Figure 4

Zinc supplementation decreased neutrophil-derived cytokine production via decreased NF-κB activation. A. Bone marrow-derived neutrophils were isolated and treated with GC frass (1 μg/ml) or LPS (1 μg/ml) with or without zinc gluconate (1 μM) and pyrithione (10 μM) for 18 h. Supernatants were clarified and analyzed by ELISA for TNFα production. Data are means ± SEM (n = 3 separate experiments) and statistical differences assessed by ANOVA (*p < 0.05). B. HL-60 cells were treated as in A and analyzed by ELISA for TNFα production. Data are expressed as mean ± SEM for four experiments (*p < 0.001). C. Zinc supplementation decreased NF-κB-DNA binding. HL-60 cells were stimulated with GC frass or LPS in the presence of zinc and pyrithione for 4 h. Nuclear extracts were analyzed for NF-κB-DNA binding by EMSA. D. Zinc supplementation decreased IKK activity. HL-60 cells were cultured with or without zinc gluconate and pyrithione prior to treatment with GC frass for 1 h. IKK activation was assessed by in vitro kinase assay using recombinant IκBα as a substrate. PBS treated (P) and GC frass treated (F) cells cultured with and without zinc.