Iron and HM NF-kappa B activation in alcohol model

Our earlier study showed that hepatic macrophages (HM) from rats fed ethanol and high fat diet had a significant 70% increase in the nonheme iron content as compared to controls [1]. This study also suggested enhanced heme turnover as a cause of the increased iron storage in HM. To test this notion, an increase in HM iron content was recapitulated in vitro by phagocytosis of heat-treated autologous red blood cells. To extend this observation to the whole animal situation, the effects of splenectomy on alcohol-fed animals were also examined. The most intriguing and critical finding from these cellular or animal model experimentations, was that activation of NF-kappa B was tightly correlated with the increased non-heme iron content in HM, suggesting the priming role of iron in NF-kappa B activation and proinflammatory cytokine expression by HM in alcoholic liver disease.

Direct iron induction of TNF-alpha in cultured HM

Direct addition of ferrous but not ferric iron in cultured HM increased TNF-alpha release 8 fold at 10 and 50 micromolar during a 4 hr treatment period without cell toxicity. Cuprous (Cu1+) but not cupric (Cu2+) copper also stimulated TNF-alpha release at 50 micromolar to less extent. Thus, these results demonstrate direct stimulation of HM TNF-alpha release by iron and copper in a redox status dependent manner. We then tested whether Fe2+ stimulates TNF-alpha promoter in cultured HM. The promoter activity was indeed increased 2~3 fold with 10~50 micromolar Fe2+. Cu1+ (50 micromolar) also slightly increased TNF-alpha promoter activity but not Cu2+ or Fe3+ (Figure 1). Co-transfection of a super repressor I-kappa B-alpha vector completely abrogated the stimulation with 50 micromolar Fe2+ (Figure 1). The enhanced promoter activity with 50 micromolar Fe2+ was about half of the maximal response achieved with LPS (500 ng/ml) in a serum-free condition. These results establish that Fe2+ activates TNF-alpha promoter in a NF-kappa B dependent manner.

Figure 1
figure 1

(A.) Cultured HM were transfected with a TNF-alpha promoter-lucigerase construct followed by the treatment with Fe2+, Fe3+, Cu1+ or Cu2+. The data were normalized by co-transfected Renilla luciferase activity. Note the Fe2+ induces the promoter activity by 2–3 fold at 10 and 50 micromolar. Cu1+ slightly induces but oxidized metals (Fe3+ and Cu2+) do not. (B.) HM were co-transfected with the promoter-luciferase construct with a vector of super-repressor I-kappa B-alpha (DN-I-kappa B), followed by addition of Fe2+. Note DN-I-kappa B completely blocks iron induced promoter activity.

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Iron-induced activation of IKK and NF-kappa B and ROS generation in cultured HM

As shown in the top panel of Figure 2, IKK activity, as assessed by phosphorylation of GST-I-kappa B-alpha, was increased at 15 min after addition of ferrous iron. The timing of IKK activation preceded a disappearance of cytosolic I-kappa B-alpha and an increase in the nuclear level of p65 at 30~45 min. Iron did not induce JNK and p38 activities (unpublished results). These results correlated well with induced NF-kappa B binding in iron-treated HM at 30 min and no effects on AP-1 binding in these cells (data not shown). Thus, these results unequivocally demonstrate that ferrous iron can directly and selectively stimulates the signaling leading to IKK and NF-kappa B activation in cultured HM. Addition of ferrous iron to these cells resulted in an enhancement of the hydroxyl and methyl-POBN adduct signals. Both signals increased with incubation time up to a maximum at 15–20 min, which coincided with IKK activation at 15~30 min and preceded activation of NF-kappa B at 30 min, suggesting the possible signaling role of the former in the latter events. In fact, this notion was made by previous studies which demonstrated activation of NF-kappa B by hydroxyl radical generating systems and a reversal of this effect by hydroxyl radical scavengers or metal chelators in Jurkat cells [2].

Figure 2
figure 2

I-kappa B kinase (IKK) and c-Jun NH2-terminal kinase (JNK) activity were determined on HM lysate collected at different time points after FeSO4 treatment. Note that IKK activity as assessed by phosphoylation of GST-I-kappa B-alpha (P-I-kappa B-alpha), is increased at 15 min after addition of iron while no activation of JNK is evident.

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Discussion and Conclusion

Our studies to date strongly suggest the causal link between iron and activation of NF-kappa B in HM in both normal and alcohol-fed rats. These findings raise a question as to how iron signals to activate NF-kappa B. Since NF-kappa B is a redox sensitive transcription factor and ROS are implicated in its activation [3, 4], it is reasonable to speculate that iron stimulates ROS production in HM and in turn ROS activates NF-kappa B. Conversely, stimulation of HM with an agonist such as LPS, induces ROS generation and ROS may initiate intracellular signaling that is dependent on a chelatable pool of iron. Nitric oxide (NO) is known to cause mobilization of intracellular iron [5] and to inhibit enzymes with catalytically active iron-sulfur groups [6]. Superoxide anion can also release iron from ferritin [7]. Indeed, our preliminary results demonstrate that a selective inhibitor of iNOS and Cu/Zn SOD overexpression abolish a LPS-mediated transient rise in the intracellular level of chelatable iron and NF-kappa B activation (unpublished observation). We propose that iron acts as a proinflammatory effector molecule via selective induction of the intracellular signaling for NF-kappa B activation and that dysregulation of this signaling mechanism may prime HM for chronic liver inflammation and injury.