NF-kappaB activation induced by lipopolysaccharide (LPS) in cultured hepatic macrophages (HM) may be abrogated by pretreatment of cells with a lipophilic iron chelator, 1,2-dimethyl-3-hydroxypyrid-4-one (L1, deferiprone), suggesting a role for iron in this molecular event [M. Lin, M., R. A. Rippe, O. Niemelä, G. Brittenham, and H. Tsukamoto, Am. J. Physiol. 272 (Gastrointest. Liver Physiol. 35): G1355-G1364, 1997]. To ascertain the relevance in vivo of this hypothesis, HM from an experimental model of alcoholic liver injury were examined for the relationship between nuclear factor (NF)-kappaB activation and iron storage. HM showed a significant increase in nonheme iron concentration (+70%), accompanied by enhanced generation of electron paramagnetic resonance-detected radicals (+200%), NF-kappaB activation (+100%), and tumor necrosis factor-alpha (+150%) and macrophage inflammatory protein-1 (+280%) mRNA induction. Treatment of the cells ex vivo with L1 normalized all these parameters. HM content of ferritin protein, ferritin L chain mRNA, and hemeoxygenase-1 mRNA and splenic content of nonheme iron were increased, suggesting enhanced heme turnover as a cause of the increased iron storage and NF-kappaB activation. To test this possibility, increased iron content in HM was reproduced in vitro by phagocytosis of heat-treated red blood cells. Treatment caused a 40% increase in nonheme iron concentration and accentuated LPS-induced NF-kappaB activation twofold. Both effects could be abolished by pretreatment of cells with zinc protoporphyrin, a hemeoxygenase inhibitor. To extend this observation, animals were splenectomized before 9-wk alcohol feeding. Splenectomy resulted in further increments in HM nonheme iron storage (+60%) and NF-kappaB activation (+90%) and mononuclear cell infiltration (+450%), particularly around the iron-loaded HM in alcohol-fed animals. These results support the pivotal role of heme-derived iron in priming HM for NF-kappaB activation and expression of proinflammatory genes in alcoholic liver injury.

TBX6-associated congenital scoliosis (TACS) as a clinically distinguishable subtype of congenital scoliosis: further evidence supporting the compound inheritance and TBX6 gene dosage model

Iron exacerbates various types of liver injury in which nuclear factor (NF)-kappaB-driven genes are implicated. This study tested a hypothesis that iron directly elicits the signaling required for activation of NF-kappaB and stimulation of tumor necrosis factor (TNF)-alpha gene expression in Kupffer cells. Addition of Fe2+ but not Fe3+ (approximately 5-50 microM) to cultured rat Kupffer cells increased TNF-alpha release and TNF-alpha promoter activity in a NF-kappaB-dependent manner. Cu+ but not Cu2+ stimulated TNF-alpha protein release and promoter activity but with less potency. Fe2+ caused a disappearance of the cytosolic inhibitor kappaBalpha, a concomitant increase in nuclear p65 protein, and increased DNA binding of p50/p50 and p65/p50 without affecting activator protein-1 binding. Addition of Fe2+ to the cells resulted in an increase in electron paramagnetic resonance-detectable.OH peaking at 15 min, preceding activation of NF-kappaB but coinciding with activation of inhibitor kappaB kinase (IKK) but not c-Jun NH2-terminal kinase. In conclusion, Fe2+ serves as a direct agonist to activate IKK, NF-kappaB, and TNF-alpha promoter activity and to induce the release of TNF-alpha protein by cultured Kupffer cells in a redox status-dependent manner. We propose that this finding offers a molecular basis for iron-mediated accentuation of TNF-alpha-dependent liver injury.

A redox-sensitive nuclear factor, NF-kappa B, induces transcription of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in macrophages. The present study has investigated the role of iron in NF-kappa B activation and TNF-alpha and IL-6 expression by rat hepatic macrophages (HM). As an in vivo model, cholestatic liver injury was induced in rats by ligation of the common bile duct (BDL). During the first 2 wk after BDL, there was an increase in the hepatic level of thiobarbituric acid-reactive substances (TBARS) that was accompanied by the appearance of protein-malondialdehyde adducts in the periportal region. This increase was reduced after 3 wk. TNF-alpha and IL-6 mRNA levels in HM from the BDL rats were increased at 1 and 2 wk and attenuated at 3 wk. Gel mobility shift assay of HM nuclear extracts demonstrated the similar temporal pattern of enhanced NF-kappa B binding activity. Treatment of the BDL animals with 1,2-dimethyl-3-hydroxypyrid-4-one (L-1), a lipophilic iron chelator, suppressed the increases in hepatic TBARS by 64%, plasma alanine aminotransferase by 45%, and HM TNF-alpha and IL-6 mRNA by > 84%. Concomitantly, the HM NF-kappa B binding activity was reduced close to the level observed in sham-operated rats. Treatment of cultured HM with L-1 also blocked lipopolysaccharide-stimulated NF-kappa B activation and TNF-alpha and IL-6 expression at mRNA and protein levels. These results demonstrate that the iron chelator effectively blocks NF-kappa B activation and coordinate TNF-alpha and IL-6 gene upregulation by HM in cholestatic liver injury or under in vitro lipopolysaccharide stimulation. These findings support a pivotal role for iron in activation of NF-kappa B and cytokine gene expression by HM in vitro and in vivo.