MATERIALS AND METHODS

Isolation of monocytes and cell culture. Monocytes were obtained from buffy coats from healthy blood donors. Written informed consents were obtained from all donors in accordance with the ethical principles set out in the declaration of Helsinki. This study was approved by the Medical Ethics Committee of the Academic Medical Center and the Ethics Advisory Body of the Sanquin Blood Supply Foundation in Amsterdam, The Netherlands. Peripheral blood mononuclear cells were isolated from buffy coats using Lymphoprep (Axis-Shield, Oslo, Norway) density gradient centrifugation. Monocytes were isolated by adherence to plastic and cultured in Iscove’s modified Dulbecco’s medium (Lonza, Basel, Switzerland) supplemented with 10%[vol/vol] heat-inactivated human pooled serum, penicillin (100 U/ml; Invitrogen, Carlsbad, CA), streptomycin (100 g/ml, Invitrogen) and ciproxin (5 g/ml; Bayer, Leverkusen, Germany) for 5 days in the presence of different cytokines: IFN-at 50 U/ml (Sigma-Aldrich), in combination with TNF-at 12.5 ng/ml (Peprotech, Rocky Hill, NJ), IL-4 at 50 ng/ml (Peprotech), IL-10 at 50 ng/ml (Peprotech), or medium alone at 37 C in a humidified atmosphere supplemented with 5% CO2.