IL-6 mediates hypoferremia of inflammation by inducing the synthesis of the iron regulatory hormone hepcidin (original) (raw)

Macrophage culture. Monocytes from peripheral blood were isolated by centrifugation through Ficoll-Paque (Amersham Pharmacia Biotech, Piscataway, New Jersey, USA) and 46% iso-osmotic Percoll gradient (12, 20) (Sigma-Aldrich, St. Louis, Missouri, USA) and their purity confirmed by Wright’s stain. Kupffer cells were isolated from collagenase-digested livers (Liver Tissue Procurement and Distribution System, University of Minnesota, Minneapolis, Minnesota, USA, NIH contract N01-DK-9-2310) by repetitive washing and centrifugation at 19 g and 820 g, followed by selective adherence to plastic. Immunostaining with anti-human CD68 Ab’s (PharMingen, San Diego, California, USA) showed that the Kupffer cell culture was 80% pure. Monocyte-derived macrophages and Kupffer cells were cultured at 106 cells/ml in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% human serum. The cells were incubated with 100 ng/ml LPS (Escherichia coli 055:B5) and the cell-free medium from unstimulated macrophages (M) or from LPS-stimulated macrophages (M-LPS) collected after 24 hours.

IL-6 neutralization and cytokine assays. Human hepatocytes (Liver Tissue Procurement and Distribution System) were cultured in human hepatocyte maintenance medium (Clonetics Corp., San Diego, California, USA) at 37°C, 5% CO2. Hep3B cells (American Tissue Culture Collection, Manassas, Virginia, USA) were cultured in IMDM with 10% FCS at 37°C, 5% CO2. Primary hepatocytes and Hep3B were treated for 24 hours with 100 ng/ml LPS, with M-LPS (final concentration 16.6%), or with the indicated cytokines (R&D Systems Inc., Minneapolis, Minnesota, USA). The treatments were performed with or without the addition of IL-6 neutralizing Ab’s or TNF-α neutralizing Ab’s (6 ∝g/ml; R&D Systems Inc.).

Animals. Animal studies were performed in conformity with applicable laws and guidelines and were approved by the Animal Research Committee at UCLA. Two pairs of IL-6–/– (IL-6 KO) mice on a C57BL/6 background (The Jackson Laboratory, Bar Harbor, Maine, USA) were bred in our rodent facility. WT C57BL/6 mice were either obtained directly from The Jackson Laboratory or from The Jackson Laboratory–derived breeders in our UCLA facility. All mice were maintained on either NIH 31 rodent diet (iron content 336 mg/kg; Harlan Teklad, Indianapolis, Indiana, USA) or Prolab RMH 2000 breeder diet (iron content 440 mg/kg; PMI Nutrition International Inc., Brentwood, Missouri, USA). Based on our pilot studies, hepatic hepcidin mRNA was high and not inducible by LPS, parenteral iron, or turpentine in C57Bl6 mice maintained on such a high-iron content diet (compared with estimated minimal daily requirement of 35 mg/kg diet) (21). Therefore, 12–15 days prior to the experiment, mice were switched to an iron-deficient diet containing 2–4 ppm iron (Harlan Teklad).

Turpentine abscess. Mice were anesthetized with isoflurane (Abbott Laboratories, North Chicago, Illinois, USA), and the interscapular area was shaved and scrubbed with 70% ethanol. Mice then received a 100-∝l injection of pure gum spirits of turpentine (Sunnyside Corp., Wheeling, Illinois, USA) or 0.9% sterile PBS in the interscapular fat pad. Mice were sacrificed 16 hours later.

Mouse tissue collection. Blood was collected by cardiac puncture and stored on ice for at least 30 minutes in polypropylene tubes to induce clotting. Serum was then obtained by two-stage centrifugation. The abdomen was opened, and a small piece of liver (less than 100 mg) was immediately homogenized in TRIzol (Invitrogen Corp., Carlsbad, California, USA).

Quantitative RT-PCR. RNA was prepared using TRIzol reagent according to the manufacturer’s instructions. The cDNA was synthesized using iScript cDNA Synthesis Kit (Bio-Rad Laboratories Inc., Hercules, California, USA). Human hepcidin and G3PDH, as well as mouse haptoglobin expression, were analyzed using iQ SYBR Green Supermix (Bio-Rad Laboratories Inc.). Mouse hepcidin-1 and β-actin expression were analyzed using a multiplex approach with a degradable quenched probe. The primers were as follows: human hepcidin, 5′-CCTGACCAGTGGCTCTGTTT-3′ and 5′-CACATCCCACACTTTGATCG-3′; human G3PDH, 5′-TGGTATCGTGGAAGGACTC-3′ and 5′-AGTAGAGGCAGGGATGATG-3′; mouse hepcidin-1, sense 5′-CCTATCTCCATCAACAGATG-3′, antisense 5′-AACAGATACCACACTGGGAA-3′, and probe 5′-FAM-CCCTGCTTTCTTCCCCGTGCAAAG-Black Hole Quencher-3′; mouse β-actin, sense 5′-ACCCACACTGTGCCCATCTA-3′, antisense 5′-CACGCTCGGTCAGGATCTTC-3′, and probe 5′-Texas red-ATGCTCTCCCTCACGCCATCCTGC-Black Hole Quencher-3′; mouse haptoglobin, 5′-AACTCCCCGAATGTGAGGCA-3′ and 5′-CGTGGCGGGAGATCATCTTG-3′. Amplification was performed at 52–58°C for 40 cycles in iCycler Thermal Cycler (Bio-Rad Laboratories Inc.), and data were analyzed using iCycler iQ Optical System Software. The relative expression in each sample was calculated by a mathematical method based on the real-time PCR efficiencies (22) using as references mRNA mouse β-actin for murine cells and tissues and human G3PDH for human cells. Because of the wide distribution of the relative expressions in mice, medians and nonparametric statistics were used to describe the data.

Human studies. All human studies were performed in accordance with local regulations and the Declaration of Helsinki. Informed consent was obtained from all subjects.

IL-6 infusion in humans. The study was approved by the Ethical Committee of Copenhagen and Frederiksberg Communities, Denmark. Six healthy volunteers were infused with recombinant human IL-6 (rhIL-6) (Sandoz Ltd., Basel, Switzerland) for 3 hours at the rate of 30 ∝g/hour (23). Urine and serum samples were collected prior to infusion, at the end of a 3-hour infusion, and 2 hours after infusion. Urine was also collected 24 hours after the start of the treatment.

Serum IL-6 measurement. We measured serum IL-6 levels in subjects infused with rhIL-6 using a human IL-6 ELISA assay (BioSource International, Camarillo, California, USA) according to the manufacturer’s recommendations.

Iron supplementation in humans. The study was approved by the Human Subjects Protection Committee at UCLA. Six healthy volunteers collected first morning urine for 9 days. In the morning of days 3, 4, and 5, the subjects ingested 65 mg of iron as ferrous sulfate (Nature Made, Mission Hills, California, USA).

Urinary hepcidin assay. Urinary creatinine concentrations were measured by UCLA clinical laboratories. Cationic peptides were extracted from urine using CM Macro-prep (BioRad Laboratories Inc.) (2) eluted with 5% acetic acid, lyophilized, and resuspended in 0.01% acetic acid. Hepcidin urinary concentrations were determined by immunodot assay. Urine extracts equivalent to 0.1–0.5 mg of creatinine were dotted on Immobilon-P membrane (Millipore Corp., Bedford, Massachusetts, USA), along with a range of synthetic hepcidin standards (0–40 ng). Hepcidin was detected on the blots using rabbit anti-human hepcidin Ab’s (12) with goat anti-rabbit HRP as a secondary Ab. Dot blots were developed by the chemiluminescent detection method (SuperSignal West Pico Chemiluminescent Substrate; Pierce Chemical Co., Rockford, Illinois, USA) and quantified with the Chemidoc cooled camera running Quantity One software (BioRad Laboratories Inc.).

Serum iron measurement. Human and mouse serum iron and unsaturated iron-binding capacity (UIBC) were determined using a colorimetric assay (Diagnostic Chemicals Ltd., Oxford, Connecticut, USA), which was modified for the microplate format and verified by testing serial dilutions of the iron standard supplied by the manufacturer. Briefly, 50 ∝l of serum was used for each of the serum iron and UIBC measurements. The total iron-binding capacity (TIBC) was calculated as the sum of serum iron and UIBC, and the percentage of transferrin saturation as serum iron/TIBC ∞ 100.

Statistics. We used Sigma Stat version 3.0 for statistical analyses (Systat Software Inc., Point Richmond, California, USA).