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Introduction
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Dendritic cells (DCs) are a distinct population of bone marrow–derived leukocytes that initiate primary and secondary immune responses 1. DCs migrate from the blood to peripheral tissues, where they reside in an immature state, awaiting antigen encounter. Upon antigen capture, DCs process them into peptides, which are loaded onto MHC molecules for presentation to T cells. As a result of pathogen invasion, inflammation, and tissue damage, DCs receive additional activating signals, which induce a profound change in DC phenotype and functions, known as maturation 123. Mature DCs express the chemokine receptor CCR7, which interacts with the chemokines CCL19 (also known as EBI-1 ligand chemokine [ELC], or macrophage inflammatory protein 3 β [MIP-3β]) and CCL21 (also known as secondary lymphoid tissue chemokine [SLC], or 6-C-Kine) 456789. These chemokines are crucial for guiding DCs from peripheral tissues to draining lymph nodes, as demonstrated in mice with natural or targeted genetic deletions of CCL19, CCL21, or CCR7 1011121314. In addition, mature DCs express high levels of stable MHC-peptide complexes on the cell surface, upregulate costimulatory and adhesion molecules, and downregulate antigen-capturing molecules. Thus, mature DCs can efficiently present antigens and stimulate virgin-T cells located in the T cell–rich areas of lymph nodes 115. Here, DCs receive further activating signals from cognate Th cells, which express CD40 ligand (CD40L) 16, OX40 1718, and TNF-related activation-induced cytokine (TRANCE) 19202122. These stimuli trigger IL-12 secretion by antigen presenting DCs thus promoting Th1 type T cell responses 202324252627.
Activating signals induce DC maturation through several distinct signaling pathways. LPS, other bacterial and viral components, as well as products released by damaged tissues, activate DCs through Toll-like receptors (TLRs) 2829. TLRs trigger downstream signaling pathways, which activate the nuclear factor (NF)-
B. NF-B promotes the transcription of a variety of genes mediating maturation 30313233. In addition, TLRs activate mitogen-activated protein kinases (MAPKs), such as the stress-activated protein kinase p38 (P38/SAPK) and the extracellular signal–regulated kinase (ERK), which concur to DC activation 2829. Inflammatory cytokines, such as IL-1, IL-18, and TNF-
, and T cell surface molecules, such as CD40L, OX40, and TRANCE, bind specific receptors that activate NF-B as well 12034353637. A second pathway of DC maturation is initiated by the receptors for the Fc portion of IgG (FcRs), which bind antibody-opsonized pathogens 38. FcRs lack intracellular signaling motifs, but display a charged residue in the transmembrane domain, which mediates association with the 
chain of FcR (FcR) 39. FcR contains a cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM), which recruits protein tyrosine kinases (PTKs) of the src and syk families. PTKs trigger protein tyrosine phosphorylation, Ca2+ mobilization, and phosphorylation of several MAPKs 4041. While FcR is essential for FcR-mediated DC maturation 42, the role of downstream PTKs and MAPKs is yet unknown.
Recent observations suggest that DC activation is controlled by yet another signaling pathway, which involves the adaptor molecule DAP12 (also called KARAP). DAP12 is associated with several NK and myeloid cells activating receptors 434445464748495051525354. Like FcR, DAP12 contains a cytoplasmic ITAM, recruits the PTKs ZAP70 and p72/syk, and promotes activation of ERK 44455556. Knock-in mice bearing a nonfunctional mutation within the ITAM of DAP12 showed a dramatic accumulation of DCs in mucocutaneous epithelia and were resistant to hapten-specific contact sensitivity 57. In addition, DAP12-deficient mice were resistant to experimental autoimmune encephalomyelitis (EAE) induced by immunization with myelin oligodendrocyte glycoprotein peptide 58. These phenotypes suggested a role of DAP12 in regulating migration and antigen presentation capacity of DCs. Three DAP12-associated receptors have been identified in myeloid cells. One of these, myeloid DAP12-associating lectin-1 (MDL-1), is a member of the C-type lectin superfamily 50. The others, signal-regulatory protein β (SIRP-β) and triggering receptor expressed on myeloid cells-1 (TREM-1), belong to the Ig superfamily 5359. TREM-1 is preferentially expressed on neutrophils and a subset of blood monocytes 53. SIRP-β and MDL-1 are mainly expressed on blood monocytes and macrophages 5060. When monocytes are differentiated toward DCs by culturing them in vitro in the presence of GM-CSF and IL-4, expression of MDL-1, SIRP-β, and TREM-1 is completely downregulated 505360.
Recently, we have cloned a cell surface receptor distantly related to TREM-1 called TREM-2. TREM-2 is a member of the Ig-superfamily characterized by a single V-type extracellular domain, a transmembrane region with a charged residue of lysine and a short cytoplasmic tail with no signaling motifs 53. Here we found that TREM-2 is associated with DAP12 and, in contrast to TREM-1, SIRP-β, and MDL-1, is not expressed on monocytes, but it is strongly upregulated on human DCs derived in vitro from monocytes. This observation provided the opportunity to investigate the role of TREM2/DAP12-mediated signaling pathways in DC migration and maturation.
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Materials and Methods
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Production of TREM-2 Human IgM Fusion Protein.
Soluble TREM-2 was produced as a chimeric protein consisting of TREM-2 extracellular domain and human IgM constant regions (TREM-2 human IgM [TREM-2-HuIgM]), as previously described 61. TREM-2 extracellular domain was amplified from the cloned full length cDNA by polymerase chain reaction using the following oligonucleotides: 5'-ACTCTGCTTCTGCCCTTGGCTGGGG, 3'-tagtagGTCGACATACTTACCGGGTGGGAAAGGGATTTCTCCTTCCAA. Purification of TREM-2-HuIgM from culture supernatants was performed by affinity chromatography on Sepharose-coupled mouse anti–human IgM mAb (Sigma-Aldrich) according to manufacturer's protocols.
Transfections.
293 cells were transiently transfected with a cDNA encoding human TREM-2 as a FLAG peptide NH2-terminal fusion protein (Eastman Kodak Co.) using cytofectene (Bio-Rad Laboratories).
Production and Modifications of Anti–TREM-2 and Control mAbs.
6-wk-old BALB/c mice (Iffa-Credo) were immunized with purified TREM-2-HuIgM. Spleen cells were fused with the SP2/0 myeloma cells and hybridoma supernatants were screened by ELISA using TREM-2-HuIgM as capturing protein and human-adsorbed horseradish peroxidase (HRP)-labeled goat anti–mouse IgG (BD PharMingen) as detecting Ab. ELISA-positive hybridoma supernatants were then tested by flow cytometry for staining 293 cells expressing FLAG-tagged TREM-2. mAb 29E3 (anti–TREM-2, IgG1,), mAb 21C7 (control IgG1, , anti–TREM-1) 53, and mAb 1B7.11 (control IgG1, , anti-2,4,6 TNP; American Type Culture Collection) were purified using GammaBind-Sepharose (Amersham Pharmacia Biotech). Purified mAbs were either biotinylated (Roche) or labeled with Cy5 (Amersham Pharmacia Biotech) according to manufacturer's protocols. F(ab') or F(ab')2 fragments of mAb 29E3 and mAb 21C7 were prepared using the F(ab')/F(ab')2 Kit (Pierce Chemical Co.). F(ab') and F(ab')2 were separated from the Fc portion by affinity chromatography on protein G-sepharose, followed by gel filtration on a Superdex 75 HR10/30 (Amersham Pharmacia Biotech). F(ab') and F(ab')2 preparations were tested for the absence of Fc fragments by immunoassay. F(ab') and F(ab')2 fragments were biotinylated allowing for crosslinking by ExtrAvidine (Sigma-Aldrich) or flow cytometry by Streptavidin-allophycocyanin (APC) or -PE (BD PharMingen). Alternatively, F(ab')2 fragments were crosslinked using a goat anti–mouse IgG F(ab')2 specific antibody (The Jackson Laboratory).
Cells.
PBMCs were purified from human blood by gradient density centrifugation on lymphocyte separation medium (LSM; ICN Biomedicals/Cappel). CD14+ monocytes were purified from PBMCs by magnetic cell sorting (MACS) using CD14 MicroBeads (Miltenyi Biotec). Monocyte-derived DCs were prepared from purified monocytes as described previously 6263.
Antibodies and Flow Cytometry.
Before staining, all cells were preincubated with PBS-20% human serum for 1 h on ice to block Fc receptors (FcR). Monocytes cultured in M-CSF or GM-CSF and IL-4 were stained with either mAb 29E3, mAb 21C7, or mAb 1B7.11, followed by human-adsorbed PE-conjugated goat anti–mouse IgG (Southern Biotechnology Associates, Inc.). In three-color stainings, immature DCs cultured with LPS (100 ng/ml), TNF- (10 ng/ml), or CD40L-transfected J558L cells 64 were incubated with Cy5-labeled mAbs 29E3 and FITC-conjugated anti-CD83 mAb (Immunotech). Cells were analyzed on a FACSCaliburTM cytometer using CELLQuestTM software (Becton Dickinson). Dead cells were excluded by gating on PI-negative cells.
Stimulation of DCs by LPS, F(ab')2 Anti–TREM-2 mAb, or Human IgG in the Presence or Absence of Inhibitors.
Human IgG, F(ab')2 29E3 (anti–TREM-2 mAb), or control F(ab')2 (21C7 anti–TREM-1 mAb) were coated for 6 h at 37°C on 96-well flat-bottom plates with a final concentration of 20 µg/ml in PBS. LPS was used at a final concentration of 1 µg/ml. Immature DCs were plated at a concentration of 5 x 105 cells/well and simultaneous contact to the plate was induced by short centrifugation (400 g, 1 min, 25°C). Supernatants and cells were collected after 6, 12, 24, 36, 48, or 72 h and tested by ELISA or flow cytometry. In blocking experiments, inhibitors (PD98059 [20 µM], LY294002 [10 µM], SB203580 [2 µM], PP2 [1 µM]; all from Calbiochem), and TPCK (20 µM; Sigma-Aldrich) were added 60 min before stimulation.
Measurement of Cytokines, Chemokines, and Cell Surface Activation Markers.
To measure stimulation-dependent changes in the expression of cell surface markers and cytokine secretion, monocyte-derived DCs were stimulated as described above for 6, 12, 24, 48, and 72 h. Supernatants were collected and tested for production of IL-6, IL-8, IL-10, TGF-β, IL-12p40, IL-12p75, IL-13, IL-15, IL-18, IL-1, IL-1β, TNF-, and MCP-1 by ELISA (BD PharMingen). Cells were harvested and stained with anti–TREM-2, -MHC class I, -MHC class II, -CD1a, -CD11a, -CD11b, -CD11c, -CD29, -CD32, -CD35, CD38-, CD40-, -CD41, -CD54, -CD61, -CD64, -CD80, -CD83, -CD86, -CD89, -CD103, -CD115, -CD116, -CCR5, -CCR6, -CXCR4, or -Mannose receptor conjugated with Cy5-, PE-, or FITC (all from Immunotech and BD PharMingen). Anti-CCR7 mAb (BD PharMingen) was followed by F(ab')2 PE-labeled goat anti–mouse IgM Ab (Southern Biotechnology Associates, Inc.). Stained cells were analyzed by flow cytometry.
Measurement of Cytosolic Ca2+.
Monocyte-derived DCs were loaded with Indo-1 AM (Sigma-Aldrich) for 30 min at 37°C, washed three times, and resuspended in RPMI/10 mM HEPES/5% FCS. Cytoplasmic Ca2+ levels were monitored in individual cells by measuring 405/525 spectral emission ratio of loaded Indo-1 dye by flow cytometry. After a baseline was acquired for at least 30 s, 29E3, 21C7, F(ab') 29E3, F(ab') 21C7, F(ab')2 29E3, or F(ab')2 21C7 were added to a final concentration of 1 µg/ml and analysis was continued up to 512 s. All antibodies and antibody fragments were biotinylated. In some experiments, ExtraAvidine (Sigma-Aldrich) was added as crosslinker together with the biotinylated primary antibodies or Ab fragments.
Determination of ERK, JNK, and p38/SAPK Activation.
Monocyte-derived DCs (106 cells per time point) were stimulated as described above. After 0 (unstimulated control), 1, 2, 5, 10, and 20 min cells were harvested and chilled on ice. Cells were spun down and lysed in reducing sample buffer. Specific induction of tyrosine phosphorylation and phosphorylation of ERK, p38/SAPK, and JNK was determined by reducing Western blot analysis using anti–phospho-ERK, anti-ERK, anti–phospho-p38/SAPK, anti-p38/SAPK, anti-phospho-JNK, and anti-JNK antibodies (all from New England Biolabs, Inc.).
Surface Biotinylation and Pervanadate Treatment.
Monocyte-derived DCs were washed three times in PBS followed by incubation with sulfo-NHS-biotin according to the manufacturer's protocol (Pierce Chemical Co.). For pervanadate treatment, cells were incubated with 200 µM pervanadate and 200 µM H2O2 at 37°C for 5 min. Biotinylation or pervanadate stimulation was stopped by washing the cells three times in PBS/10% FCS/200 µM pervanadate and one time with ice cold PBS/200 µM pervanadate, respectively.
Immunoprecipitations.
Surface-biotinylated cells were lysed in 1% digitonin, 100 mM Tris-HCl, pH 7.4, 150 mM NaCl, protease inhibitors (Complete; Roche Molecular Biochemicals). After overnight preclearing with normal mouse serum coupled to protein G Sepharose 4B (Amersham Pharmacia Biotech), lysates were subjected to immunoprecipitation with 5 µg/ml of 29E3, 21C7, or 1B7.11 at 4°C for 3 h. Immunocomplexes were precipitated by addition of protein-G-Sepharose 4B for 3 h at 4°C. Precipitates were washed four times with lysis buffer, followed by a final wash with 0.5% digitonin, 100 mM Tris-HCl, pH7.4, 150 mM NaCl. After separation by SDS-PAGE, precipitates were analyzed by Western blot with HRP-conjugated streptavidin. In deglycosylation experiments the precipitates were incubated for 18 h with or without N-Glycanase F (Roche) according to the manufacturer's protocol. Pervanadate-treated cells were subjected to immunoprecipitation as described above. Immunoprecipitates were analyzed by Western blot with antiphosphotyrosine PY20-HRP (Transduction Laboratories) or anti-DAP12 rabbit antiserum followed by human/mouse-adsorbed anti–rabbit IgG-HRP (Southern Biotechnology Associates, Inc.).
Chemotaxis Assay.
Monocyte-derived human DCs (107) were treated for 24 h with F(ab')2 21C7, F(ab')2 29E3 coated on plastic (20 µg/ml), or LPS (1 µg/ml). Cells (5 x 105 in 100 µl IMDM/0.5% BSA) were incubated for 1 h at 37°C. Cells were subsequently loaded into collagen-coated Transwells (Costar; 3-µm pore filter), which were placed onto 24-well plates containing 450 µl medium supplement with 100 ng/ml CCL19 (ELC/MIP-3β) or CCL20 (6-C-Kine/SLC) (both from PeproTech). After an incubation period of 4 h at 37°C, cells that had migrated to the lower chamber were collected and counted on a cytofluorimeter (FACSCaliburTM, constant time acquisition; Becton Dickinson). In blocking experiments cells were preincubated with anti-CCR7 mAb (10 µg/ml) and added to the Transwell.
Detection of Apoptosis.
Determination of DNA fragmentation was performed as described previously 65. Inhibitors of kinases or serine proteases (PD98059 [20 µM], LY294002 [10 µM], TPCK [20 µM]) were added 60 min before stimulation. Inhibitors had no effect on cell viability or the rate of constitutive apoptosis at the indicated concentrations.
Nuclear Extracts and Electrophoretic Mobility Shift Assays.
Nuclear extracts were prepared according to the method of Schreiber et al. 66 with some modifications. Stimulation of monocyte-derived human DCs (107) with control or anti–TREM-2 antibody or with LPS was performed for 0.5 or 4 h at 37°C as described above. Cells were washed in PBS, resuspended in 10 ml of ice-cold buffer A (10 mM Tris-HCl, pH 7.9, 60 mM KCl, 1 mM EDTA, 0.75 mM spermidine, 0.15 mM spermine, 1 mM DTT, 0.5 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml pepstatin), and incubated for 15 min on ice. Nonidet P-40 was added from a 10% stock solution to a final concentration of 0.6%, and samples were vortexed for 10 s. After incubation for 3 min on ice, samples were centrifuged at 3,000 rpm for 10 min at 4°C. Nuclei were washed in 10 ml of ice-cold buffer A and resuspended in 30 ml of ice-cold buffer C (20 mM Tris-HCl, pH 8, 0.4 M NaCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 25% glycerol). Nuclei were incubated for 30 min at 4°C, and nuclear extracts were separated from debris by centrifugation at 15,000 g for 15 min at 4°C. Protein concentrations were determined by Bradford assay using Bio-Rad protein assay (Bio-Rad Laboratories). NF-B consensus and mutant binding sites were 5'-AGTTGAGGGGACTTTCCCAGGC-3' and 5'-AGTTGAGGCGACTTTCCCAGGC-3', respectively. Annealed binding sites were radiolabeled using polynucleotide T4 kinase and [32P]-ATP. Radiolabeled oligonucleotides were purified by electrophoresis through an 8% polyacrylamide gel containing 22.5 mM Tris-borate and 0.5 mM EDTA, overnight elution from gel slices at 37°C, concentration using Elutip-d columns (Schleicher & Schuell), and ethanol precipitation. Electrophoretic mobility assays (EMSAs) were performed as described previously 67 with some modifications. Nuclear extracts (2 µg) were incubated with 1 µg of poly(dI-dC) carrier and 1 µg of BSA in a 25 µl reaction mix containing 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM DTT, 1 mM EDTA, and 5% glycerol for 10 min at 4°C in the presence or absence of 25-fold excess of unlabeled oligonucleotide competitors. Labeled binding-site probes (15 fmols, 
5 x 104 cpm) were then added for an additional 20 min of incubation at 4°C. Samples were electrophoresed through a 4% polyacrylamide gel containing 22.5 mM Tris-borate and 0.5 mM EDTA at 4°C.
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Results
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Human Immature Monocyte-derived DCs Express TREM-2, a 40 kD Glycoprotein which Is Associated with DAP12.
In initial studies, TREM-2 transcript was selectively detected in monocyte-derived DCs by reverse transcriptase (RT)-PCR (data not shown). To precisely investigate the cellular distribution of TREM-2 as well as its biochemical characteristics and functions, we produced an anti–TREM-2 mAb (29E3). This antibody stained TREM-2–transfected 293 cells specifically, compared with control transfectants (Fig. 1 A). In agreement with RT-PCR data, TREM-2 was highly expressed on DCs derived from peripheral blood monocytes upon in vitro culture with GM-CSF and IL-4 (Fig. 1 B). DC maturation induced by LPS, TNF, CD40L-expressing cells (Fig. 1 C), IL-1β, CpG oligonucleotides, or aggregated IgG (data not shown) led to complete downregulation of TREM-2. TREM-2 was undetectable on macrophages obtained by culturing monocytes up to 14 d with M-CSF (Fig. 1 B) and on primary DCs of peripheral blood (data not shown). Thus, TREM-2 is preferentially expressed on immature monocyte-derived DCs.
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