Plenary paper

Brief report

Hepcidin, a putative mediator of anemia of inflammation, is a type II acute-phase protein

Elizabeta Nemeth, Erika V. Valore, Mary Territo, Gary Schiller, Alan Lichtenstein, and Tomas Ganz

Hepcidin is a liver-made peptide proposed to be a central regulator of intestinal iron absorption and iron recycling by macrophages. In animal models, hepcidin is induced by inflammation and iron loading, but its regulation in humans has not been studied. We report that urinary excretion of hepcidin was greatly increased in patients with iron overload, infections, or inflammatory diseases. Hep-

Introduction

cidin excretion correlated well with serum ferritin levels, which are regulated by similar pathologic stimuli. In vitro iron loading of primary human hepatocytes, however, unexpectedly down-regulated hepcidin mRNA, suggesting that in vivo regulation of hepcidin expression by iron stores involves complex indirect effects. Hepcidin mRNA was dramatically induced by interleukin-6 (IL-6) in vitro, but

The recently discovered peptide hepcidinmay be the key mediator of anemia of inflammation.2,3 It is a conserved 25–amino acid peptide produced in the liver and detectable in blood and urine.1,4 Mice lacking hepcidin mRNAdeveloped iron overload affecting the liver and pancreas, with iron deficit in the macrophage-rich spleen.Transgenic mice overexpressing hepcidin died at birth of severe iron deficiency.These studies suggested that hepcidin inhibits iron absorption in the small intestine, the release of recycled iron from macrophages,and transport of iron across the placenta.In agreement with the animal studies, patients with large hepatic adenomas and otherwise unexplained iron-refractory anemia overexpressed hepcidin mRNA in their tumors.Studies of hepcidin mRNAregulation showed increase in iron-overloaded miceand decrease in mice with anemia from bleeding or hemolysis.Mice injected with lipopolysaccharideor turpentine,8and fish with bacterial infectionalso had elevated hepcidin mRNA in the liver. Together, the data suggest hepcidin is induced by iron stores and inflammation, and functions as a signal inhibiting iron absorption in the small intestine and sequestering iron in macrophages.However, all findings on hepcidin regulation have come from animal models. We explored the regulation of hepcidin synthesis in human patients and tissues.

Study design

Subjects

Approval was obtained from the UCLA institutional review board for these studies. Informed consent was provided according to the Declaration of Helsinki. The patients’ total iron-binding capacity (TIBC), and levels of

From the Departments of Medicine and Pathology, David Geffen School of Medicine, and the West Los Angeles Veterans Administration Hospital, University of California, Los Angeles (UCLA), Los Angeles, CA.

Submitted October 25, 2002; accepted November 5, 2002. Prepublished online as Blood First Edition Paper, November 14, 2002; DOI 10.1182/blood-2002-103235.

Supported by the Will Rogers Fund (T.G.).

BLOOD, 1 APRIL 2003 · VOLUME 101, NUMBER 7

not by IL-1 or tumor necrosis factor a (TNF-a), demonstrating that human hepcidin is a type II acute-phase reactant. The linkage of hepcidin induction to inflammation in humans supports its proposed role as a key mediator of anemia of inflammation. (Blood. 2003;101:2461-2463)

© 2003 by The American Society of Hematology

serum iron, ferritin, hemoglobin, hematocrit, and urinary creatinine were determined at the UCLA Hospital Laboratory.

Reagents

Hepcidin antibody was prepared by rabbit immunization with synthetic hepcidinconjugated to keyhole limpet hemocyanin (KLH, no. 7762; Pierce, Rockford, IL).

Urinary hepcidin assay

Cationic peptides were extracted from urine using CM Macroprep (Bio-Rad, Hercules, CA).Urine extracts equivalent to 4 mg of creatinine were analyzed along with synthetic hepcidin standards (0.05, 0.15, 0.5, and 1.5 fg) by sodium dodecyl sulfate (SDS)–Tricine–polyacrylamide gel electrophoresis (PAGE) and Western blotting. Hepcidin was detected on the blots using rabbit antihuman hepcidin antibody.

Hepatocyte culture

Human hepatocytes (Liver Tissue Procurement and Distribution System, Minneapolis, MN) were cultured in human hepatocyte maintenance medium (Clonetics, San Diego, CA) at 37°C, 5% CO2. Hepatocyte treatments included 10 fM ferric-ammonium citrate (FAC; Sigma, St Louis, MO), 30 fM diferric transferrin (Sigma), 20 ng/mL interleukin-1O (IL-1O; R&D Systems, Minneapolis, MN), 20 ng/mL IL-6 (PeproTech, Rocky Hill, NJ), 20 ng/mL tumor necrosis factor O (TNF-O; R&D Systems), 100 ng/mL lipopolysaccharide (LPS; Escherichia coli 055:B5), medium conditioned by monocytes incubated with LPS (Mo-LPS; final concentration 12.5%), and 200 ng/mL IL-1 receptor antagonist (R&D Systems).

Monocyte culture

Blood monocytes were isolated by centrifugation at 400g for 20 minutes through Ficoll-Paque (Amersham Pharmacia Biotech, Piscataway, NJ) and

Reprints: Tomas Ganz, 37-055 CHS, Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, CA 90095-1690; e-mail:[email protected]

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.

© 2003 by The American Society of Hematology