Effect of acute phase cytokines on iron uptake in hepatocytes and differential localization of Lipocalin-2 and Transferrin receptors in rat hepatic and extra hepatic organs

Abstract

Acute phase response (APR) is the systemic reaction to tissue injury and inflammation. It is clinically characterized by systemic symptoms such as fever, weakness, and anemia, somnolence loss of appetite and cytokines release. In the blood, it results in an increase in the plasma levels of a number of positive acute phase proteins (APPs), including clotting proteins, transport proteins, anti-proteases, and complement factors, with a concomitant decrease in negative APPs such as albumin. In addition, a decrease of serum iron-levels and consecutive increase of hepatic iron levels is also a hallmark of APR. This reaction is mediated by acute phase cytokines (APC) such as interleukin-1β (IL-1β), tumor necrosis factor-alpha (TNF-α) and IL-6 cytokines through the activation of different transcription factors. The mechanism of liver iron uptake during APR is poorly understood. Therefore, current study aimed to investigate the exact mechanism of iron uptake with relation to iron regulatory proteins and their signaling pathway under the influence of APC in liver and liver cells. Furthermore, sub-cellular localization of these proteins in hepatic and extra-hepatic organs during APR is also examined. In vitro: primary cultures of rat isolated hepatocytes were stimulated with APC (IL-1β, IL-6 and TNF-α) in the presence/absence of different concentrations of iron (FeCl3: 0.01mM, 0.1mM). APR was induced by injecting turpentine-oil (TO) in hind limbs of rat and mice (wild type and IL-6 knock-out). Hepatocytes cell lysates and cell supernatants were prepared for iron measurement, RT-PCR and Western blotting. Tissue samples (liver, spleen and heart) were re-moved to use for immunobloting and immunohistochemical analysis. In hepatocytes treated with iron in the presence/absence of cytokines, a significant iron up-take was detected with 0.1mM iron administration with a maximum (133µg/g protein) at 24h compared to control and other iron concentrations administrated. This iron uptake was further enhanced in the presence of acute phase cytokines with a maximum iron uptake (481µg/g of pro-tein) after concomitant administration of IL-6 and 0.1mM iron. Concomitantly, gene expression of LCN-2 and ferritin subunits (FTH, FTL) were up-regulated by iron and further induced by acute phase cytokines both, at mRNA and protein level. The maximum increase was seen for the combination of IL-6 with iron. TfR2 was increased by IL-6 and iron alone, however TfR1 was decreased. Combination of iron and APC abrogated the down-regulation of TfR1. Iron as well as APC stimulation led to LCN-2 release into supernatant, with increased release in the presence of iron and APC (IL-6 mainly) combined. Similar to in vitro data, liver tissue of rat and wild type mice also showed an increase in iron transport (TfR1, TfR2 and LCN-2) and storage proteins (FTH, FTL) during APR whereas such an increase was not detected for TfR2, LCN-2 and FTH in IL6-KO mice. In parallel to in-duction of iron transporters, a phosphorylation of STAT3 was also observed in WT-mice howev-er such a change was not noticed in IL-6 KO mice. Immunohistochemistry of hepatic and extra hepatic tissues indicated organ dependent local-ization of TfR1 and TfR2. TfR1 was primarily localized in the membrane and cytoplasm of liver, and spleen cells whereas, in both organs, TfR2 showed nuclear expression mostly. However, in heart, both TfR1 and TfR2 were detected to be surplus in membrane, cytoplasm and nucleus. In all the organs studied, TfR1, TfR2 and LCN-2 were colocalized. The iron storage protein FTL was detected in cytoplasm while a strong nuclear positivity for FTH was evident in the liver. Similarly, in spleen and heart FTL was detected mainly in the cytoplasm while FTH demonstrat-ed intense nuclear and a weak cytoplasmic expression. By summarizing, results of current study demonstrate that liver behaves as a “sponge” for iron during APR. Iron uptake in hepatocytes is tightly controlled by already present iron and this uptake can be further enhanced by APC, mainly by IL-6. Hepatocytes need more iron to respond the massive increase of protein synthesis under such stress conditions. These changes could ex-plain iron retention in hepatocytes during APR. Furthermore, portal blood iron is taken up by liver cells mostly through importer proteins (TfRs and LCN-2) and these proteins are regulated mainly through IL-6 activated STAT3 pathway. The differential localization pattern of iron regu-latory proteins in hepatic and extra-hepatic organs suggests their organ specific diverse role dur-ing APR.