(A) Analysis of TNF-α, IL-1β, IL-6, and IL-10 by ELISA from HIV-1 nef-transfected HMØs. Purified HMØs were cultured in macrophage media in the presence of IL-3 (5 ng/mL) and GM-CSF (20 ng/mL). After 4 days in culture, cells were transfected with HIV-1 nef cDNA conjugated with transferrin-poly(L-lysine) in the presence of AD, dl312 and incubated for 48 hours. Cells were washed to remove growth factors and transferrin. HMØs were cultured in macrophage media containing 5% FBS and treated with 20 ng/mL PMA for donors no. l, 2, and 4 and 1 ng/mL for donor no. 3 for 72 hours. Conditioned media were analyzed for TNF-α (a), IL-1β (b), IL-6 (c), and IL-10 (d) by ELISA. HMØs from 4 donors were analyzed and represented individually. (B) Comparative analysis of cytokines produced by Ad-d1312-mediated nef-transfected HMØ treated with LPS and PMA. Cells were transfected and maintained as described in (A). HMØs were treated with LPS (100 ng/mL) or PMA (20 ng/mL) for 72 hours; conditioned media were analyzed for the presence of cytokines by ELISA. Values presented are the average of four different experiments.