REGULATION OF IRON UPTAKE IN PRIMARY CULTURE RAT HEPATOCYTES: THE ROLE OF ACUTE-PHASE CYTOKINES Shakil Ahmad, Sadaf Sultan, Naila Naz, Ghayyor Ahmad, Salamah Mohammad Alwahsh, Silke Cameron, Federico Moriconi, Giuliano Ramadori, and Ihtzaz Ahmed Malik Division of Gastroenterology and Endocrinology, Department of Internal Medicine, University Medical Center, Georg-August University, Goettingen, Germany Received 13 Sep 2013; first review completed 2 Oct 2013; accepted in final form 2 Dec 2013 ABSTRACT—Decreased serum and increased hepatic iron uptake is the hallmark of acute-phase (AP) response. Iron uptake is controlled by iron transport proteins such as transferrin receptors (TfRs) and lipocalin 2 (LCN-2). The current study aimed to understand the regulation of iron uptake in primary culture hepatocytes in the presence/absence of AP mediators. Rat hepatocytes were stimulated with different concentrations of iron alone (0.01, 0.1, 0.5 mM) and AP cytokines (inter- leukin 6 [IL-6], IL-1", tumor necrosis factor !) in the presence/absence of iron (FeCl 3 : 0.1 mM). Hepatocytes were harvested at different time points (0, 6, 12, 24 h). Total mRNA and proteins were extracted for reverse transcriptaseYpolymerase chain reaction (RT-PCR) and Western blot. A significant iron uptake was detected with 0.1 mM iron administration with a maxi- mum (133.37 T 4.82 Hg/g of protein) at 24 h compared with control and other iron concentrations. This uptake was further enhanced in the presence of AP cytokines with a maximum iron uptake (481 T 25.81 Hg/g of protein) after concomitant administration of IL-6 + iron to cultured hepatocytes. Concomitantly, gene expression of LCN-2 and ferritin subunits (light- and heavy-chain ferritin subunits) was upregulated by iron or/and AP cytokines with a maximum at 24 h both at mRNA and protein levels. In contrast, a decreased TfR1 level was detected by IL-6 and iron alone, whereas combination of iron and AP cytokines (mainly IL-6) abrogated the downregulation of TfR1. An increase in LCN-2 release into the supernatant of cultured hepatocytes was observed after addition of iron/AP cytokines into the medium. This increase in secretion was further enhanced by combi- nation of IL-6 + iron. In conclusion, iron uptake is tightly controlled by already present iron concentration in the culture. This uptake can be further enhanced by AP cytokines, mainly by IL-6. KEYWORDS—Transferrin receptors, lipocalin 2 (LCN-2), acute-phase cytokines (IL-1", IL-6, TNF-!), FeCl 3 INTRODUCTION Despite the abundance of iron in nature and in the human body, iron absorption, transport, storage, and excretion are tightly regulated. Within the cell, iron is mainly stored in the form of ferritin (1). In human, ferritin is composed of two subunits: the light-chain ferritin subunit (FTL; with 125 amino acids, 19 KDa) and the heavy-chain ferritin subunit (FTH; 183 amino acids, 21 KDa). Both subunits are highly conserved (2); nevertheless, they are genetically separate (3) and maintain distinct functions (4). Iron homeostasis is controlled by a large group of iron regu- latory proteins including ferroportin 1 (5), transferrin receptors (TfR1, TfR2) (6), hepcidin (7), and hemojuvelin (8). In fact, transferrin (Tf)Ybound iron is imported into the reticuloendo- thelial system, to liver parenchymal cells and to all proliferating cells in the body after binding to TfRs. Interaction of diferric-Tf with TfRs and internalization of the complex by receptor- mediated endocytosis leads to iron uptake into the cells (9). As a result, Tf efficiently transports the majority of iron into the cells (10). However, there has been convincing evidence that, in situations of disrupted iron homeostasis, iron can also be delivered to cells by alternative, Tf-independent mechanisms. This alternative to Tf iron is called nonYTf-bound iron (NTBI). This pool has been documented in a variety of iron overload syndromes when Tf is saturated (11), including hemochroma- tosis (69% of patients) and end-stage renal disease (22% of patients) (12); the identification of the components of NTBI, however, remains elusive. Lipocalin 2 (LCN-2; human ortholog neutrophil gelatinase- associated lipocalin) has been proposed to be a mediator of the Tf-independent iron delivery pathway (13). It belongs to the lipocalin family, which is known to be involved in the regu- lation of immune responses, modulation of cell growth and metabolism, prostaglandin synthesis, and iron transportation (13). In fact, it is a bacteriostatic agent and capable of se- questering iron in the form of siderophores (14). A recent study has shown that LCN-2 siderophoreYiron complexes can transport iron into cells during kidney development (10). Acute-phase response (APR) is the systemic reaction to tissue injury and inflammation. It is clinically characterized by sys- temic symptoms such as fever, weakness, anemia, somnolence loss of appetite, and cytokines release (15). In the blood, it re- sults in an increase in the plasma levels of a number of positive acute-phase proteins (APPs), including clotting proteins, trans- port proteins, antiproteases, and complement factors, with a concomitant decrease in negative APPs such as albumin (16). In addition, a decrease in serum iron levels and consecutive in- crease in hepatic iron levels are also a hallmark of APR. This reaction is mediated by both interleukin 1 (IL-1)Ylike cytokines (IL-1, tumor necrosis factor ! [TNF-!]) and IL-6Ylike cytokines (IL-6, oncostatin M, and others), through the activation of dif- ferent transcription factors. Address reprint requests to Ihtzaz Ahmed Malik, PhD, Department of Gastroenterology and Endocrinology, University Medical Center, Goettingen, Robert-Koch-Strasse 40, D-37099, Goettingen, Germany. E-mail: i.malik@med.uni-goettingen.de. DOI: 10.1097/SHK.0000000000000107 Copyright Ó 2014 by the Shock Society 337 SHOCK, Vol. 41, No. 4, pp. 337Y345, 2014 Copyright © 2014 by the Shock Society. Unauthorized reproduction of this article is prohibited.