Hepatic macrophages (HM) function as the primary effector cells in alcoholic liver injury via the release of cytotoxic and pro-inflammatory mediators. Expression of many of these mediators are transcriptionally regulated by a redox-sensitive trans-acting factor, NF-kappaB. We have shown that the treatment of cultured Kupffer cells with a lipophilic iron chelator, 1,2-dimethyl-3-hydroxypyrid-4-one (L1) abolishes LPS-stimulated NF-kappaB activation and expression of TNFalpha and IL-6. Administration of L1 to rats with cholestatic liver injury leads to abrogation of HM NF- kappaB activation and TNFalpha mRNA expression with resultant amelioration of liver injury. HM from rat5s with alcoholic liver injury, exhibit NF-kappaB activation, TNFalpha up-regulation, and an increased non-heme iron content. Ex vivo treatment of the cells with L1 normalizes all thee changes. The expanded iron storage in HM of these animals was accompanied by the increased content of ferritin L-chain mRNA and ferritin protein, as well as splenic iron accumulation. These cells also show enhanced hemeoxygenase -I mRNA expression, suggesting increased heme metabolism. Based on these results, we hypothesize that an increased iron storage primes HM for NF-kappaB mediated responses in alcoholic liver injury. We further advance the hypothesis to state that intracellular chelatable iron which is liberated from stored iron by oxidative stress, participates in NF-kappaB activation an that the activities of iron-regulated mRNA binding proteins, IRP (iron regulatory proteins), provide feed back regulation for this process via their known control over a catalytically active iron pool. To test these hypotheses, we will examine: 1) in vitro effects of iron status on temporal changes in the levels of chelatable iron, NF-kappaB and IRP mediated responses in LPS-stimulated cultured Kupffer cells; 2) in vivo temporal changes in cellular iron, NF-kappaB and IRP mediated responses in HM during evolution of alcoholic liver injury; 4) n vivo effects of HM iron depletion with liposome encapsulated iron chelators on NF- kappaB mediated responses and alcoholic liver injury; and 5) effects of splenectomy on iron storage and NF-kappaB activation by HM in alcoholic liver injury.
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