Media and Reagents.
The medium used was RPMI 1640 supplemented with 2 mM l-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% heat-inactivated FCS (all from GIBCO BRL, Gaithersburg, MD). Recombinant human GM-CSF was provided by Sandoz AG (Bale, Switzerland), recombinant human IL-4 was purchased from Genzyme Corp. (Cambridge, MA), and recombinant human TGF-β1, polyclonal chicken anti–human TGF-β1 neutralizing antibodies, and control chicken Ig were all from R&D Systems (Minneapolis, MN).
Culture of Peripheral Blood Monocytes.
monocytes were isolated from peripheral blood mononuclear cells of healthy volunteers by negative magnetic depletion using hapten-conjugated CD3, CD7, CD19, CD45RA, CD56, and anti-IgE antibodies (monocyte isolation kit, MACS; Miltenyi Biotec, Berglsch Gladbach, Germany) and a magnetic cell separator (MACS) according to the manufacturer's instructions, routinely resulting in
95% purity of CD14+
Cells were cultured in 6- or 24-well tissue culture plates (Costar Corp., Cambridge, MA) in fresh complete medium supplemented with 250 ng/ml GM-CSF + 100 ng/ml IL-4. TGF-β1 was added at the beginning or, in some experiments, at day 2, 4, or 6 of the culture. At days 2 and 4, half of the medium was removed and an equivalent volume of fresh medium, supplemented with the above mentioned cytokines, was added.
Cell Proliferation and Apoptosis Studies.
For immunocytochemical detection of cell cycle–associated antigens, harvested cells were cytocentrifugated using a Cytospin 3 device (Shandon SA, Eragny, France). Slides were air-dried, fixed in acetone, and stained with anti-Ki67 antibodies (clone Mib-1, IgG1; Immunotech, Marseille, France) with an avidin-biotin-peroxidase protocol (19
) revealed by 3-3′ diaminobenzidine as chromogen (Vectasin ABC kit, Vector, CA). [3
Life Science, Buckinghamshire, UK) incorporation was measured in newly synthesized DNA over 48 h, using pulses initiated at day 0, 2, or 4 of the culture with 1 μCi/well of [3
H]thymidine. Cells were then harvested with a 96-well Harvester (Pharmacia
, St. Quentin, France), collected on glass-fiber filter (Pharmacia
) and the incorporation of thymidine was measured with a Beta-plate microscintillation counter (LKB, Pharmacia
). For apoptosis analysis by flow cytometry (20
) and cell cycle analysis, 5 × 105
cells for each point were resuspended in 500 μl PBS containing 0.1% triton and 50 μg/ml propidium iodide (PI) (Sigma Chemical Co.
, St. Louis, MO), and incubated for 20 min at 37°C. Analysis of the DNA content of 3 × 104
cells was performed immediately using a flow cytometer FACScan®
, Mountain View, CA) with the Lysis II software (Becton Dickinson
). Allogeneic mixed leukocyte reaction was performed as previously described (1
Flow Cytometry and Cytological and Immunocytological Analysis of PBMCs and PBMC-derived Cells.
For single- and two-color flow cytometry, 1.5 × 105 cells were incubated for 30 min at 4°C in PBS, 2% human AB serum, and 0.01 M NaN3, with FITC-conjugated CD1a (clone BL1, IgG1; Immunotech), CD80 (IgG1; Immunotech), CD40 (IgG1; Immunotech), HLA-I (HLA ABC, IgG2a; Immunotech), HLA-II (IgG1; Becton Dickinson), and/or PE-conjugated CD14 (Leu-M3, IgG2b; Becton Dickinson) and CD11b (IgG2a; Becton-Dickinson) mAbs at the appropriate concentration, or with control isotype-matched irrelevant mAbs at the same concentration. After washing, cells were analyzed with a FACScalibur® (Becton Dickinson) using CellQuest software (Becton Dickinson). For cytological examination, harvested cells were processed as described above and stained using the May-Grunwald-Giemsa technique (RAL, Rieux, France). For immunochemistry, slides were stained with CD1a (BL1, IgG1; Immunotech), E-cadherin (HECD-1, IgG1; R&D Systems), Lag (mouse IgG1; gift of Dr F. Furukawa, Hamamastu University, Hamdacho, Japan), and CLA (HECA-452, rat IgM; gift of Dr. L.J. Picker, University of Texas, Dallas, TX).
Cells to be processed for electron microscopy were fixed overnight at 4°C in 2.5% glutaraldehyde (Sigma Chemical Co.) in 0.1 M cacodylate buffer, pH 7.4, followed by postfixation for 1–2 h in 0.1 M cacodylate-buffered 2% osmium tetraoxide (Merck, Darmstadt, Germany). After dehydration in a series of graded ethanol and propylene oxide solutions, cells were embedded in epoxy resin (Epon 812; TAAB, Janning, Vanves, France). Ultrathin sections were stained with uranyl acetate and lead citrate and examined with a JEOL.1200 EX2 electron microscope (JEOL, Croissy sur Seine, France).