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′-tagtagGTCGAC
ATACTTACCGGGTGGGAAAGGGATTTCTCCTTCCAA. 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.
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,κ), mAb 21C7 (control IgG1, κ, anti–TREM-1) 53
, and mAb 1B7.11 (control IgG1, κ, 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).
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-α (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 FACSCalibur™ cytometer using CELLQuest™ 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 × 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-1α, IL-1β, TNF-α, 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.
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.).
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 × 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 (FACSCalibur™, 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-κB consensus and mutant binding sites were 5′-AGTTGAGGGGACTTTCCCAGGC-3′ and 5′-AGTTGAGGCGACTTTCCCAGGC-3′, respectively. Annealed binding sites were radiolabeled using polynucleotide T4 kinase and γ[32
P]-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 × 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.