(A–D) HSCs were cultured alone or cocultured with DCs. At 24 hours, DCs were washed off, and HSCs were either (A) analyzed for surface marker expression by flow cytometry or (B–D) cultured alone for an additional 24 hours before analysis for inflammatory mediator production in conditioned media. (A) FLDCs induced higher HSC expression of CD40 and ICAM-1. (B and C) HSCs that had been cocultured with FLDCs produced elevated levels of numerous inflammatory mediators, whereas NLDCs only induced HSCs to produce higher KC (P < 0.05). Furthermore, CpG-treated FLDCs induced higher HSC production of IL-1α, IL-6, G-CSF, GMCSF, MIG, and MCP-1 compared with unstimulated FLDCs (P < 0.05). Conversely, priming NLDCs with CpG did not enhance their ability to activate HSCs. (D) To determine the mechanism of DC-HSC cross-talk, in selected experiments, cellular contact was prevented between HSCs and FLDCs by using 0.4-μm transwell inserts. Alternatively, TNF-α was blocked using 5 μg/ml 2E2. Prevention of cellular contact partially abrogated HSC production of IL-1α, MIP-1α, MIP-1β, IL-6, G-CSF, MIP-2, and KC (all P < 0.05). Similarly, TNF-α blockade reduced HSC production of all inflammatory mediators except MIP-1α (P < 0.05). (E) To test DC induction of HSC proliferation, irradiated DCs were coincubated with HSCs for 48 hours. Proliferation was measured by uptake of ³H-thymidine over the final 12 hours. FLDCs induced a 6-fold increase in HSC proliferation (P < 0.05). DC-HSC experiments were repeated at least twice.