Amphotericin B and lipid A synergistically up-regulate cytokine production

Objectives: Amphotericin B is an antifungal drug used to treat candidiasis. However, amphotericin B is also reported to induce production of proinflammatory cytokines by cultured cells. Thus, we investigated the effects of amphotericin B on proinflammatory cytokine production in response to lipid A, the bioactive component of LPS in the outer cell wall of gram-negative bacteria. Methods: Human gingival fibroblasts (1x10⁵cells/well) were incubated with α-MEM containing 10% FBS in 24-well flat-bottomed plates for 24 h. Cells were then washed twice 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. Secreted human IL-6 and IL-8 levels in culture supernatants were measured by ELISA. For Smad3 inhibition assays, we pretreated cells with an inhibitor for 1 h prior to addition of amphotericin B and lipid A. Data were analyzed using one-way analysis of variance and either the Bonferroni or Dunn method. To detect Smad3 and MyD88 in human gingival fibroblasts, cells were treated with or without amphotericin B (2.5 μg/ml) and lipid A (100 ng/ml) for 24 h and then fixed with formaldehyde followed by methanol. After incubation with antibodies, collected cells were analyzed by flow cytometry. NF-κB activation and nuclear translocation were assessed by ELISA using nuclear protein extracts. Results: Treatment with amphotericin B alone slightly up-regulated IL-6 and IL-8 production by human gingival fibroblasts. Amphotericin B synergistically up-regulated lipid A-mediated production of IL-6 and IL-8. While amphotericin B minimally activated MyD88 and Smad3, it synergistically increased NF-κB activation by lipid A. Conclusion: These results suggest that amphotericin B and lipid A synergistically induce NF-κB activation and production of IL-6 and IL-8 in human gingival fibroblasts.