Figure 4. HBsAg induces the production of TNF-α and IL-10 in monocytes.

(A) Monocytes and pDCs were isolated using magnetic beads. 3×10⁴ pDCs were either co-cultured or separated by a transwell insert with 6×10⁵ monocytes in a 24 well in 10% FCS-RPMI 1640 containing IL-3 (10 ng/ml). The cells were either pretreated or not with 1 µg/ml pHBsAg and stimulated with 3 µM CpG ODN2216 for additional 24 hours. The IFN-α production was determined by ELISA. The data shown are representative of at least three experiments. (B) 2×10⁵ monocytes and 1×10⁴ pDCs were co-cultured in 10% FCS-RPMI 1640 containing IL-3 (10 ng/ml) in 96-well culture plates in a total volume of 200 µl. The cells were pretreated with 1 µg/ml pHBsAg and stimulated with 3 µM CpG ODN2216 for 24 hours. The TNF-α production was determined by an ELISA. The data are representative of at least three experiments. (C) CD14+-depleted PBMCs were cultured at a concentration of 2×10⁶/200 µl in 96-well culture plates and then pretreated with 1 µg/ml pHBsAg followed by stimulation with 3 µM CpG ODN2216 for 24 hours. The TNF-α production was determined by an ELISA. The data are representative of at least three experiments. (D) PBMCs and CD14+-depleted PBMCs were cultured at a concentration of 2×10⁶/200 µl in 96-well culture plates and pretreated with 1 µg/ml pHBsAg followed by stimulation with 3 µM CpG ODN2216 for 24 hours. The IL-10 production was determined by an ELISA. The data are representative of at least three experiments. (E) Monocytes were isolated by magnetic bead treatment and cultured in 96-well plates at a concentration of 1×10⁵/200 µl. pHBsAg at a concentration of 1 µg/ml was added to the culture supernatant for 24 hours, and the production of TNF-α and IL-10 was determined by an ELISA.