Cell culture and transfection
CHO cells were maintained in RPMI 1640 + 10% FCS, transfected using Lipofectamine 2000 (Invitrogen), and used 24 h after transfection. BMDMs were derived from femurs of sex- and age-matched wt or knockout mice (C57BL/6 background) as described previously (Roach et al., 1997
). DAP12-deficient mice (provided by L. Lanier, University of California, San Francisco, San Francisco, CA) and TREM-2–deficient mice were previously described (Bakker et al., 2000
; Turnbull et al., 2006
). Syk-deficient cells were obtained as described previously (Mocsai et al., 2006
). Cells were maintained in DME with 10% FCS and 10% CMG14-12 cell culture supernatant as a source of macrophage colony-stimulating factor.
Retrovirus-mediated transduction in BMDMs
cDNAs (D12, T1D12, and T2D12) were introduced into BMDMs by using retroviral infection as described previously (Hamerman et al., 2006
). The packaging cell line Plat-E (provided by T. Kitamura, University of Tokyo, Tokyo, Japan; Morita et al., 2000
) was transfected with retroviral constructs using Lipofectamine 2000. Virus-containing supernatants were collected 48 h later, added onto 3-d marrow cells in the presence of 4 µg/ml polybrene, and incubated for at least 3 d before assays. Retrovirus-mediated depletion of endogenous TREM-2 was obtained as previously described (Hamerman et al., 2006
TREM antibodies were purchased from R&D Systems. Syk antibody was purchased from Cell Signaling Technology. Cyto D and bafilomycin A1 were obtained from Sigma-Aldrich. Genistein, PP2, C. difficile toxin, LY29004, ML-7, and DNA–protein kinase inhibitor were purchased from EMD. Alexa Fluor particles and dye and E. coli antibodies were obtained from Invitrogen.
TREM–DAP12 and DAP12 cDNAs were previously described (Hamerman et al., 2006
), and Flag-Syk cDNA (provided by I. Frasier, California Institute of Technology, Pasadena, CA) was previously described (Zavzavadjian et al., 2007
). Myc-tagged dn RhoA, Rac1, and Cdc42 were generated in the laboratory of G. Bokoch (The Scripps Research Institute, LA Jolla, CA) and distributed by Addgene.
Alexa Fluor 594 or 488 particles (zymosan, E. coli, and S. aureus) were purchased from Invitrogen. AsRed– and GFP–E. coli were provided by the Sandia National Laboratories. F. tularensis strain U112 was provided by D. Monack (Stanford University, Stanford, CA). All cultures were grown to midlog phase. Alexa Fluor 594 staining of F. tularensis was performed according to the manufacturer's instructions. Bacterial viability after staining was assessed by dilution plating and found to be comparable with that of unstained bacteria.
Cells were transferred into HBSS + 0.1% BSA for 1 h. 1 µM bafilomycin A1 was added to prevent lysosomal degradation of internalized particles. To block phagocytosis and to assess bacteria binding, 2 µM cyto D was added in control wells for 10 min. Alternatively, cells were transferred to 4°C to allow particle binding but not internalization. Particles at an MOI of 10:1 (BMDM) or 50:1 (CHO) were spun onto cells at 500 g for 3 min, and phagocytosis assays were performed for 60 min in a 37°C incubation chamber. For analysis by microscopy, cells challenged with 594–E. coli were fixed with 3.7% PFA and stained with an E. coli antibody (Invitrogen). The number of extracellular cell-bound (double stained) and intracellular (single stained) bacteria was scored on 50 cells. For analysis by flow cytometry (FACSCalibur; BD), the mean fluorescence intensity of cell-associated bacteria was determined. Analysis was performed on the whole population with parental cells or was restricted to transduced BMDMs or transfected CHO cells (expressing GFP in each case) as indicated. A quantitative estimation of phagocytosis was obtained by subtracting extracellular fluorescence from total bacterial fluorescence.
Bacteria colocalization with cells () was analyzed with a fluorescence microscope (Eclipse TE300; Nikon) with a 60× objective. Images were acquired with a camera (CoolSNAP HQ2; Photometrics) using Simple PCI software (Compix). Triple-staining experiments () were analyzed with a fluorescence confocal microscope (LSM510; Carl Zeiss, Inc.) using a 63× objective.
TREM surface staining
CHOs were fixed with PFA and incubated with 5 µg/ml TREM-1 or TREM-2 antibodies for 30 min at 4°C followed by 2 µg/ml Alexa Fluor 647 secondary antibodies for 30 min at 4°C. TREM surface staining was analyzed by flow cytometry.
Syk and myc intracellular staining
CHO cells transfected with Flag-Syk or myc-tagged RhoA, Rac1, and Cdc42 were detached, fixed in PFA, permeabilized with −20°C methanol, incubated with 5 µg/ml anti-Syk or 2.5 µg/ml anti-myc for 30 min at 4°C, and then incubated with 2 µg/ml Alexa Fluor 647 secondary antibodies. Intracellular staining was measured by flow cytometry.
T2D12 cells incubated with E. coli for 60 min were fixed in Karnovsky's fixative, postfixed in reduced OsO4, and stained with uranyl-acetate. After ethanol dehydration and clearing in propylene oxide, cells were embedded in eponate 12 (Ted Pella Co.). Thin sections were examined under an electron microscope (Tecnal 10; Philips).
All samples were analyzed in duplicates. Experiments were repeated at least three times, and statistical significance was determined by using the two-tailed paired Student's t test. Results were considered significant when P ≤ 0.05.