Production and subcellular localization of IL-18 in iDCs and mDCs. (A) Western blot analysis of IL-18 (top blot) and IL-1β (bottom blot) in cell lysates from iDCs cultured 24 hours without (-; lane 1) or with 50 ng/mL TNF-α (lane 2), 1 μg/mL LPS (lane 3) or 1 μg/mL HMGB1 (lane 4). (B) Measurement of IL-12 production. IL-12 concentrations in culture supernatants of 4- or 24-hour incubations of iDCs or LPS-treated DCs (5 × 105 cells) were measured using a human IL-12 ELISA kit. Results are expressed as picograms per milliliter. Error bars represent standard error of a single experiment performed in triplicate. (C) Subcellular localization of IL-18 in iDCs and mDCs; iDCs (lanes 1, 3, 5, and 7) and mDCs (treated 24 hours with TNF-α; lanes 2, 4, 6, and 8) were homogenized and postnuclear supernatants (PNS; lanes 1 and 2, 1/20 of total) were subjected to differential ultracentrifugation giving rise to 2 pellets enriched in lysosomes (Ly; lanes 5 and 6) and endosomes (End; lanes 7 and 8), respectively. Lanes 3 and 4 show soluble cytosol (cyt). Fractions were blotted and hybridized with anti-IL-18 (top blot) and anti-cathepsin D (CD; bottom blot). One representative experiment of 4 performed is shown.
