PBMC from normal unidentified donors were isolated by Ficoll-Hypaque (Pharmacia Corporation, Peapack, NJ) density gradient centrifugation and were cultured in RPMI 1640 medium with 2 mM L-glutamine (BioWhittaker, Inc., Walkersville, MD) supplemented with 10% heat-inactivated fetal calf serum (FCS) (Gemini Bio-Products, www.gembio.com). Human monocytes were isolated from PBMC preparations by positive selection with CD14 microbeads from Miltenyi Biotec (www.miltenyibiotec.com) as described by the manufacturer, and were cultured at 1×106 cells per well in 6-well plates in Macrophage-SFM medium (Gibco, www.invitrogen.com) supplemented with 15 ng/ml recombinant human MCSF (R&D, www.rndsystems.com) and 5% heat-inactivated human AB serum (Nabi, Boca Raton, FL). The cell cultures were incubated at 37°C and 5% CO2 for 5 days, after which supernatant was replaced with fresh medium lacking MCSF before manipulation. More than 95% of the adherent cells obtained with this technique were CD14+ macrophages as verified by flow cytometry. THP-1 cells were obtained from ATCC (www.atcc.org) and cultured in RPMI 1640 medium supplemented with 10% FCS (BioWhittaker, www.lonzabio.com). 293T cells were obtained from ATCC (www.atcc.org) and were maintained in Dulbecco's Modified Eagle's medium (DMEM) (Gibco, www.invitrogen.com) supplemented with 10% FCS.