems) was used at
an optimal concentration of 25 ng/ml. IL-4 (sp. act.:
107 U/mg; Schering-Plough Research Institute) was
used at 50 U/ml. In some experiments, cells were activated with 1 ng/ml
PMA (Sigma, St. Louis, MO) and 1 µg/ml ionomycin (Calbiochem, La
Jolla, CA) or with 25 ng/ml LPS (Sigma).
Cell preparations
Umbilical cord blood samples, peripheral blood samples, and tonsils were obtained according to institutional guidelines. PBMC were purified from human peripheral blood by Ficoll-Hypaque centrifugation. Monocytes were purified from PBMC by centrifugation over a 50% Percoll gradient followed by immunomagnetic depletion of contaminating T, B, and NK cells using a mixture of mAbs composed of OKT3 (CD3), OKT8 (CD8), and 4G7 (CD19, all ascites from our laboratory), NKH-1 (CD56) (Coulter, Miami, FL), ION16 (CD16) and anti-glycophorin A (both from Immunotech, Marseille, France), and goat anti-mouse Ig beads (Dynabeads M450; Dynal, Oslo, Norway). The isolated cells were >95% CD14+ as judged by staining with anti-CD14 mAb and flow cytometric analysis. T lymphocytes were purified from PBMC by immunomagnetic depletion using a mixture composed of mAbs IOM2 (CD14), ION16 (CD16), IOT17 (CD35), and ION2 (HLA-DR) from Immunotech, mAb 89 (CD40, produced in our laboratory) (32), NKH-1 (CD56), and 4G7 (CD19). The purity of CD3+ T cells (CD4+ and CD8+) was >95%. Granulocytes were purified from whole blood by centrifugation in Ficoll to pellet RBCs and polymorphonuclear cells. The granulocyte fraction was then purified with Polymorphprep (Nycomed, Oslo, Norway). B cells were isolated from tonsils essentially as previously described (33). Briefly, T lymphocytes were first depleted by rosetting with SRBCs. The residual non-B cells were further depleted with T cell-specific (CD2, CD3, and CD4) and monocyte-specific (CD14) Abs, followed by immunomagnetic depletion. The resulting population was >98% CD19+ B cells. CD34+ hematopoietic progenitors were purified from umbilical cord blood as previously described (34). Briefly, cells bearing CD34 Ag were isolated from mononuclear fraction through positive selection using anti-CD34 mAb (Immu-133.3; Immunotech) and goat anti-mouse IgG-coated microbeads (Miltenyi Biotec, Bergish Gladbach, Germany). Isolation of CD34+ progenitors was achieved using Minimacs separation columns (Miltenyi Biotec). In all experiments, the isolated cells were 80–99% CD34+ as judged by staining with anti-CD34 mAb. After purification, CD34+ cells were cryopreserved in 10% DMSO.
Granulocytes and macrophages were generated in vitro from CD34+ hematopoietic progenitors in the presence of G-CSF and SCF for 12 days, and M-CSF and SCF for 12 days, respectively. Aliquots of cells were further treated with 1 ng/ml PMA and 1 µg/ml ionomycin for 1 and 6 h, and then pooled. Activated and nonactivated cells were lysed for RNA extraction.
Generation of DC from CD34+ progenitors and from monocytes
Cultures of CD34+ cells were established
in the presence of SCF, GM-CSF, TNF-, and 5%
AB+ pooled human serum, as described (34, 35). By day 5–6, human serum was removed and cells were further
cultured in the presence of GM-CSF and TNF- until day 12. At this
time point, aliquots of cells were activated with PMA and ionomycin for
1 and 6 h, then pooled and lysed for RNA extraction. For analysis
of DCIR expression by flow cytometry, cells were collected at the time
points indicated within the text. In some experiments
CD1a+ and CD14+ DC
precursor subsets were separated at day 6 by flow cytometry and further
cultured until day 12 in the presence of GM-CSF, with or without
TNF-. Monocyte-derived DC were produced by culturing purified blood
monocytes for 6 days in the presence of GM-CSF and IL-4
(36). In some experiments, 5 x
105 monocyte-derived DC/well (24-well culture
plate) were further activated with LPS at the concentration of 25 ng/ml
for 72 h or by coculture with 4 x 104
irradiated (7,500 rads) murine fibroblastic L cells transfected with
the cDNA for CD40 ligand (33).
Northern and Southern blot analyses
Human mRNA adult tissue blots were used (MTN blots 7760-1, 7759-1, and 7767-1 and Immune system (MTN) blot 7754-1; Clontech, Palo Alto, CA). Southern blots were prepared from human genomic DNA isolated from PBMC (37). Genomic DNA was cut with the enzymes BamHI or SacI under standard conditions, and fragments were separated on a 1% TAE agarose gel, then transferred to Hybond N (Amersham, Uppsala, Sweden) overnight in 0.4 M NaOH and 10x SSC. Hybridization of Southern and Northern blots was with a 391-bp DNA fragment from the 3' end of the DCIR cDNA, produced by PCR amplification of a region defined by the oligonucleotides 5'-GATAAACACTCAAGAAGAGCAG (forward primer) and 5'-CCTAAATTCTATGGACCTCCCTTACAC (reverse primer). This fragment was labeled with [32P]dCTP using the High Prime kit (Boehringer Mannheim, Meylan, France). Unincorporated nucleotides were removed by spin column chromatography (Chromaspin-100, Clontech). Membranes were prehybridized and hybridized under standard conditions (37). Low and high stringency washes were at 2x SSC/0.2% SDS and 0.2x SSC/0.2% SDS respectively, each done twice for 30 min. The membranes were incubated with Biomax MR film (Kodak, Rochester, NY) for 21 days.
RNA, DNA, and RT-PCR analysis
Cells were lysed and total RNA was extracted (38) and used for the preparation of first-strand cDNAs. First-strand cDNAs were prepared after DNase I treatment (in the presence of RNase inhibitor) of 5 µg of total RNA using oligo(dT) primers (Pharmacia, Uppsala, Sweden) and the Superscript kit. Synthesis of cDNAs was controlled by performing RT-PCR using ß-actin primers. RT-PCR with the primers 5'-CTGAGGAAAGGGCTTCTGTGAACTGCG (forward primer) and 5'-CATTCTAGCACAGTCCTTCTCACTGTC (reverse primer) specific for human DCIR (hDCIR) cDNA (1 ng/ml) was performed using the AmpliTaq enzyme and buffer (Perkin-Elmer, Paris, France), dNTPs at 0.8 mM, and DMSO at a 5% final concentration. Cycle conditions were 92°C for 1 min, 60°C for 2 min, and 72°C for 3 min for 35 cycles. PCR products were cloned using the pCRII vector (TA cloning kit, Invitrogen, San Diego, CA). Double-stranded plasmid DNA was sequenced on an ABI 373A sequencer (Applied Biosystems, Foster City, CA) using dye terminator technology. Sequencher (Gene Codes, Ann Arbor, MI) and Lasergene (DNA-STAR, London, U.K.) software was used to analyze sequences. Comparisons against the GenBank databases were using the BLAST algorithm.
Chromosomal localization of hDCIR gene
Chromosomal localization was performed with the Stanford G3 RH medium resolution panel (Research Genetics, Huntsville, AL). PCR was as described above using oligonucleotides which amplify a 211-bp fragment specific for the hDCIR gene (forward 5'-GTGCCTATCTGGTGCCTCTGCT, reverse 5'-GTGTTGATGCCTGAGGACTTG). The results were scored manually and analysis was performed with the RHMAPPER program (http://shgc-www.stanford.edu).
Production of recombinant DCIR and generation of anti-DCIR mAbs
hDCIR exodomain-IgG and HRP fusion proteins were produced for production and screening of mAbs, respectively. A XhoI fragment was derived from PCR with hDCIR cDNA as template and primers designed to amplify hDCIR residues 448–951, which encode the extracellular domain, incorporating a XhoI site distal to the transmembrane region and a XhoI site distal to the C-terminal codon. The fragment was inserted into a pCDM8-based human IgG1/Fc fusion vector that had been modified further by insertion of a HindIII-XhoI fragment encoding MDPKGSLSWRILLFLSLAFELSYG (corresponding to the signal sequence for mouse SLAM; our unpublished results). The hDCIR-HRP fusion expression plasmid (hDCIR-HRP vector) was derived from the above plasmid by replacing the XhoI-NotI hIgG region with residues encoding HRP (residues 14–937 of SYNHRPER, GenBank accession no. J05552). The hDCIR-Ig fusion protein was produced by electroporating COP5 cells (39) with the DCIR-Ig vector. After 7–10 days of culture, the Ig fusion protein was purified from supernatant by HiTrapA chromatography (Pharmacia). For production of the hDCIR-HRP fusion protein, COP5 cells were transfected with the hDCIR-HRP vector using the FuGENE 6 transfection reagent (Boehringer Mannheim) according to the manufacturer’s instructions. After 4–5 days of culture in RPMI 1640 medium without phenol red and FCS, the cell supernatant was collected and used as source of the protein.
mAbs against hDCIR were produced by immunizing BALB/c mice (Iffa Credo,
Les Oncins, France) with three i.p. injections of 2 µg purified
DCIR-Ig fusion protein in Freund’s adjuvant (Sigma). Spleens were
removed 3 days after a final i.v. injection of DCIR-Ig, and splenocytes
were fused with murine SP2 myeloma cell line using polyethylene
glycol-1000 and cultured in 96-well plates using standard procedures.
Hybridoma supernatants were screened for their reactivity against
hDCIR-HRP fusion protein by ELISA. Briefly, supernatants were incubated
in microtiter plates (Nunc, Glostrup, Denmark) previously coated with
goat anti-mouse IgG (Fc fragment-specific; Jackson
ImmunoResearch, West Grove, PA). After two washes, 100 µl of
supernatants of COP5 cells transfected with the DCIR-HRP vector were
added and plates were incubated for 2 h at room temperature.
Plates were then washed twice, and the presence of anti-DCIR mAbs
complexed to DCIR-HRP fusion protein was determined by adding 100 µl
of TMB Microwell peroxidase substrate (Kirkegaard & Perry Laboratories,
Gaithersburg, MD). After cloning of selected hybridomas, mAbs were
produced as ascites fluids and purified. By flow cytometric analysis,
all mAbs recognized COP5 cells transfected with the full-length cDNA
encoding hDCIR, but not mock-transfected cells (data not shown). Data
presented in the present study were obtained with the anti-DCIR mAb
111F8 of isotype IgG1, kappa.
Flow cytometric analysis and cell sorting
Cell-surface expression of DCIR was determined by immunofluorescence staining and flow cytometric analysis with a FACSCalibur (Becton Dickinson, Mountain View, CA). For single staining, cells were incubated for 30 min at 4°C with 10 µg/ml purified anti-DCIR mAb 111F8, then washed twice in PBS, 1% BSA, and 0.1% NaN3, and labeled with PE-conjugated F(ab')2 goat anti-mouse Ig (Dako, Glostrup, Denmark) or with biotin-conjugated F(ab')2 goat anti-mouse IgG (H+L) (Jackson ImmunoResearch) followed by PE-conjugated streptavidin (SA-PE; Becton Dickinson). Nonspecific staining was determined by using isotype-matched control mAb. For double staining, cells were first labeled with anti-DCIR mAb 111F8 or isotype control mAb and further with biotin-conjugated goat anti-mouse IgG, then washed and incubated for 15 min in 5% normal mouse serum (Dako) for saturation. Cells were then labeled with SA-PE and with FITC-conjugated anti-CD3, anti-CD19, anti-CD16, anti-CD56, anti-CD14, or anti-CD15 mAbs (all from Becton Dickinson). Dead cells were excluded during cytometric analysis according to their forward and right angle scatter parameters. To analyze expression of DCIR on CD1a+ and CD14+ cells derived from CD34+ progenitors, cells were labeled as described above but DCIR was revealed with Tricolor (TC)-conjugated streptavidin (Caltag, Burlingame, CA).
Separation of CD1a+ and
CD14+ DC precursor subsets from cultured
CD34+ cord blood cells was performed essentially
as previously described (40). Briefly, after 6 days of
culture in the presence of SCF, GM-CSF, TNF-, and 5% human serum,
cells were collected and labeled with FITC-conjugated OKT6 (CD1a; Ortho
Diagnostic Systems, Raritan, NJ) and PE-conjugated Leu-M3 (CD14; Becton
Dickinson). Cells were separated according to CD1a and CD14 expression
into CD14+CD1a- and
CD14-CD1a+ populations
using a FACStarPlus (Becton Dickinson). All the
procedures of staining and sorting were performed in the presence of
0.5 mM EDTA to avoid cell aggregation. Reanalysis of the sorted
populations showed a purity higher than 98%. Sorted cells were seeded
in the presence of GM-CSF plus or minus TNF- (1–2 x
105 cells/ml) for 6–7 additional days. Cells
were routinely collected between day 11 and day 14.
Immunohistological localization of DCIR positive cells
Microscope slides of acetone-fixed cryocut tonsil sections were incubated with 10 µg/ml anti-DCIR mAb 111F8 for 60 min, and subsequently with sheep anti-mouse IgG1 (The Binding Site, Birmingham, U.K.) followed by mouse anti-alkaline phosphatase-alkaline phosphatase (APAAP technique; Dako). Alkaline phosphatase activity was revealed using the Fast Blue substrate (Sigma). For double staining of DCIR and CD1a, tonsil sections were incubated with the mouse IgG1 anti-DCIR mAb 111F8 and the IgG2b anti-CD1a (Becton Dickinson) and then revealed by sheep anti-mouse IgG1 and APAAP technique (as described above) and biotinylated sheep anti-mouse IgG2b (The Binding Site) followed by ExtrAvidin-peroxidase (Sigma). Alkaline phosphatase activity and peroxidase activity were respectively demonstrated using Fast Blue substrate and 3-amino-ethyl carbazole (Sigma).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Alignment of amino acid sequences of type II membrane proteins of the calcium-dependent lectin family reveals a number of conserved motifs in the C-type lectin domain. A motif (SCYWFSH) shared between the hepatic ASGPRs 1 and 2 and the macrophage lectin was used to search the Human Genome Sciences (HGS) databases using the tblastn algorithm (41), and a number of cDNAs were identified. Two sequences from DC libraries were selected and reanalyzed against the public and HGS libraries to detect homologous or identical cDNAs using the blastn algorithm. A single contiguous sequence (contig) of 1299 bp, composed of 5' sequences from 14 different clones and containing an entire open reading frame, was isolated.
A representative cDNA clone was sequenced in both strands to give the
full-length sequence of the DCIR cDNA. This clone is 1272 bp in length
(excluding the poly(A) tail) and contains a putative open reading frame
of 711 bp (Fig. 1
). The potential start
codon is not in a consensus Kozak sequence (42) but
because it is not preceeded by an alternative ATG, and because stop
codons exist at upstream positions, we predicted that this was the
start of the encoded protein. A polypeptide of 237 aa was deduced from
this sequence. No signal sequence was detected, but a putative
hydrophobic signal anchor sequence of 25 aa extending from nt 374
to 448 was identified, thus indicating that this clone encodes a type
II transmembrane protein. The predicted m.w. was 27.5 kDa. The 3' UTR
contains a number of potential rapid degradation signals, including
three repeats of the consensus sequence ATTTA (43).
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Analysis of the predicted amino acid sequence of DCIR indicated
that DCIR belongs to the type II Ca2+-dependent
lectin family with the presence of a single CRD (8, 44).
The C-type lectin domain of DCIR has the greatest homology to the
macrophage lectin (42%), the hepatic ASGPR-1 and ASGPR-2 (35% and
37%, respectively), and CD23 (32%) (Fig. 2A). The conserved cysteine
residues (arrows in Fig. 2A) of the C-type lectin fold are
clearly conserved across the members of this family; however, a number
of distinguishing features can be seen. Like the macrophage and hepatic
lectins, DCIR has a double cysteine motif at the start of the lectin
domain (circle in Fig. 2A). The function of this
supplementary cysteine is unknown, as there is apparently no other
cysteine in the lectin domain that may form a disulfide bridge with
this residue. It is possible that this residue may be involved in
intermolecular disulfide bridge formation, although there is another
cysteine in DCIR at position 91 (Fig. 1
) that probably fulfils this
function. The calcium-binding domain is conserved in DCIR including the
EPS motif (residues 195–197), glutamate (E) at position 201, and
asparagine aspartate (ND) at positions 218–219 (asterisks in Fig. 2A). These motifs are notably absent from the NKG2 receptors
(NKG2E shown here) as well as in other NK cell receptors with C-type
lectin-like domain (17). The recent crystal structure of
CD94 (45) gives a clear demonstration of the differences
in the two types of CRD, with notably a reduced loop at the position of
the Ca2+ binding domain. Thus CD94 and the NKG2
receptors do not bind calcium and probably have an alternate ligand
binding structure to that of the C-type lectins that fix calcium, DCIR
included. A single potential N-glycosylation site is
present at position 185 (Fig. 1). Phylogenic analysis of the CRD
domains (Fig. 2B) further indicates that DCIR is
intermediate in structure between the group of macrophage/hepatic
lectins and CD23 (Fig. 2B).
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) predicts a 49-aa
intracellular domain that contains a tyrosine at position 7 centered in
the sequence ITYAEV that perfectly matches the consensus sequence
I/VXYXXL/V of the ITIM (28, 29, 30, 46, 47). This type of
domain has been shown to act as an inhibitory motif in molecules of the
lectin family such as human NKG2A/B (24) and murine Ly49
(48, 49). Identification of a mouse homologue of hDCIR
Bioinformatics searches in the EST databases (GenBank dbEST) using
the predicted polypeptide sequence of hDCIR (tblastn algorithm)
revealed mouse clones encoding a homologous protein. One IMAGE
consortium (LLNL) cDNA clone AA170532 mouse spleen (50),
estimated to be a full-length clone by sequence analysis, was selected
and DNA sequenced. This clone showed features similar to hDCIR. The
full-length clone was 1418 bp, excluding the poly(A) sequence, and
contained a 5' UTR of 278 bp (data not shown). As for hDCIR, the
putative start codon was not contained within a consensus Kozak region,
but was preceded by an upstream stop codon, and the 3' UTR also
contained sequences similar to rapid degradation signals, including
four consensus ATTTA sites (data not shown). The predicted
polypeptide is 238 residues in length and codes for a type II membrane
protein with an ITIM and a C-type lectin domain. Alignments with the
human protein (Fig. 3) showed 54%
identity, 65% homology over the whole sequence. Notably, the ITIM
domain is localized within a highly conserved region (13 of 15 residues
are identical). There are three potential N-glycosylation
sites at positions 91, 131, and 136. Of interest is the conserved
membrane-proximal glutamine motif (FQKYSQLLE), and the cysteine residue
potentially implicated in disulfide bridge formation (boxed in Fig. 3).
Equally, the C-type lectin domains show blocks of conservation,
including the EPS motif at positions 197–199. Differences seen between
hDCIR and the human macrophage and hepatic lectins are retained in the
mouse sequence, notably phenylalanine (F) instead of tryptophan (W) at
position 167, glutamine (Q) at positions 179 and 183, the addition of
tryptophan-glycine (WG) at positions 216–217, and serine (S) instead
of tryptophan at position 229 (filled circles in Fig. 3). We thus
propose that this clone is the mouse homologue of hDCIR.
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Southern blot analysis of genomic DNA with a probe consisting of a
391-bp DNA fragment from the 3' end of DCIR cDNA showed a single band
at 
7 kb after digestion with the enzymes
BamHI and SacI (data not shown).
These data indicate a unique gene for DCIR. Chromosomal localization
with the Stanford G3 panel gave as closest marker SHGC-12041, with a
lod score of 7.7. This marker, which is the gene coding for M130
Ag/CD163 (51), is localized to chromosome 12p13. This
region is host to a number of genes encoding receptors of the C-type
lectin-like family, notably CD94, the NKG2 molecules, CD69, AICL, and
NKR-P1 that belong to the NK gene complex (15, 16).
DCIR is expressed in immune tissues
Northern blot analysis (Fig. 4) of
DCIR showed a band at 1.3 kb. This band is slightly smeared and may
indicate the presence of a second mRNA at 1.5 kb. The high stringency
washes used for this Northern blot tend to suggest that this is not due
to a related gene, but rather to the presence of two transcripts. mRNA
was detected in greatest quantity in peripheral blood leukocytes, with
moderate quantities in spleen, lymph node, and bone marrow, and at very
low levels in thymus (Fig. 4) as well as in spinal cord and trachea
(data not shown). However, DCIR mRNA was not detected in heart, brain,
placenta, lung, liver, skeletal muscle, kidney, pancreas, prostate,
testis, ovary, small intestine, colon, stomach, and thyroid (data not
shown). Taken together, these data indicate a preferential expression
of DCIR in hematopoietic tissues.
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As DCIR was expressed preferentially in immune tissues, we further
investigated its cellular distribution by RT-PCR analysis in various
myelo-monocytic and lymphoid cell types. As shown in Fig. 5A, DCIR mRNA could be
amplified from DC generated in vitro by culturing
CD34+ cord blood progenitors for 12 days in the
presence of GM-CSF plus TNF- (35). Expression was also
detected in macrophages and granulocytes derived from
CD34+ progenitors in the presence of M-CSF or
G-CSF, respectively. This expression did not appear to alter in
response to treatment with PMA/ionomycin. In freshly isolated cells,
DCIR was expressed in PBMC and strongly detected in blood monocytes,
whereas a low expression was seen in tonsillar B cells. Interestingly,
DCIR mRNA was not detected in purified blood T cells nor in blood NK
cells (Fig. 5A). Furthermore, DCIR mRNA could not be
detected in a number of cell lines including U937, HL60, THP1
(myelo-monocytic cells), JY, RAMOS, DAUDI, BL2 (B cell lines), Jurkat
and MOLT4 (T cell lines), TF1 (erythro-leukemia), CHA (kidney
carcinoma), MRC5 (fetal lung fibroblasts) and SW620, and HT29 (colon
carcinoma cell lines) (data not shown).
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B). DCIR
was found to be expressed by all circulating
CD14+ monocytes and, at lower intensity, by the
majority (88 ± 5%, n = 3) of
CD19+ B lymphocytes. Flow cytometric analysis
showed strong expression of DCIR by freshly isolated
CD15+ blood granulocytes (Fig. 5B),
thus confirming the expression of DCIR mRNA observed on in
vitro-derived granulocytes (Fig. 5A). Finally, DCIR could
not be detected on the surface of circulating
CD3+ T cells nor on CD16+
or CD56+ NK cells (Fig. 5B). In vitro, DCIR is differentially expressed on DC subsets and its expression is regulated during DC maturation
Human DC generated in vitro from cord blood
CD34+ progenitors represent an heterogenous
population of cells that arise from different precursors and possess
different functions (34, 52, 53). Because DCIR mRNA was
detected in total CD34+-derived DC at day 12
(Fig. 5A), we further analyzed its distribution on different
DC subsets and activation states. By two-color flow cytometry (Fig. 6A), DCIR was found to be
expressed on both CD14+ and
CD1a+ DC precursor cells obtained by culturing
cord blood CD34+ progenitor cells for 5–6
days with GM-CSF and TNF- in the presence of human serum
(34). The intensity of DCIR expression was generally
stronger on the CD14+ subset of cells. To
determine whether DCIR expression is equally affected by further
differentiation of these two precursors into DC,
CD1a+/CD14- and
CD1a-/CD14+ cells were
flow cytometry-sorted at day 6 of culture and further recultured for 6
days in the presence of GM-CSF with or without TNF-. As shown in
Fig. 6B, CD14+-derived DC expressed
considerably higher levels of DCIR than their
CD1a+-derived counterparts. Analysis of DCIR mRNA
expression by RT-PCR also confirmed the lower levels of DCIR mRNA in
CD1a+-derived DC (Fig. 6C). Notably,
for both subsets, DCIR expression was significantly higher upon removal
of TNF-, between days 6 and 12 (Fig. 6B).
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A, monocyte-derived DC
strongly express DCIR after 7 days of culture with GM-CSF and IL-4,
indicating that DCIR expression is maintained during the
differentiation of monocytes into DC. At this stage monocyte-derived DC
are considered to be immature as they express low levels of CD80, CD86,
MHC class II molecules, CD25, and CD83 (Ref. 4 and data
not shown). Of interest, DCIR expression decreased when those cells
were further cultured with LPS or CD40 ligand (Fig. 7A), two
signals that induce DC activation and maturation (4). The
down-regulation of DCIR expression was also observed at the mRNA level,
as shown in Fig. 7B in the case of CD40 activation, where
DCIR mRNA expression was totally lost by 24 h of coculture with
CD40 ligand-expressing L cells. It should be noted that DCIR mRNA
expression could not be altered by treatment of the same cells with a
combination of PMA and Ionomycin (Fig. 7B).
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To determine the pattern of expression of DCIR in vivo, we
performed immunohistological studies. In tonsil, DCIR-positive cells
were found beneath the epithelium and around the B cell follicles, but
not observed in germinal centers (Fig. 8,
A and B). A weaker expression was seen in cells
of the mantle zone of the follicles, principally composed of naive B
cells (Fig. 8B). At higher magnification (Fig. 8C), cells that strongly express DCIR display a dendritic
morphology. Double staining of DCIR and CD1a (Fig. 8D)
showed that the Langerhans-like cells in the tonsil epithelium that are
stained with anti-CD1a mAb are not stained with anti-DCIR mAb,
whereas DCIR-positive (CD1a negative) cells are mostly present within
the subepithelial region.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) or immature Langerhans cells in skin epidermis
(data not shown). DCIR expression can be decreased by signals inducing
DC maturation (e.g. CD40 ligand, LPS, and TNF-) that would
correspond to a stage subsequent to migration into secondary lymphoid
tissues in response to proinflammatory factors and to contact with
Ag-specific T cells expressing CD40 ligand. Taken together, these
results indicate that DCIR is selectively expressed on DC
subpopulations according to their origin and stage of maturation.
Additional experiments will be required to gain insight into the
functional significance of this particular expression pattern of
DCIR. The chromosomal localization of hDCIR close to the NK gene complex on chromosome 12p13 is interesting, particularly as DCIR was not detected in NK and T cells. The complex of C-type lectin family members at this locus includes CD69, AICL, CD161, CD94, and the NKG2 family (15, 16, 57), as well as the recently described MAFA-like receptor gene (58). In the mouse the NK gene complex, exemplified by the Ly49 lectin-like family, is localized to the syntenic region on chromosome 6 (59); thus, it will be of interest to determine the localization of the DCIR mouse gene homologue. Although the hDCIR gene maps close to the NK complex, the sequence encoding the extracellular carboxyl-terminal portion of the molecule is not typical of the NK cell receptors. Characteristics of the C-type lectin CRDs are only partially conserved in the NK cell receptors, with a partial or total lack of the Ca2+-ligating residues that are associated with sugar-binding sites (17). In striking contrast, the Ca2+-ligating residues are well conserved between DCIR and other C-type lectin CRDs. The CRD of DCIR thus displays the closest homology with the hepatic ASGPRs. Yet, the localization of the latter genes on chromosome 17 (60) could suggest that DCIR represents an evolutionary intermediate between the NK cell receptors and the hepatic lectins.
The cytoplasmic domain of DCIR contains one ITIM motif (ITYAEV). This type of domain has been shown to inhibit signal transduction via activation receptors (e.g., ITAM-bearing receptors) and is present in the cytoplasmic tail of C-type lectin-like molecules including the human NKG2A/B and murine Ly49 family members expressed by NK cells (18, 59). Inhibition is mediated by the recruitment of SHP-1 (SH2-containing phosphatase-1) and/or SHP-2 phosphatases to the phosphorylated ITIM domain (28). By analogy to these known inhibitory receptors, DCIR may be implicated in inhibition of molecular events associated with cellular activation. It is proposed that receptors with two cytoplasmic tyrosine motifs, like NKG2A/B and the Ig-like superfamily members KIR or gp49, may interact simultaneously with the tandem SH2 domains of SHP-1, thereby producing a more stable interaction, whereas receptors with a single tyrosine motif such as mouse Ly49A may form homodimers for recruiting SHP-1 (46, 61). The presence of a single ITIM in the intracytoplasmic tail of DCIR may indicate that this molecule also forms a dimer (as suggested by the presence of a supplementary cysteine in its extracellular domain) to recruit SHP-1 or SHP-2.
The presence of a putative inhibitory motif in the cytoplasmic tail of
DCIR and its expression on monocytes and some immature DC subsets
suggest that DCIR may play a role in modulating DC differentiation
and/or maturation. This has been recently described for the p40/LAIR1
molecule, an ITIM-containing receptor of the Ig-like superfamily
(62), the cross-linking of which blocks GM-CSF-induced
differentiation of mature monocytes and CD34+
peripheral blood precursors into DC (63). DCIR may also be
involved in Ag uptake by immature DC because the consensus for
tyrosine-based internalization domain Yxx
(where is any bulky
hydrophobic residue) (64, 65, 66) is also contained in the
ITIM sequence. Motifs of this nature may act as internalization signals
in different membrane receptors including type II lectins such as the
hepatic ASGPRs and macrophage lectin (12, 13, 14, 67, 68) as
well as in the ITIM-bearing Ig-like ILT3 molecule that is involved in
Ag capture and delivery into an Ag-processing compartment of DC
(69). To address this question, we have compared the time
kinetics and levels of internalization of anti-DCIR mAb with that
of an anti-mannose receptor (MMR) mAb used as positive control for
Ag internalization through receptor-mediated endocytosis. Following
cross-linking, DCIR was only slowly and weakly internalized in
monocyte- and CD34+-derived DC, in contrast to
the rapid kinetics observed with the MMR (data not shown). This finding
suggests that Ag capture by receptor-mediated endocytosis is not the
principal function of DCIR.
The expression of DCIR on B lymphocytes also appears to be regulated
according to their stage of activation. We found that while the
majority of blood B cells express relatively high levels of DCIR, only
a subset of freshly isolated tonsillar B cells were stained with
anti-DCIR mAb, and at lower intensity (data not shown).
Furthermore, two-color flow cytometry demonstrated that
DCIR+ tonsillar B cells coexpressed CD44 but not
CD38 (data not shown), two markers that discriminate germinal center B
cells (CD38+/CD44-) from
naive and memory B cells
(CD38-/CD44+)
(70). In keeping with this, anti-DCIR mAb did not
stain germinal center B cells on tonsil sections, but weakly stained B
cells within the mantle zone of the B follicles (Fig. 8). These data
indicate that DCIR is mostly expressed by resting naive and memory
circulating B cells and suggest that DCIR expression is down-regulated
by activation. It is interesting to note that the expression of DCIR on
B cells paralleled that of ILT2, an Ig-like superfamily member
containing ITIM motifs, that has been shown to inhibit B cell
activation through the B cell Ag receptor (71). We are
presently investigating whether DCIR may also negatively regulate B
cell activation.
The natural ligand(s) for DCIR have not been yet identified. Carbohydrate ligands are likely because DCIR belongs to the C-type lectin superfamily. The amino acid sequence present in the CRDs of animal lectins provides information about saccharide-binding specificity. For example, the sequence EPN is found in CRDs known to bind mannose or glucose derivatives, and is present in two domains of the MMR (8). In contrast, the sequence QPD is characteristic of CRDs that bind galactose and N-acetylgalactosamine and is present in the hepatic ASGPR-1 and -2 (8). Both human and mouse DCIR display a variant motif (EPS) in place of EPN or QPD. An EPS sequence is present in the CRD-2 of the MMR but is believed to contribute only weakly to binding of polyvalent ligands (72).
In conclusion, both human and mouse DCIR are type II proteins containing a consensus ITIM in their intracytoplasmic tail. hDCIR is encoded by a gene localized close to the NK complex on chromosome 12. Unlike the type II lectin-like members of this complex, hDCIR contains a bona fide extracellular lectin CRD with calcium-binding residues. Moreover, unlike immunoreceptors of the NK complex, hDCIR is not expressed by NK and T cells. DCIR may thus represent a molecule intermediate in structure and function(s) between the NK cell receptors and typical CRD-bearing lectins such as the ASGPRs that function in ligand internalization. The expression of DCIR on discrete DC subsets and maturation stages strongly suggests a particular role in directing the ontogeny and/or the Ag-handling potential of this pivotal cell for initiation of specific immunity.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Elizabeth E. M. Bates, Schering-Plough, Laboratory for Immunological Research, 27 chemin des Peupliers, BP11, 69571 Dardilly cedex, France. E-mail address:
3 E.E.M.B. and N.F. contributed equally to this paper.
4 Abbreviations used in this paper: DC, dendritic cells; DCIR, DC immunoreceptor; hDCIR, human DCIR; MMR, macrophage-mannose receptor; ASGPR, asialoglycoprotein receptor; CRD, carbohydrate recognition domain; EST, expressed sequence tag; ITIM, immunoreceptor tyrosine-based inhibitory motif; SCF, stem cell factor; RACE, rapid amplification of cDNA ends; UTR, untranslated region.
Received for publication March 15, 1999. Accepted for publication June 9, 1999.
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), human hepatic ASPGR-1 and -2, human CD23, human CD69,
and human NKG2E. Amino acids conserved between DCIR and other type II
lectins are shaded. Residues conserved among human C-type lectins are
boxed. The six conserved cysteine residues forming disulfide bridges
are marked by arrows. The double cysteine motif at the start of the
lectin domain is indicated by circles. Residues conserved in the
calcium-binding domain are labeled by asterisks. Accession numbers for
these molecules are as follows: macrophage lectin (D50532), ASPGR-1
(P07306), ASPGR-2 (P07307), CD23 (P06734), CD69 (Z22576), and NKG2-E
(Q07444). B, Phylogenetic tree showing the relationship
between hDCIR and other C-type lectins. The C-type lectin domains of
hDCIR, ASGPR-1, ASGPR-2, macrophage lectin, CD23, CD69, and NKG2E were
analyzed using the MegAlign function of Lasergene (DNAstar).
