Hepcidin, a master regulator of iron homeostasis, is produced in small amounts by inflammatory monocytes/macrophages. Chronic immune activation leads to iron retention within monocytes/macrophages and the development of anemia of chronic disease (ACD). We questioned whether monocyte-derived hepcidin exerts autocrine regulation toward cellular iron metabolism. Monocyte hepcidin mRNA expression was significantly induced within 3 hours after stimulation with LPS or IL-6, and hepcidin mRNA expression was significantly higher in monocytes of ACD patients than in controls. In ACD patients, monocyte hepcidin mRNA levels were significantly correlated to serum IL-6 concentrations, and increased monocyte hepcidin mRNA levels were associated with decreased expression of the iron exporter ferroportin and iron retention in these cells. Transient transfection experiments using a ferroportin/EmGFP fusion protein construct demonstrated that LPS inducible hepcidin expression in THP-1 monocytes resulted in internalization and degradation of ferroportin. Transfection of monocytes with siRNA directed against hepcidin almost fully reversed this lipopolysaccharide-mediated effect. Using ferroportin mutation constructs, we found that ferroportin is mainly targeted by hepcidin when expressed on the cell surface. Our results suggest that ferroportin expression in inflammatory monocytes is negatively affected by autocrine formation of hepcidin, thus contributing to iron sequestration within monocytes as found in ACD.

A dysregulated iron homeostasis is a cornerstone of acute and chronic inflammatory processes involving cell-mediated immunity and frequently leads to the development of anemia, termed as anemia of chronic disease (ACD), or anemia of inflammation.1,2  ACD is a multifactorial disease, where immune mechanisms play key pathogenetic roles. These include cytokine-mediated suppression of erythropoiesis,3,4  a blunted erythropoietin response,5,7  and an increased iron accumulation in and a defective iron recycling from the reticuloendothelial system.8,,,,13  The liver-derived acute phase protein hepcidin, which is induced by cytokines and iron, plays a key role in this concert.14,15  It causes anemia when overexpressed,16,17  whereas hepcidin mutations lead to hepatic iron overload,18,19  which can be referred to its regulatory effects on cellular iron efflux. This is exerted after binding of hepcidin to the only known cellular iron exporter ferroportin,20,22  leading to ferroportin internalization and blockade of duodenal iron absorption and macrophage iron recycling.23  Because hepcidin expression is induced by inflammatory stimuli, including interleukin-6 (IL-6) or lipopolysaccharide (LPS),24,,,,29  an increased expression of this acute phase protein has been found to be associated with macrophage iron retention in ACD patients.30,31  In addition, hepcidin-independent inhibition of ferroportin mRNA expression by inflammatory cytokines also contributes to macrophage iron retention under inflammatory conditions.32,33 

Interestingly, tissues other than the liver can also synthesize hepcidin, including the kidney, the right heart atrium, and the spinal cord.14,34  Moreover, significant hepcidin expression has been found in spleen, alveolar, and bone marrow-derived murine macrophages.14,35,37  Although the basal expression is relatively low in comparison to liver hepcidin expression, LPS, group A Streptococcus strains and Pseudomonas aeruginosa can induce a 20- to 80-fold increase of hepcidin expression in these cells by a TLR-4–dependent pathway.35,36 

Based on these observations and being aware of a strong correlation between pro-hepcidin serum levels, macrophage ferroportin expression, and iron sequestration within the reticuloendothelial system,30  we now investigated whether monocyte-derived hepcidin may effectively modulate ferroportin expression and thus monocyte iron homeostasis under inflammatory conditions in an autocrine fashion.

The study was approved by the ethics board of the Medical University, Innsbruck, Austria.

Patients

A total of 25 patients were included in the study. Informed consent for obtaining additional blood samples for scientific purposes during routine blood drawing was obtained before the procedure from each subject in accordance with the Declaration of Helsinki. Patients were considered to suffer from ACD when (1) they had a chronic infection or autoimmune disease, (2) when they were anemic with a hemoglobin of less than 13 g/dL for men and less than 12 g/dL for women, and (3) when they had low transferrin saturation (TfS < 16%) but normal or increased serum ferritin concentrations (> 100 ng/mL).1  Among the 12 patients with ACD, 5 had recurrent pneumonia, 3 had rheumatoid arthritis, 1 had large cell vasculitis, and 3 had chronic osteomyelitis. Although some patients received antibiotics at enrollment, none of the patients with newly diagnosed autoimmune disorders had been treated with immunosuppressive drugs before study enrollment. We also studied a group of age-matched controls (n = 13) with no signs of anemia, normal serum iron status, and no signs of inflammation (normal serum concentrations of C-reactive protein < 0.7 mg/dL). We did not include patients with malignancies because radio- and/or chemotherapeutic regimens as well as bone marrow infiltration by the tumor alter the pathophysiology of the anemia compared with subjects with ACD on the basis of an autoimmune or infectious disease.1,38 

None of our patients was under treatment with iron, recombinant human erythropoietin, and/or received blood transfusion before study entry.

The baseline characteristics of the different patient groups are shown in Table 1.

Blood samples were drawn on a routine basis and laboratory parameters (eg, alanine aminotransferase, aspartate aminotransferase, hemoglobin, blood cell count, and serum iron parameters) were examined by routinely used automated laboratory tests, ferritin concentration by an immunoassay, and transferrin concentration by a turbidimetric method. Serum specimens were drawn during this routine examination and stored at −70°C until cytokine assays were performed. Determination of serum IL-6 concentrations was carried out using a commercially available enzyme-linked immunosorbent assay kit obtained from R&D (Quantikine HS ELISA Kit; Minneapolis, MN). The detection limit was 0.04 pg/mL.

Monocyte isolation and cell culture

Peripheral blood mononuclear cells were freshly isolated from whole blood by Ficoll-Paque separation (Pharmacia, Uppsala, Sweden) as previously described.39  For monocyte isolation by plastic adherence, peripheral blood mononuclear cells were resuspended with RPMI 1640 (Biochrom, Berlin, Germany) supplemented with 2 mM l-glutamine, 10% (v/v) heat inactivated fetal calf serum (PAA Laboratories, Pasching, Austria), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (Biochrom). Cells were seeded into a 100-mm dish (BD Biosciences, Franklin Lakes, NJ) and allowed to adhere in a 5% CO2 incubator at 37% for 45 minutes. Nonadherent cells were removed, and the adherent cells were washed carefully, at least twice, with prewarmed phosphate-buffered saline (PBS; Biochrom) before being harvested. The purity of the resulting cells suspension was randomly tested by fluorescent-activated cell sorting analysis and yielded more than 96% monocytes.

THP-1 cells, a human monocytic cell line, were cultured in RPMI 1640 medium with the same supplements as used for monocytes. Cells were seeded in 6-well plates at passage 5 to 15 at a density of 1.5 × 106 cells per well in 3 mL of medium. After allowing them to rest for 12 hours, cells were stimulated with 1 μg/mL LPS, 10 ng/mL IL-6, or 50 μM of ferric chloride for 3, 6, 12, and 24 hours, respectively.

Determination of hepcidin mRNA in human monocytes by TaqMan RT-PCR

Total RNA was extracted from monocytes and THP-1 cells using a guanidinium-isothiocyanate-phenol-chloroform-based procedure as previously described.33  Reverse transcription was performed with 1 μg of total RNA, 10 ng/μL random hexamer primers (Roche Diagnostics, Mannheim, Germany), dNTPs (Roche Diagnostics), 500 μM each, and 200 U M-MLV reverse transcriptase (Invitrogen, Vienna, Austria) in 1 times reverse transcription buffer for 50 minutes at 37°C. TaqMan reverse-transcribed polymerase chain reaction (RT-PCR) primers and probes were designed, and quantification of targets genes by RT-PCR was carried out exactly as described.40  The following hepcidin primers and TaqMan probe were used: forward primer 5′-TTTCCCACAACAGACGGGAC-3′, reverse primer 5′-AGCTGGCCCTGGCTCC-3′, probe 5′-FAM-CAGAGCTGCAACCCCAGGACAGAGC-BHQ-3′, purchased from Microsynth (Balgach, Switzerland). For quantification of the human housekeeping gene beta-actin, the PerkinElmer Life and Analytical Sciences (Waltham, MA) predeveloped assay kit was used.

Western blotting

Membrane protein extracts were prepared from THP-1 and human monocytes as described40  and run on a 10% SDS-polyacrylamide gel. Proteins were transferred onto a nylon membrane (Hybond-P; GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom) and after blocking incubated either with a rabbit anti-GFP-antibody (2 μg/mL, Invitrogen), a mouse monoclonalbiotin conjugated anti-c-Myc-antibody (Santa Cruz Biotechnology, Heidelberg, Germany), a rabbit antihuman ferritin-antibody (2μg/mL, DakoCytomation, Vienna, Austria), a rabbit antihuman ferroportin antibody (1:250; kindly provided by Andrew McKie), or a mouse antihuman β-actin antibody (2 μg/mL; Sigma-Aldrich, St Louis, MO), which was used as a loading control.41 

Immunofluorescence

THP-1 cells were harvested and cytospun at 320g for 5 minutes. For fixation, cells were washed twice with ice-cold PBS and treated with a 4% paraformaldehyde PBS solution for 20 minutes on ice. Afterward, cells were washed twice with PBS again and treated with methanol/acetone (1:1) for 6 minutes. Fixed cells were rinsed 2 times with PBS and incubated with blocking solution (PBS supplemented with 10 mg/mL bovine serum albumin) for 1 hour. THP-1 cells were transfected with ferroportin tagged with fluorescent EmGFP to visualize ferroportin and PKH26 RED Fluorescence Cell Linker (Sigma-Aldrich) was used to visualize the membrane localizing the cell boarder. For microscopic analysis, the cells were covered into a mounting medium to prevent fading on glass slides and observed under an inverted confocal microscope from Carl Zeiss (Jena, Germany). The base frame is an Axiovert 100M with a highly integrated LSM 510 scanning head. Acquisition was done with the Carl Zeiss LSM imaging software SP2 version 2.81. The following objectives were used: Plan-Apochromat objective, ×63 oil, numerical aperture of 1.4, and EC Plan Neofluar ×40, numerical aperture of 0.75. Laser lines at 488 nm and 543 nm were used for excitation. The emission filter for GFP was 505 to 530 nm and for PKH26 RED LP 585 nm.

Purification of native human hepcidin

For the purification of hepcidin, human urine was centrifuged at 3000g for 30 minutes and filtered through a 0.45-μm filter, before it was diluted with PBS. Hepcidin purification was essentially carried out as previously described14  with a minor modification of the first purification step. In the original protocol, CM Sepharose was used, which was substituted for copper charged chelating Sepharose (GE Healthcare) in our modification.

After loading the diluted urine sample, the chelating resin was eluted with 0.1% aqueous trifluoroacetic acid (TFA) and the elution fraction was collected. A 1-μL aliquot of the elution fraction was subsequently analyzed for hepcidin by MALDI-TOF MS (Ultraflex MALDI TOF-TOF; Bruker Daltonics, Bremen, Germany) using an equal volume of saturated α-cyano-4-hydroxy-cinnamic acid in 50% acetonitrile/0.1% TFA as matrix. After spotting onto a stainless steel target and drying in air, fractions were analyzed on a Bruker Ultraflex mass spectrometer operated in reflectron mode. All spectra were recorded by summarizing 400 laser shots, using a 337-nm nitrogen laser with a pulse of 50 Hz. The instrument was calibrated externally using the peptide calibration standard II, purchased from Bruker. The Flex Analysis version 2.4 software packages provided by the manufacturer were used for data processing.

Subsequently, hepcidin was purified by RP HPLC, where the lyophilized and redissolved eluate from the first purification step was further separated on a semipreparative 10 × 250-mm C18 column (218TP510; Grace Vydac, Hesperia, CA) using a gradient from 0.1% TFA in water to 0.1% TFA in acetonitrile. The chromatography was carried out on a Shimadzu High Performance Liquid Chromatography LC-10Avp instrument equipped with an autosampler, gradient pumps, a UV-VIS photodiode array detector, and a fraction collector (Shimadzu, Duisburg, Germany). Data were recorded and handled using LabSolutions software provided by the manufacturer.

To analyze for the presence of hepcidin isoforms, 0.5-mL fractions were collected and 1 μL of each fraction was analyzed by MALDI-TOF MS as described above. Fractions containing hepcidin were pooled and relyophilized, being finally redissolved in distilled water at a concentration of 1 mg/mL.

Plasmid construction and transient transfection

Human wild-type pro-hepcidin was amplified from HepG2 cDNA using 5′-cagggcaggtaggttctagc-3′ as forward and 5′-acagacggcacgatggcact-3′as reverse primer. The PCR product was cloned into the PCR2.1 vector using the TA cloning Kit (Invitrogen). For subcloning into pcDNA3, a eukaryotic expression vector, both plasmids were digested with HindIII/XbaI restriction enzymes.

For generation of an EmGFP N-terminally tagged ferroportin expression clone, we used the Gateway recombination system (Invitrogen). As entry vector, pENTR 221 containing the ferroportin open reading frame was obtained from Invitrogen. LR recombination reaction was performed for transfer of ferroportin open reading sequence into a Vivid Colors pcDNA 6.2/EmGFP-DEST vector to create an EmGFP N-terminally tagged ferroportin expression clone. As control vector, we used Vivid Colors pcDNA 6.2 EmGFP/CAT (chloramphenicol acetyltransferase) plasmid provided by Invitrogen.

pcDNA3.1/c-Myc expression vectors containing the gene encoding ferroportin, and the N144H and A77D ferroportin mutants were designed as described elsewhere.42 

Transfection of THP-1 cells was performed using the Amaxa (Cologne, Germany) system after a routine protocol.43  In brief, THP-1 cells were seeded in 12-well plates at a density of 106 cells per well in 2-mL medium 6 hours before transfection. Cells were centrifuged at 90g for 10 minutes and thereafter resuspended in Cell Line Nucleofector Solution V (Amaxa) to a final concentration of 106 cells/100 μL; 100 μL of cell suspension was mixed with 5 μg of plasmid DNA and transferred to an Amaxa certified cuvette. For transfection, we used the program V-01. Transfection efficiency was between 70% and 80% as checked by immunofluorescence.

Transient hepcidin knockdown by siRNA

Target-specific and control siRNA double-stranded molecules were purchased from Dharmacon RNA Technologies (Lafayette, CO); 3 μg siRNA was nucleofected in 106 cells because this was the optimal amount for protein knockdown. Efficiency of nucleofection was measured by flow cytometry after nucleofecting a TAMRA-labeled siRNA into the same cells under the same conditions. To exclude unspecific effects, each experiment was controlled by the additional nucleofection of a scrambled siRNA. This is defined as reference.

Transfection of THP-1 cells was performed using the Amaxa system after a routine protocol; 100 μL of cell suspension, which was prepared as for transient transfection, was mixed with 3 μg of siRNA and transferred to an Amaxa-certified cuvette. For transfection, we used the program V-01. Transfection efficiency was between 70% and 80% as checked by immunofluorescence. The effect of siRNA knockdown on hepcidin mRNA levels was quantified by real-time PCR.

Intracellular iron determination by graphite furnace atomic absorption spectrometry

Graphite furnace atomic absorption spectrometry was used for quantitative iron determination and carried out with a Unicam Model Solaar, 939 QZ, atomic absorption spectrometer, equipped with a Zeeman-effect background corrector, an FS90 furnace autosampler, and a longitudinally heated atomizer with extended lifetime graphite tubes (Thermo Electron, Waltham, MA). Slit 0.2 and wavelength λ 271.9 nm were used as spectrometer parameters. A Thermo hollow cathode iron lamp (15 mA maximum operating current) was run at 100% maximum current. The calibration solutions (25.0 μg/L to 1.5 μg/L) were prepared by adequate dilution of a 1000 mg/L Titrisol (Merck, Darmstadt, Germany) stock solution with 0.1% Ultrapure nitric acid (Merck), 0.2% Triton X-100, and high-purity water (Milli-Q system, Millipore, Billerica, MA). Samples were suspended by ultrasonication for 40 seconds in 0.1% Ultrapure nitric acid and 0.2% Triton X-100. A pyrolysis temperature of 1000°C and an atomization temperature of 2000°C were used. Argon was used as inert gas at a constant flow of 1 L/min throughout the heating program, except during the atomization step, when the gas flow was interrupted. The accuracy of the procedure was assessed through the analysis of lyophilized control samples (Recipe Chemicals and Instruments, Munich, Germany), which were reconstituted according to the manufacturer's instruction with Milli-Q water and further diluted with 0.1% Ultrapure nitric acid and 0.2% Triton X-100.

Data analysis

Statistical analysis was carried out using SPSS statistics package. Calculations for statistical differences between the various groups were carried out by Student t test or by nonparametric Kruskal-Wallis test. Associations among the various parameters in the different groups were calculated using Spearman's rank correlation technique and Bonferroni correction for multiple tests.

Hepcidin mRNA in primary monocytes and THP-1 cells is induced by LPS and IL-6 but not by iron

Basal levels of hepcidin mRNA could be detected in both freshly isolated human monocytes and THP-1 cells (Figure 1). In time course experiments, we found that hepcidin mRNA expression in human monocytes is most prominently induced after 3 hours of LPS stimulation. Thereafter, hepcidin mRNA expression declines and returns to baseline levels (Figure 1A). Interestingly, IL-6 had a similar kinetic effect (Figure 1B). The same also holds true for THP-1 cells (Figure 1C,D). However, hepcidin induction by LPS and IL-6 in THP-1 cells was less pronounced than in primary monocytes and reached maximum levels 6 hours after stimulation (Figure 1C,D). Interestingly, supplementation of THP-1 cells with ferric chloride had no significant regulatory effect on hepcidin mRNA expression in monocytes (Figure 1E).

Figure 1

Expression and regulation of hepcidin mRNA in primary monocytes and THP-1 cells by LPS, IL-6, and iron. THP-1 cells (C-E) were stimulated for 3, 6, 12, and 24 hours with 1μg/mL LPS (C), 10 ng/mL IL-6 for (D), or 50μM ferric chloride (E). Freshly isolated human monocytes were stimulated for 6 hours with 1 μg/mL LPS (A) or 10 ng/mL IL-6 (B). After that, cells were subjected to RNA preparation, followed by reverse transcription and quantitative TaqMan PCR. Specific values of target genes were normalized to those of beta-actin. Data are depicted as lower quartile, median and upper quartile (boxes), and minimum/maximum ranges (whiskers; *P < .05, control cells vs stimulated cells by Student t test).

Figure 1

Expression and regulation of hepcidin mRNA in primary monocytes and THP-1 cells by LPS, IL-6, and iron. THP-1 cells (C-E) were stimulated for 3, 6, 12, and 24 hours with 1μg/mL LPS (C), 10 ng/mL IL-6 for (D), or 50μM ferric chloride (E). Freshly isolated human monocytes were stimulated for 6 hours with 1 μg/mL LPS (A) or 10 ng/mL IL-6 (B). After that, cells were subjected to RNA preparation, followed by reverse transcription and quantitative TaqMan PCR. Specific values of target genes were normalized to those of beta-actin. Data are depicted as lower quartile, median and upper quartile (boxes), and minimum/maximum ranges (whiskers; *P < .05, control cells vs stimulated cells by Student t test).

Close modal

Monocyte hepcidin expression in ACD patients and association with cellular iron retention

To study monocyte hepcidin expression in vivo and its possible association with monocyte iron retention during inflammation, we investigated primary monocytes of 12 ACD patients and 13 age-matched controls. Hepcidin mRNA expression was detected in monocytes of both groups and was significantly higher in ACD subjects than in controls (P < .05; Figure 2A). Accordingly, as ACD patients suffered from inflammatory diseases, we found a strong positive correlation (R = −0.892, P = .001) between monocyte hepcidin mRNA expression and serum IL-6 levels (Figure 2B), as a marker of inflammation.

Figure 2

Monocyte hepcidin mRNA expression and correlation to serum IL-6 and ferritin levels in ACD patients. (A) Freshly isolated blood monocytes from control (n = 13) and ACD (n = 12) patients were subjected to RNA preparation, followed by reverse transcription and quantitative TaqMan PCR. Specific values of target genes were normalized to those of beta-actin. Data are depicted as lower quartile, median and upper quartile (boxes), and minimum/maximum ranges (whiskers; *P < .05, monocytes from control patients vs monocytes from ACD patients by Student t test). (B) Correlation between monocyte hepcidin mRNA and IL-6 (R = −0.892, P = .001) in ACD patients as determined by Spearman Rank correlation technique. The regression line and the 95% confidence interval are plotted. (C) Correlation between monocyte hepcidin mRNA and serum ferritin (R = −0.027, P = .937) in ACD patients as determined by Spearman Rank correlation technique. The regression line and the 95% confidence interval are plotted.

Figure 2

Monocyte hepcidin mRNA expression and correlation to serum IL-6 and ferritin levels in ACD patients. (A) Freshly isolated blood monocytes from control (n = 13) and ACD (n = 12) patients were subjected to RNA preparation, followed by reverse transcription and quantitative TaqMan PCR. Specific values of target genes were normalized to those of beta-actin. Data are depicted as lower quartile, median and upper quartile (boxes), and minimum/maximum ranges (whiskers; *P < .05, monocytes from control patients vs monocytes from ACD patients by Student t test). (B) Correlation between monocyte hepcidin mRNA and IL-6 (R = −0.892, P = .001) in ACD patients as determined by Spearman Rank correlation technique. The regression line and the 95% confidence interval are plotted. (C) Correlation between monocyte hepcidin mRNA and serum ferritin (R = −0.027, P = .937) in ACD patients as determined by Spearman Rank correlation technique. The regression line and the 95% confidence interval are plotted.

Close modal

In primary monocytes from ACD patients, we observed decreased ferroportin protein levels and increased ferritin levels, a marker of cellular iron retention, compared with controls (Figure 3A). The decrease in ferroportin expression was paralleled by a corresponding increase in monocyte hepcidin mRNA expression (Figure 3A).

Figure 3

Association of monocyte hepcidin mRNA levels with monocyte ferroportin/ferritin expression and intracellular iron levels. (A) Freshly isolated blood monocytes were subjected to protein and RNA preparation for subsequent Western blotting and TaqMan PCR for determination of protein levels of ferroportin, ferritin, and β-actin as well as hepcidin mRNA levels. For Western blotting, one of 4 representative experiments is shown. (B) Freshly isolated blood monocytes were used for intracellular iron measurement by atom absorption technique as described in “Intracellular iron determination by graphite furnace atomic absorption spectrometry.” Data are depicted as lower quartile, median and upper quartile (boxes), and minimum/maximum ranges (whiskers; *P < .05, iron content in monocytes of control vs ACD patients by Student t test).

Figure 3

Association of monocyte hepcidin mRNA levels with monocyte ferroportin/ferritin expression and intracellular iron levels. (A) Freshly isolated blood monocytes were subjected to protein and RNA preparation for subsequent Western blotting and TaqMan PCR for determination of protein levels of ferroportin, ferritin, and β-actin as well as hepcidin mRNA levels. For Western blotting, one of 4 representative experiments is shown. (B) Freshly isolated blood monocytes were used for intracellular iron measurement by atom absorption technique as described in “Intracellular iron determination by graphite furnace atomic absorption spectrometry.” Data are depicted as lower quartile, median and upper quartile (boxes), and minimum/maximum ranges (whiskers; *P < .05, iron content in monocytes of control vs ACD patients by Student t test).

Close modal

In parallel to the increased ferritin levels, we detected enhanced intracellular iron concentrations in monocytes of ACD patients compared with control subjects as measured by atom absorption (Figure 3B).

Monocyte-derived hepcidin causes ferroportin degradation in vitro

To specifically study the effects of monocyte hepcidin on ferroportin expression, an N-terminal EmGFP/ferroportin fusion protein was overexpressed in THP-1 cells as described in “Plasmid construction and transient transfection.” The EmGFP/ferroportin fusion protein was well expressed in transiently transfected THP-1 cells as estimated by Western blot analysis (Figure 4A). Exogenous addition of purified human hepcidin to the cells resulted in a dramatic reduction in EmGFP/ferroportin protein expression (Figure 4A lane 7).

Figure 4

Monocyte-derived hepcidin causes ferroportin degradation in vitro. (A) Western blot of THP-1 cells transfected with EmGFP/CAT expression vector (lanes 1–4; as control) or EmGFP/ferroportin expression vector (lanes 5–8). Cells were either left untreated (control, lanes 1 and 5), cotransfected with a hepcidin expression vector for another 12 hours (lanes 2 and 6), treated with natural human hepcidin (1 μg/mL) for 3 hours (lanes 3 and 7), or stimulated with LPS 1 μg/mL for 6 hours (lanes 4 and 8). Monocytes were subjected to cell membrane extract preparation and subsequent Western blotting for quantification of EmGFP protein expression. Western blotting for β-actin was used as an internal control. One of 4 representative experiments is shown. (B) Immunofluorescence of THP-1 cells: Cells were transfected with ferroportin/EmGFP expression vector and left untreated (lane 1) or stimulated with LPS (1 μg/mL) for 6 hours (lane 2). PKH-26, a red fluorescence cell linker, was used for cell membrane staining. Ferroportin/EmGFP fusion protein showed green fluorescence. Colocalization of both results in a yellow staining. The size of the images is 360 × 360 μm displayed in original resolution of 1024 × 1024 pixel colored in 8 bit using the ×630 magnification.

Figure 4

Monocyte-derived hepcidin causes ferroportin degradation in vitro. (A) Western blot of THP-1 cells transfected with EmGFP/CAT expression vector (lanes 1–4; as control) or EmGFP/ferroportin expression vector (lanes 5–8). Cells were either left untreated (control, lanes 1 and 5), cotransfected with a hepcidin expression vector for another 12 hours (lanes 2 and 6), treated with natural human hepcidin (1 μg/mL) for 3 hours (lanes 3 and 7), or stimulated with LPS 1 μg/mL for 6 hours (lanes 4 and 8). Monocytes were subjected to cell membrane extract preparation and subsequent Western blotting for quantification of EmGFP protein expression. Western blotting for β-actin was used as an internal control. One of 4 representative experiments is shown. (B) Immunofluorescence of THP-1 cells: Cells were transfected with ferroportin/EmGFP expression vector and left untreated (lane 1) or stimulated with LPS (1 μg/mL) for 6 hours (lane 2). PKH-26, a red fluorescence cell linker, was used for cell membrane staining. Ferroportin/EmGFP fusion protein showed green fluorescence. Colocalization of both results in a yellow staining. The size of the images is 360 × 360 μm displayed in original resolution of 1024 × 1024 pixel colored in 8 bit using the ×630 magnification.

Close modal

A similar effect was also seen when THP-1 cells expressing the EmGFP/ferroportin fusion protein were transiently transfected with a hepcidin expression plasmid. Twelve hours after transfection, we found EmGFP/ferroportin levels to be significantly reduced (Figure 4A lane 6), compared with controls (Figure 4A lane 5) or cells transfected with the parent plasmid not containing hepcidin cDNA (not shown).

Finally, we stimulated EmGFP/ferroportin protein expressing cells with LPS for 6 hours because this resulted in maximum hepcidin mRNA expression in these cells (Figure 1). Again, this resulted in reduced EmGFP/ferroportin protein surface expression compared with controls (Figure 4A lane 8).

To further study the effect of monocyte-derived hepcidin on ferroportin surface expression, a double staining of cells was used. Therefore, PKH-26, a red fluorescence dye, was used for membrane staining. As EmGFP/ferroportin shows green fluorescence, colocalization of both was shown by yellow fluorescence (Figure 4Bi). Stimulation of cells with LPS (1 μg/mL) reduced yellow fluorescence at the cell surface, indicating internalization of ferroportin (Figure 4Bii).

Hepcidin knockdown inhibits ferroportin degradation in vitro

To examine whether the reduced ferroportin surface expression after LPS stimulation is indeed mediated by an increased endogenous formation of hepcidin mRNA in THP-1 cells, we performed hepcidin knockdown experiments using a hepcidin siRNA approach. After nucleofection of THP-1 cells with the specific siRNA, we found highly significant reduction of hepcidin mRNA levels (P = .001) compared with control cells and cells transfected with the scrambled siRNA, respectively (Figure 5A).

Figure 5

Hepcidin knockdown inhibits ferroportin degradation in vitro. (A) Determination of relative abundance of hepcidin expression in THP-1 cells. Cells were left untreated, as a control, or nucleofected with 3 μg of hepcidin siRNA, or, to exclude unspecific effects, nucleofected with a scrambled siRNA. At 36 hours after nucleofection, the mRNA degradation was monitored by real-time PCR. Data are shown as mean plus or minus SD for relative abundances (*P < .001, THP-1 cells nucleofected with 3 μg of hepcidin siRNA vs THP-1 cells transfected with a scrambled siRNA by Student t test). (B) Western blot of THP-1 cells transfected with EmGFP/FP-1 expression vector (lanes 1–4) or mock transfected (lane 5) as a control. Cells were left untreated (lane 1), cotransfected with hepcidin siRNA (lane 2), treated with LPS (1 μg/mL) for 6 hours (lane 3), or cotransfected with hepcidin siRNA and stimulated with LPS (1 μg/mL) for 6 hours (lane 4). Monocytes were subjected to cell membrane extract preparation and subsequent Western blotting for quantification of EmGFP protein expression. Western blotting for β-actin was used as an internal control. One of 4 representative experiments is shown.

Figure 5

Hepcidin knockdown inhibits ferroportin degradation in vitro. (A) Determination of relative abundance of hepcidin expression in THP-1 cells. Cells were left untreated, as a control, or nucleofected with 3 μg of hepcidin siRNA, or, to exclude unspecific effects, nucleofected with a scrambled siRNA. At 36 hours after nucleofection, the mRNA degradation was monitored by real-time PCR. Data are shown as mean plus or minus SD for relative abundances (*P < .001, THP-1 cells nucleofected with 3 μg of hepcidin siRNA vs THP-1 cells transfected with a scrambled siRNA by Student t test). (B) Western blot of THP-1 cells transfected with EmGFP/FP-1 expression vector (lanes 1–4) or mock transfected (lane 5) as a control. Cells were left untreated (lane 1), cotransfected with hepcidin siRNA (lane 2), treated with LPS (1 μg/mL) for 6 hours (lane 3), or cotransfected with hepcidin siRNA and stimulated with LPS (1 μg/mL) for 6 hours (lane 4). Monocytes were subjected to cell membrane extract preparation and subsequent Western blotting for quantification of EmGFP protein expression. Western blotting for β-actin was used as an internal control. One of 4 representative experiments is shown.

Close modal

We then could demonstrate that transfection of EmGFP/ferroportin expressing THP-1 cells with a hepcidin siRNA already results in a higher EmGFP/ferroportin expression compared with cells transfected with the scrambled siRNA. Moreover, the hepcidin specific siRNA almost fully prevented the LPS-induced reduction in EmGFP/ferroportin expression (Figure 5B).

Effects of hepcidin challenge on the expression of ferroportin mutation variants

To specifically study whether hepcidin affects ferroportin only on the cell surface or also intracellularly, we transiently transfected THP-1 cells with 2 different ferroportin mutants. Whereas the A77D variant is not expressed on the cell surface and does not show any response to extracellular hepcidin,44  the N144H mutant is found on the cell surface but only partially internalizes in response to hepcidin exposure.45 

As can be seen in Figure 6 (left panel), wild-type ferroportin as well as the 2 mutant ferroportin variants are expressed in transiently transfected THP-1 cells and can be detected by Western blot. The lower basal expression of the A77D mutant after transfection is in accordance with previous observations.42  When treating cells with LPS for 6 hours, wild-type ferroportin expression was clearly reduced compared with baseline levels. Whereas a small but in comparison to wild-type ferroportin much less pronounced reduction in the expression of A77D ferroportin was observed compared with baseline levels, no change could be detected for the N144H mutant on LPS treatment. Accordingly, addition of purified human hepcidin (1 μg/mL) to the culture medium reduces wild-type ferroportin expression, whereas almost no effect is observed toward the 2 ferroportin mutation variants.

Figure 6

Effects of hepcidin challenge on the expression of ferroportin mutation variants. Western blots of THP-1 cells transfected with ferroportin/c-Myc expression vector (lanes 1, 4, and 7), A77D ferroportin mutant/c-Myc expression vector (lanes 2, 5, and 8), and N144H ferroportin mutant/c-Myc expression vector (lanes 3, 6, and 9). Cells were either left untreated (lanes 1–3), treated with LPS (1 μg/mL) for 6 hours (lanes 4–6) or exposed to native human hepcidin (1 μg/mL) for 3 hours (lanes 7–9). Monocytes were subjected to protein extract preparation and subsequent Western blotting for quantification of c-Myc protein expression. Western blotting for β-actin was used as an internal control. One of 3 representative experiments is shown.

Figure 6

Effects of hepcidin challenge on the expression of ferroportin mutation variants. Western blots of THP-1 cells transfected with ferroportin/c-Myc expression vector (lanes 1, 4, and 7), A77D ferroportin mutant/c-Myc expression vector (lanes 2, 5, and 8), and N144H ferroportin mutant/c-Myc expression vector (lanes 3, 6, and 9). Cells were either left untreated (lanes 1–3), treated with LPS (1 μg/mL) for 6 hours (lanes 4–6) or exposed to native human hepcidin (1 μg/mL) for 3 hours (lanes 7–9). Monocytes were subjected to protein extract preparation and subsequent Western blotting for quantification of c-Myc protein expression. Western blotting for β-actin was used as an internal control. One of 3 representative experiments is shown.

Close modal

Herein we examined the endogenous formation of hepcidin in human monocytic cells and primary monocytes from patients with ACD and investigated its impact on monocyte iron homeostasis.

We found that both freshly isolated human monocytes and THP-1 cells, a monocytic cell line, express significant levels of hepcidin mRNA, which is in accordance with data obtained in mice.35,37  LPS as well as IL-6 significantly induced hepcidin formation in both cell types, but the inducibility of hepcidin by these inflammatory stimuli was more pronounced in primary monocytes compared with THP-1 cells. These results are at least in part in contrast to macrophage results as published by Nguyen et al37  and Liu et al,35  as they found no induction of hepcidin mRNA by IL-6. This may be referred to the well-known differences in immune response pattern and metabolic pathways between monocytes and macrophages occurring during differentiation and cell maturation. Interestingly, after a single stimulus of LPS and/or IL-6, the induction of hepcidin mRNA in human monocytes was very rapid, showing a maximum expression already 3 hours after stimulation. Thereafter, hepcidin mRNA levels returned to baseline within a few hours, indicating either low stability of hepcidin mRNA and/or the presence of potent negative regulators of hepcidin gene expression within monocytes.

Accordingly, hepcidin mRNA expression was increased in monocytes of ACD patients, and its relationship to inflammation was supported by a strong correlation with IL-6 levels, which could not be found in control patients. This is in accordance with data obtained from mice, indicating that hepcidin formation by cells of the reticuloendothelial system is primarily regulated by inflammatory stimuli35,37  rather than by iron status.37 

So far it is not clear whether the same signals regulate hepcidin formation in the liver and/or myeloid cells. In murine macrophages, hepcidin is induced by a TLR-4–dependent pathway,36  whereas in human monocytes, as shown here, it can also be induced via IL-6 induction, which is TLR-4 independent and involves STAT-3-dependent activation.46,48  Moreover, the induction kinetics of hepcidin appears to differ between monocytes/macrophages and hepatocytes. Whereas in vivo LPS stimulation results in a 2-phase kinetic of liver hepcidin formation, showing a rapid induction after a few hours, a subsequent decline thereafter, and a second peak after more than 24 hours,49  hepcidin formation by monocytes is short and self-limited as shown herein. These observations may be traced back to variations in the hepcidin promoter region or different signaling pathways between these tissues.

The functionality of endogenous hepcidin on monocyte iron homeostasis was confirmed by experiments using an EmGFP/ferroportin fusion protein expressed in THP-1 cells. In accordance with Nemeth et al,23  we found that overexpression of endogenous hepcidin or exogenous administration of human hepcidin significantly reduced ferroportin expression (Figure 4A) and the localization of EmGFP/ferroportin at the cell surface as indicated by immunofluorescence (Figure 4B).

The functional importance of monocyte-derived hepcidin for ferroportin expression was further confirmed by our results, demonstrating that siRNA directed against hepcidin impairs the LPS-mediated reduction of ferroportin levels.

It needs to be considered that the hepcidin mRNA content of monocytes is approximately 1000 times lower than that of hepatocytes.36  However, whereas liver-derived hepcidin is the master regulator of iron homeostasis in the circulation, we propose that hepcidin formation by activated monocytes/macrophages results in a biologically significant accumulation of this peptide in the inflammatory environment, which then affects iron homeostasis of monocytes/tissue macrophages in an autocrine and paracrine fashion. This may be of specific importance at inflammatory sites with poor perfusion, such as the interstitium, where circulating hepcidin is not sufficiently abundant.

To study whether endogenously formed hepcidin acts as a secreted peptide on monocyte ferroportin or directly within the cell by affecting the posttranslational processing and trafficing of ferroportin to the outer membrane, we performed transient transfection experiments using 2 ferroportin mutation variants. Thereby, we found that neither LPS nor exogenous addition of purified hepcidin significantly affected the expression of the N144H variant, which is expressed on the cell surface but only partially internalizes on hepcidin treatment. When using the cytoplasmatic ferroportin mutant A77D, which is not exposed on the cell surface, we found no change in its expression on addition of hepcidin to the culture medium, whereas a small reduction was observed on LPS treatment. These results indicate that hepcidin produced by monocytes targets membrane bound ferroportin primarily as a secreted peptide in an autocrine way; however, hepcidin also affects, to an albeit much lesser extent, ferroportin expression within the cell. These latter data are in accordance with a recent study50  indicating 2 pathways of hepcidin trafficking within macrophages.

Formation of hepcidin by monocytes may contribute to the development of iron retention seen in ACD. This is indicative from our observations of increased hepcidin formation by monocytes of ACD patients together with the association between increased hepcidin mRNA, monocyte ferritin levels, and monocyte iron concentrations on the one side and reduced monocyte ferroportin expression. As cytokines and LPS have also been shown to down-regulate the mRNA expression of ferroportin, thus acting upstream of the hepcidin-ferroportin interaction,32,33  we hypothesize that cytokines and hepcidin collaborate in modulating monocyte iron homeostasis in the following way.

In the case of an infectious challenge, monocytes/macrophages are aimed to restrict iron to invading pathogens to limit their growth.51,52  As monocytes/macrophages express ferroportin at their cell membrane to maintain iron recirculation, the rapid induction of hepcidin by cytokines and LPS would result in immediate blockage of iron export by ferroportin because of hepcidin-mediated degradation of the protein, thus resulting in iron restriction within macrophages/monocytes. This would fit to the rapid iron sequestration in macrophages seen within hours after LPS stimulation.33 

Subsequently, LPS and cytokines, such as interferon-γ inhibit ferroportin mRNA expression to keep iron export low and to withhold iron from pathogens. These series of events ultimately lead to iron accumulation in monocytes/macrophages, hypoferremia, and an iron-restricted erythropoiesis contributing to the development of ACD.

Thus, the formation of hepcidin by monocyte/macrophage and its autocrine effects on iron homeostasis of these cells would be part of the innate immune defense to reduce the availability of the essential nutrient iron from pathogens.

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 USC section 1734.

The authors thank Dr A. McKie (Kings College, London, United Kingdom) for his kind gift of ferroportin antibody and Dr Lisa Schimanski (Institute for Molecular Medicine, Oxford, United Kingdom) for generously providing the ferroportin mutation plasmids.

This work was supported by the Austrian Research Funds FWF (P-19 664; G.W.), the TWF (0404/237; I.T.), and Research Funds from the OENB (P-12 558; I.T.).

Contribution: I.T. and G.W. designed the research; I.T., M.T., M.S., S.M., M.N., H.Z., R.B.-W., H.N., H.T., and G.W. performed research and examination of patients; I.T., M.T., and G.W. controlled and analyzed data; I.T. and G.W. wrote the paper; I.T., M.T., M.S., S.M., M.N., H.R., H.Z., R.B.-W., H.N., H.T., and G.W. checked the final version.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Günter Weiss, Medical University, Departmentof General Internal Medicine, Clinical Immunology and Infectious Diseases, Anichstr 35, A-6020 Innsbruck, Austria; e-mail: guenter.weiss@i-med.ac.at.

1
Weiss
G
Goodnough
LT
Anemia of chronic disease.
N Engl J Med
2005
352
1011
1023
2
Oppenheimer
SJ
Iron and its relation to immunity and infectious disease.
J Nutr
2001
131
suppl
616S
633S
discussion 633S–635S
3
Means
RT
Jr
Recent developments in the anemia of chronic disease.
Curr Hematol Rep
2003
2
116
121
4
Wang
CQ
Udupa
KB
Lipschitz
DA
Interferon-gamma exerts its negative regulatory effect primarily on the earliest stages of murine erythroid progenitor cell development.
J Cell Physiol
1995
162
134
138
5
Jelkmann
W
Proinflammatory cytokines lowering erythropoietin production.
J Interferon Cytokine Res
1998
18
555
559
6
Means
RT
Jr
Krantz
SB
Inhibition of human erythroid colony-forming units by gamma interferon can be corrected by recombinant human erythropoietin.
Blood
1991
78
2564
2567
7
Beguin
Y
Clemons
GK
Pootrakul
P
Fillet
G
Quantitative assessment of erythropoiesis and functional classification of anemia based on measurements of serum transferrin receptor and erythropoietin.
Blood
1993
81
1067
1076
8
Alvarez-Hernandez
X
Liceaga
J
McKay
IC
Brock
JH
Induction of hypoferremia and modulation of macrophage iron metabolism by tumor necrosis factor.
Lab Invest
1989
61
319
322
9
Torti
FM
Torti
SV
Regulation of ferritin genes and protein.
Blood
2002
99
3505
3516
10
Knutson
M
Wessling-Resnick
M
Iron metabolism in the reticuloendothelial system.
Crit Rev Biochem Mol Biol
2003
38
61
88
11
Weiss
G
Iron and immunity: a double-edged sword.
Eur J Clin Invest
2002
32
Suppl 1
70
78
12
Weiss
G
Bogdan
C
Hentze
MW
Pathways for the regulation of macrophage iron metabolism by the anti-inflammatory cytokines IL-4 and IL-13.
J Immunol
1997
158
420
425
13
Mulero
V
Brock
JH
Regulation of iron metabolism in murine J774 macrophages: role of nitric oxide-dependent and -independent pathways following activation with gamma interferon and lipopolysaccharide.
Blood
1999
94
2383
2389
14
Park
CH
Valore
EV
Waring
AJ
Ganz
T
Hepcidin, a urinary antimicrobial peptide synthesized in the liver.
J Biol Chem
2001
276
7806
7810
15
Pigeon
C
Ilyin
G
Courselaud
B
et al
A new mouse liver-specific gene, encoding a protein homologous to human antimicrobial peptide hepcidin, is overexpressed during iron overload.
J Biol Chem
2001
276
7811
7819
16
Nicolas
G
Bennoun
M
Porteu
A
et al
Severe iron deficiency anemia in transgenic mice expressing liver hepcidin.
Proc Natl Acad Sci U S A
2002
99
4596
4601
17
Weinstein
DA
Roy
CN
Fleming
MD
Loda
MF
Wolfsdorf
JI
Andrews
NC
Inappropriate expression of hepcidin is associated with iron refractory anemia: implications for the anemia of chronic disease.
Blood
2002
100
3776
3781
18
Roetto
A
Papanikolaou
G
Politou
M
et al
Mutant antimicrobial peptide hepcidin is associated with severe juvenile hemochromatosis.
Nat Genet
2003
33
21
22
19
Pietrangelo
A
Hemochromatosis: An endocrine liver disease.
Hepatology
2007
46
1291
1301
20
McKie
AT
Marciani
P
Rolfs
A
et al
A novel duodenal iron-regulated transporter, IREG1, implicated in the basolateral transfer of iron to the circulation.
Mol Cell
2000
5
299
309
21
Donovan
A
Brownlie
A
Zhou
Y
et al
Positional cloning of zebrafish ferroportin1 identifies a conserved vertebrate iron exporter.
Nature
2000
403
776
781
22
Abboud
S
Haile
DJ
A novel mammalian iron-regulated protein involved in intracellular iron metabolism.
J Biol Chem
2000
275
19906
19912
23
Nemeth
E
Tuttle
MS
Powelson
J
et al
Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization.
Science
2004
306
2090
2093
24
Nemeth
E
Rivera
S
Gabayan
V
et al
IL-6 mediates hypoferremia of inflammation by inducing the synthesis of the iron regulatory hormone hepcidin.
J Clin Invest
2004
113
1271
1276
25
Lee
P
Peng
H
Gelbart
T
Wang
L
Beutler
E
Regulation of hepcidin transcription by interleukin-1 and interleukin-6.
Proc Natl Acad Sci U S A
2005
102
1906
1910
26
Lee
P
Peng
H
Gelbart
T
Beutler
E
The IL-6- and lipopolysaccharide-induced transcription of hepcidin in HFE-, transferrin receptor 2-, and beta 2-microglobulin-deficient hepatocytes.
Proc Natl Acad Sci U S A
2004
101
9263
9265
27
Nemeth
E
Valore
EV
Territo
M
Schiller
G
Lichtenstein
A
Ganz
T
Hepcidin, a putative mediator of anemia of inflammation, is a type II acute-phase protein.
Blood
2003
101
2461
2463
28
Nicolas
G
Chauvet
C
Viatte
L
et al
The gene encoding the iron regulatory peptide hepcidin is regulated by anemia, hypoxia, and inflammation.
J Clin Invest
2002
110
1037
1044
29
Kemna
E
Pickkers
P
Nemeth
E
van der Hoeven
H
Swinkels
D
Time-course analysis of hepcidin, serum iron, and plasma cytokine levels in humans injected with LPS.
Blood
2005
106
1864
1866
30
Theurl
I
Mattle
V
Seifert
M
Mariani
M
Marth
C
Weiss
G
Dysregulated monocyte iron homeostasis and erythropoietin formation in patients with anemia of chronic disease.
Blood
2006
107
4142
4148
31
Andrews
NC
Anemia of inflammation: the cytokine-hepcidin link.
J Clin Invest
2004
113
1251
1253
32
Yang
F
Liu
XB
Quinones
M
Melby
PC
Ghio
A
Haile
DJ
Regulation of reticuloendothelial iron transporter MTP1 (Slc11a3) by inflammation.
J Biol Chem
2002
277
39786
39791
33
Ludwiczek
S
Aigner
E
Theurl
I
Weiss
G
Cytokine-mediated regulation of iron transport in human monocytic cells.
Blood
2003
101
4148
4154
34
Kulaksiz
H
Theilig
F
Bachmann
S
et al
The iron-regulatory peptide hormone hepcidin: expression and cellular localization in the mammalian kidney.
J Endocrinol
2005
184
361
370
35
Liu
XB
Nguyen
NB
Marquess
KD
Yang
F
Haile
DJ
Regulation of hepcidin and ferroportin expression by lipopolysaccharide in splenic macrophages.
Blood Cells Mol Dis
2005
35
47
56
36
Peyssonnaux
C
Zinkernagel
AS
Datta
V
Lauth
X
Johnson
RS
Nizet
V
TLR4-dependent hepcidin expression by myeloid cells in response to bacterial pathogens.
Blood
2006
107
3727
3732
37
Nguyen
NB
Callaghan
KD
Ghio
AJ
Haile
DJ
Yang
F
Hepcidin expression and iron transport in alveolar macrophages.
Am J Physiol Lung Cell Mol Physiol
2006
291
L417
L425
38
Spivak
JL
Iron and the anemia of chronic disease.
Oncology (Williston Park)
2002
16
25
33
39
Weiss
G
Murr
C
Zoller
H
et al
Modulation of neopterin formation and tryptophan degradation by Th1- and Th2-derived cytokines in human monocytic cells.
Clin Exp Immunol
1999
116
435
440
40
Ludwiczek
S
Theurl
I
Artner-Dworzak
E
Chorney
M
Weiss
G
Duodenal HFE expression and hepcidin levels determine body iron homeostasis: modulation by genetic diversity and dietary iron availability.
J Mol Med
2004
82
373
382
41
Theurl
I
Ludwiczek
S
Eller
P
et al
Pathways for the regulation of body iron homeostasis in response to experimental iron overload.
J Hepatol
2005
43
711
719
42
Schimanski
LM
Drakesmith
H
Merryweather-Clarke
AT
et al
In vitro functional analysis of human ferroportin (FPN) and hemochromatosis-associated FPN mutations.
Blood
2005
105
4096
4102
43
Theurl
I
Zoller
H
Obrist
P
et al
Iron regulates hepatitis C virus translation via stimulation of expression of translation initiation factor 3.
J Infect Dis
2004
190
819
825
44
De Domenico
I
Ward
DM
Langelier
C
et al
The molecular mechanism of hepcidin-mediated ferroportin down-regulation.
Mol Biol Cell
2007
18
2569
2578
45
Drakesmith
H
Schimanski
LM
Ormerod
E
et al
Resistance to hepcidin is conferred by hemochromatosis-associated mutations of ferroportin.
Blood
2005
106
1092
1097
46
Verga Falzacappa
MV
Vujic Spasic
M
Kessler
R
Stolte
J
Hentze
MW
Muckenthaler
MU
STAT3 mediates hepatic hepcidin expression and its inflammatory stimulation.
Blood
2007
109
353
358
47
Pietrangelo
A
Dierssen
U
Valli
L
et al
STAT3 is required for IL-6-gp130-dependent activation of hepcidin in vivo.
Gastroenterology
2007
132
294
300
48
Wrighting
DM
Andrews
NC
Interleukin-6 induces hepcidin expression through STAT3.
Blood
2006
108
3204
3209
49
Yeh
KY
Yeh
M
Glass
J
Hepcidin regulation of ferroportin 1 expression in the liver and intestine of the rat.
Am J Physiol Gastrointest Liver Physiol
2004
286
G385
G394
50
Sow
FB
Florence
WC
Satoskar
AR
Schlesinger
LS
Zwilling
BS
Lafuse
WP
Expression and localization of hepcidin in macrophages: a role in host defense against tuberculosis.
J Leukoc Biol
2007
82
934
945
51
Weinberg
ED
Iron loading and disease surveillance.
Emerg Infect Dis
1999
5
346
352
52
Nairz
M
Weiss
G
Molecular and clinical aspects of iron homeostasis: from anemia to hemochromatosis.
Wien Klin Wochenschr
2006
118
442
462

Author notes

I.T. and M.T. contributed equally to this study.

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