Methods: Human gingival fibroblasts (2×104 cells/well) were incubated with α-MEM containing 10% FBS in 96-well flat-bottomed plates for 24 h. Cells were then washed three times with serum-free α-MEM and incubated in the presence or absence of synthetic lipid A (100 ng/ml) and amphotericin B (0.4, 1, or 2.5 μg/ml) in α-MEM containing 10% FBS for 24 h. Levels of secreted human IL-6 and IL-8 in culture supernatants were measured by ELISA. For the depletion of membrane cholesterol, cells were pretreated with methyl-β-cyclodextrin (MβCD) in α-MEM containing 10% FBS for 1 h and washed three times with serum-free α-MEM before treatment with amphotericin B and lipid A. For caspase-8 and caspase-1 inhibition assays, cells were pretreated with inhibitors (10 or 25 µM) or the same volume of DMSO for 1 h, prior to the addition of amphotericin B and lipid A. Cell viability was assessed by measuring the reduction of MTS to formazan. Caspase-8 and caspase-1 activation was measured by flow cytometry. NF-κB activation was measured using an ELISA kit. Data were analyzed using one-way analysis of variance and either the Bonferroni or Dunn method.
Results: Amphotericin B synergistically upregulated lipid A-induced production of IL-6 and IL-8. While Amphotericin B minimally activated NF-κB p50 in human gingival fibroblasts, it synergistically increased lipid A-induced NF-κB p50 activation. Pretreatment with 5 mM MβCD reduced IL-6 and IL-8 production without the downregulation of cell viability. Amphotericin B activated caspase-8. However, a caspase-8 inhibitor inhibited IL-6, but not IL-8, production by amphotericin B and lipid A.
Conclusions: Our data suggest that cholesterol-rich microdomains and caspase-8 are required for the synergistic production of IL-6 by amphotericin B and lipid A in human gingival fibroblasts.