Following isolation, PBMC were cultured at a final concentration of 3 × 106 cells/ml (for ELISA measurements) or 5 × 106/ml (for gel shifts) or 1 × 106 cells/ml (for flow cytometry) in RPMI 1640 medium (BioWhittaker, Heidelberg, Germany, LPS-free), containing 10% heat-inactivated fetal calf serum (FCS, low-LPS, Invitrogen, Karlsruhe, Germany), 1% penicillin (10000 U/ml), 1% streptomycin (10000 μg/ml), and 1% 200 mM L-glutamine (BioWhittaker).